Patent application title: CYSTEINE REACTIVE PROBES AND USES THEREOF
Inventors:
Benjamin F. Cravatt (La Jolla, CA, US)
Benjamin F. Cravatt (La Jolla, CA, US)
Keriann M. Backus (La Jolla, CA, US)
Bruno E. Correia (La Jolla, CA, US)
Megan M. Blewett (San Diego, CA, US)
John R. Teijaro (San Diego, CA, US)
IPC8 Class: AG01N3368FI
USPC Class:
1 1
Class name:
Publication date: 2020-09-17
Patent application number: 20200292555
Abstract:
Disclosed herein are methods, compositions, probes, polypeptides, assays,
and kits for identifying a cysteine containing protein as a binding
target for a small molecule fragment. Also disclosed herein are methods,
compositions, and probes for mapping a biologically active cysteine site
on a protein and screening a small molecule fragment for interaction with
a cysteine containing protein.Claims:
1. A method of identifying a cysteine containing protein as a binding
target for a small molecule fragment, comprising: a) obtaining a set of
cysteine-reactive probe-protein complexes from a sample comprising a
first cell solution treated with a small molecule fragment and a cysteine
reactive probe wherein the cysteine-reactive probe comprises a reactive
moiety capable of forming a covalent bond with a cysteine residue located
on the cysteine containing protein; b) analyzing the set of
cysteine-reactive probe-protein complexes by a proteomic analysis means;
and c) based on step b), identifying a cysteine containing protein as the
binding target for the small molecule fragment.
2. The method of claim 1, further comprising determining a value of each of the cysteine containing protein from the set of cysteine-reactive probe-protein complexes for identifying a cysteine containing protein as the binding target for the small molecule fragment, wherein the value is determined based on the proteomic analysis means of step b).
3. The method of claim 1, wherein the sample further comprises a second cell solution.
4. The method of claim 1, further comprising contacting the first cell solution with a small molecule fragment for an extended period of time prior to incubating the first cell solution with a first cysteine-reactive probe to generate a first group of cysteine-reactive probe-protein complexes.
5. The method of claim 4, wherein the extended period of time is about 5, 10, 15, 20, 30, 60, 90, 120 minutes or longer.
6. The method of claim 3, further comprising contacting the second cell solution with a second cysteine-reactive probe to generate a second group of cysteine-reactive probe-protein complexes.
7. The method of claim 3, wherein the first cysteine-reactive probe and the second cysteine-reactive probe are the same.
8. The method of claim 3, wherein the first group and the second group of cysteine-reactive probe-protein complexes comprise the set of cysteine-reactive probe-protein complexes.
9. The method of claim 1, wherein the cysteine containing protein is an enzyme, a transporter, a receptor, a channel protein, an adaptor protein, a chaperone, a signaling protein, a plasma protein, transcription related protein, translation related protein, mitochondrial protein, or cytoskeleton related protein.
10. The method of claim 1, wherein the cysteine containing protein is a protein illustrated in Table 3.
11. The method of claim 1, wherein the cysteine containing protein is a protein illustrated in Table 1, Table 2, Table 8, Table 9, Table 10A, Table 10B, Table 10C, Table 10D or Table 10E.
12. The method of claim 1, wherein the small molecule fragment is a small molecule fragment of Formula (I): ##STR00105## wherein: RM is a reactive moiety selected from a Michael acceptor moiety, a leaving group moiety, or a moiety capable of forming a covalent bond with the thiol group of a cysteine residue; and F is a small molecule fragment moiety.
13. The method of claim 12, wherein the Michael acceptor moiety comprises an alkene or an alkyne moiety.
14. The method of claim 12, wherein F is obtained from a compound library.
15. The method of claim 14, wherein the compound library comprises ChemBridge fragment library, Pyramid Platform Fragment-Based Drug Discovery, Maybridge fragment library, FRGx from AnalytiCon, TCI-Frag from AnCoreX, Bio Building Blocks from ASINEX, BioFocus 3D from Charles River, Fragments of Life (FOL) from Emerald Bio, Enamine Fragment Library, IOTA Diverse 1500, BIONET fragments library, Life Chemicals Fragments Collection, OTAVA fragment library, Prestwick fragment library, Selcia fragment library, TimTec fragment-based library, Allium from Vitas-M Laboratory, or Zenobia fragment library.
16. The method of claim 12, wherein F is a small molecule fragment moiety illustrated in FIG. 3.
17. The method of claim 1, wherein the cysteine-reactive probe is a cysteine-reactive probe of Formula (II): ##STR00106## wherein: RM is a reactive moiety selected from a Michael acceptor moiety, a leaving group moiety, or a moiety capable of forming a covalent bond to the thiol group of a cysteine residue; and AHM is an affinity handle moiety.
18. The method of claim 17, wherein the Michael acceptor moiety comprises an alkene or an alkyne moiety.
19. The method of claim 17, wherein the affinity handle moiety comprises an affinity handle and a binding moiety that facilitates covalent interaction of the cysteine-reactive probe to a cysteine residue of a cysteine-containing protein.
20. The method of claim 17, wherein the affinity handle comprises a carbodiimide, N-hydroxysuccinimide (NHS) ester, imidoester, pentafluorophenyl ester, hydroxymethyl phosphine, maleimide, haloacetyl, pyridyl disulfide, thiosulfonate, vinylsulfone, hydrazide, alkoxyamine, alkyne, azide, or isocyanate group.
Description:
CROSS-REFERENCE
[0001] This application is a continuation of U.S. application Ser. No. 15/331,745, filed on Oct. 21, 2016, which claims the benefit of U.S. Provisional Application No. 62/345,710, filed on Jun. 3, 2016, and U.S. Provisional Application No. 62/244,881, filed on Oct. 22, 2015, each of which are incorporated herein by reference in their entireties.
SEQUENCE LISTING
[0003] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Oct. 19, 2016, is named 48054-702_301_SL.txt and is 372,896 bytes in size.
BACKGROUND OF THE INVENTION
[0004] Protein function assignment has been benefited from genetic methods, such as target gene disruption, RNA interference, and genome editing technologies, which selectively disrupt the expression of proteins in native biological systems. Chemical probes offer a complementary way to perturb proteins that have the advantages of producing graded (dose-dependent) gain- (agonism) or loss- (antagonism) of-function effects that are introduced acutely and reversibly in cells and organisms. Small molecules present an alternative method to selectively modulate proteins and to serve as leads for the development of novel therapeutics.
SUMMARY OF THE INVENTION
[0005] Disclosed herein, in certain embodiments, is a method of identifying a cysteine containing protein as a binding target for a small molecule fragment, comprising: (a) obtaining a set of cysteine-reactive probe-protein complexes from a sample treated with a cysteine-reactive probe wherein the cysteine-reactive probe comprises a reactive moiety capable of forming a covalent bond with a cysteine residue located on the cysteine containing protein; (b) analyzing the set of cysteine-reactive probe-protein complexes by a proteomic analysis means; (c) based on step b), identifying a cysteine containing protein as the binding target for the small molecule fragment. In some embodiments, the method further comprises assigning a value to each of the cysteine containing protein from the set of cysteine-reactive probe-protein complexes for identifying a cysteine containing protein as the binding target for the small molecule fragment, wherein the value is determined based on the proteomic analysis means of step b) In some embodiments, the sample comprises a first cell solution and a second cell solution. In some embodiments, the method further comprises contacting the first cell solution with a small molecule fragment for an extended period of time prior to incubating the first cell solution with a first cysteine-reactive probe to generate a first group of cysteine-reactive probe-protein complexes. In some embodiments, the extended period of time is about 5, 10, 15, 20, 30, 60, 90, 120 minutes or longer. In some embodiments, the method further comprises contacting the second cell solution with a second cysteine-reactive probe to generate a second group of cysteine-reactive probe-protein complexes. In some embodiments, the first cysteine-reactive probe and the second cysteine-reactive probe are the same. In some embodiments, the first group and the second group of cysteine-reactive probe-protein complexes comprise the set of cysteine-reactive probe-protein complexes. In some embodiments, cells from the second cell solution are grown in a media (e.g., an isotopically enriched media). In some embodiments, cells from the first cell solution are grown in a media (e.g., an isotopically enriched media). In some embodiments, cells from both the first cell solution and the second cell solution are grown in two different isotopically enriched media so that cells from the first cell solution is distinguishable from cells obtained from the second cell solution. In other embodiments, cells from only one of the cell solutions (e.g., either the first cell solution or the second cell solution) are grown in an isotopically enriched media. In some embodiments, the method further comprises contacting the first cell solution with a first set of small molecule fragments and a complementing set of cysteine-reactive probes wherein each small molecule fragment competes with its complementing cysteine-reactive probe for binding with a cysteine residue, and wherein each small molecule fragment and each complementing cysteine-reactive probe are different within each respective set. In some embodiments, the method further comprises contacting the second cell solution with a second set of cysteine-reactive probes wherein the second set of cysteine-reactive probes is the same as the complementing set of cysteine-reactive probes, and wherein each cysteine-reactive probe is different within the set. In some embodiments, the first set of cysteine-reactive probes generates a third group of cysteine-reactive probe-protein complexes and the second set of cysteine-reactive probes generates a fourth group of cysteine-reactive probe-protein complexes. In some embodiments, the cysteine containing protein comprises a biologically active cysteine residue. In some embodiments, the biologically active cysteine site is a cysteine residue that is located about 10 .ANG. or less to an active-site ligand or residue. In some embodiments, the cysteine residue that is located about 10 .ANG. or less to the active-site ligand or residue is an active site cysteine. In some embodiments, the biologically active cysteine site is an active site cysteine. In some embodiments, the biologically active cysteine site is a cysteine residue that is located greater than 10 .ANG. from an active-site ligand or residue. In some embodiments, the cysteine residue that is located greater than 10 .ANG. from the active-site ligand or residue is a non-active site cysteine. In some embodiments, the biologically active cysteine site is a non-active site cysteine. In some embodiments, the small molecule fragment that covalently interacts with the biologically active cysteine impairs and/or inhibits activity of the cysteine containing protein. In some embodiments, the cysteine containing protein exists in an active form. In some embodiments, the small molecule fragment and/or the cysteine-reactive probe interact with the active form of the cysteine containing protein. In some embodiments, the cysteine containing protein exists in a pro-active form. In some embodiments, the small molecule fragment and/or the cysteine-reactive probe interact with the pro-active form of the cysteine containing protein. In some embodiments, the structural environment of the biologically active cysteine residue modulates the reactivity of the cysteine residue. In some embodiments, the structural environment is a hydrophobic environment or a hydrophilic environment. In some embodiments, the structural environment is a charged environment. In some embodiments, the structural environment is a nuclcophilic environment. In some embodiments, the cysteine containing protein is an enzyme, a transporter, a receptor, a channel protein, an adaptor protein, a chaperone, a signaling protein, a plasma protein, transcription related protein, translation related protein, mitochondrial protein, or cytoskeleton related protein. In some embodiments, the cysteine containing protein is an enzyme, a transporter, a receptor, a channel protein, an adaptor protein, a chaperone, a signaling protein, transcription related protein, or translation related protein. In some embodiments, the enzyme comprises kinases, proteases, or deubiquitinating enzymes. In some embodiments, the protease is a cysteine protease. In some embodiments, the cysteine protease comprises caspases. In some embodiments, the signaling protein comprises vascular endothelial growth factor. In some embodiments, the signaling protein comprises a redox signaling protein. In some embodiments, the cysteine containing protein is a protein illustrated in Table 1. In some embodiments, the cysteine containing protein is a protein illustrated in Table 2. In some embodiments, the cysteine containing protein is a protein illustrated in Table 3. In some embodiments, the cysteine containing protein comprises a cysteine residue denoted in Table 3. In some embodiments, the cysteine containing protein is a protein illustrated in Table 8. In some embodiments, the cysteine containing protein is a protein illustrated in Table 9. In some embodiments, the cysteine containing protein is a protein illustrated in Table 10A, Table 10B, Table 10C, Table 10D or Table 10E. In some embodiments, the small molecule fragment is a small molecule fragment of Formula (I):
##STR00001##
wherein: RM is a reactive moiety selected from a Michael acceptor moiety, a leaving group moiety, or a moiety capable of forming a covalent bond with the thiol group of a cysteine residue; and F is a small molecule fragment moiety. In some embodiments, the Michael acceptor moiety comprises an alkene or an alkyne moiety. In some embodiments, F is obtained from a compound library. In some embodiments, the compound library comprises ChemBridge fragment library, Pyramid Platform Fragment-Based Drug Discovery, Maybridge fragment library, FRGx from AnalytiCon. TCI-Frag from AnCoreX, Bio Building Blocks from ASINEX, BioFocus 3D from Charles River, Fragments of Life (FOL) from Emerald Bio, Enamine Fragment Library, IOTA Diverse 1500, BIONET fragments library, Life Chemicals Fragments Collection, OTAVA fragment library. Prestwick fragment library, Selcia fragment library, TimTec fragment-based library. Allium from Vitas-M Laboratory, or Zenobia fragment library. In some embodiments, F is a small molecule fragment moiety illustrated in FIG. 3. In some embodiments, F further comprises a linker moiety that connects F to the carbonyl moiety. In some embodiments, the small molecule fragment is a small molecule fragment illustrated in FIG. 3. In some embodiments, the small molecule fragment is a specific inhibitor or a pan inhibitor. In some embodiments, the cysteine-reactive probe is a cysteine-reactive probe of Formula (II)
##STR00002##
wherein: RM is a reactive moiety selected from a Michael acceptor moiety, a leaving group moiety, or a moiety capable of forming a covalent bond to the thiol group of a cysteine residue; and AHM is an affinity handle moiety. In some embodiments, the Michael acceptor moiety comprises an alkene or an alkyne moiety. In some embodiments, the affinity handle moiety comprises an affinity handle and a binding moiety that facilitates covalent interaction of the cysteine-reactive probe to a cysteine residue of a cysteine-containing protein. In some embodiments, the binding moiety is a small molecule fragment obtained from a compound library. In some embodiments, the compound library comprises ChemBridge fragment library, Pyramid Platform Fragment-Based Drug Discovery, Maybridge fragment library, FRGx from AnalytiCon, TCI-Frag from AnCoreX, Bio Building Blocks from ASINEX, BioFocus 3D from Charles River, Fragments of Life (FOL) from Emerald Bio, Enamine Fragment Library, IOTA Diverse 1500, BIONET fragments library, Life Chemicals Fragments Collection, OTAVA fragment library. Prestwick fragment library, Selcia fragment library, TimTec fragment-based library, Allium from Vitas-M Laboratory, or Zenobia fragment library. In some embodiments, the affinity handle is a bioorthogonal affinity handle. In some embodiments, the affinity handle comprises a carbodiimide, N-hydroxysuccinimide (NHS) ester, imidoester, pentafluorophenyl ester, hydroxymnethyl phosphine, maleimide, haloacetyl, pyridyl disulfide, thiosulfonate, vinylsulfone, hydrazide, alkoxyamine, alkyne, azide, or isocyanate group. In some embodiments, the affinity handle comprises an alkyne or an azide group. In some embodiments, the affinity handle is further conjugated to an affinity ligand. In some embodiments, the affinity ligand comprises a chromophore, a labeling group, or a combination thereof. In some embodiments, the chromophore comprises fluorochrome, non-fluorochrome chromophore, quencher, an absorption chromophore, fluorophore, organic dye, inorganic dye, metal chelate, or a fluorescent enzyme substrate. In some embodiments, the fluorophore comprises rhodamine, rhodol, fluorescein, thiofluorescein, aminofluorescein, carboxyfluorescein, chlorofluorescein, methylfluorescein, sulfofluorescein, aminorhodol, carboxyrhodol, chlororhodol, methylrhodol, sulforhodol, aminorhodamine, carboxyrhodamine, chlororhodamine, methylrhodamine, sulforhodamine, thiorhodamine, cyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, merocyanine, cyanine 2, cyanine 3, cyanine 3.5, cyanine 5, cyanine 5.5, cyanine 7, oxadiazole derivatives, pyridyloxazole, nitrobenzoxadiazole, benzoxadiazole, pyren derivatives, cascade blue, oxazine derivatives, Nile red, Nile blue, cresyl violet, oxazine 170, acridine derivatives, proflavin, acridine orange, acridine yellow, arylmethine derivatives, auramine, crystal violet, malachite green, tetrapyrrole derivatives, porphin, phtalocyanine, bilirubin 1-dimethylaminonaphthyl-5-sulfonate, 1-anilino-8-naphthalene sulfonate, 2-p-touidinyl-6-naphthalene sulfonate, 3-phenyl-7-isocyanatocoumarin, N-(p-(2-bcnzoxazolyl)phenyl)maleimide, stilbenes, pyrenes, 6-FAM (Fluorescein), 6-FAM (NHS Ester), 5(6)-FAM, 5-FAM, Fluorescein dT, 5-TAMRA-cadavarine, 2-aminoacridone, HEX, JOE (NHS Ester), MAX, TET, ROX, TAMRA, TARMA.TM. (NHS Ester), TEX 615, ATTO.TM. 488. ATTO.TM. 532, ATTO.TM. 550, ATTO.TM. 565, ATTO.TM. Rho101, ATTO.TM. 590. ATTO.TM. 633, ATTO.TM. 647N, TYE.TM. 563, TYE.TM. 665, or TYE.TM. 705. In some embodiments, the labeling group is biotin moiety, streptavidin moiety, bead, resin, a solid support, or a combination thereof. In some embodiments, the affinity handle moiety further comprises a chromophore. In some embodiments, the cysteine-reactive probe is a cysteine-reactive probe illustrated in FIG. 3. In some embodiments, the second cell solution further comprises a control. In some embodiments, the control is dimethyl sulfoxide (DMSO). In some embodiments, the protcomic analysis means comprises a mass spectroscopy method. In some embodiments, the mass spectroscopy method is a liquid-chromatography-mass spectrometry (LC-MS) method. In some embodiments, the method further comprises analyzing the results from the mass spectroscopy method by an algorithm for protein identification. In some embodiments, the algorithm combines the results from the mass spectroscopy method with a protein sequence database for protein identification. In some embodiments, the algorithm comprises ProLuCID algorithm, Probity, Scaffold, SEQUEST, or Mascot. In some embodiments, the mass spectroscopy method is a MALDI-TOF based method. In some embodiments, the value assigned to each of the cysteine containing protein is obtained from the mass spectroscopy analysis. In some embodiments, the value assigned to each of the cysteine containing protein is the area-under-the curve from a plot of signal intensity as a function of mass-to-charge ratio. In some embodiments, the identifying in step c) further comprises (i) locating a first value assigned to a cysteine containing protein from the first group of cysteine-reactive probe-protein complex and a second value of the same cysteine containing protein from the second group of cysteine-ractive probe-protein complex; and (ii) calculating a ratio between the two values assigned to the same cysteine containing protein. In some embodiments, the ratio of greater than 2 indicates that the cysteine containing protein is a candidate for interacting with the small molecule fragment. In some embodiments, the ratio of greater than 3 indicates that the cysteine containing protein is a candidate for interacting with the small molecule fragment. In some embodiments, the identifying in step c) further comprises calculating a percentage of inhibition of the cysteine-reactive probe to the cysteine containing protein. In some embodiments, the percentage of inhibition of greater than 50%, 60%, 70%, 80%, 90%, or at 100% indicates that the cysteine containing protein is a candidate for interacting with the small molecule fragment. In some embodiments, the cell is obtained from a tumor cell line. In some embodiments, the cell is obtained from a MDA-MB-231, Ramos, or Jurkat cell line. In some embodiments, the cell is obtained from a tumor sample. In some embodiments, the sample is a tissue sample. In some embodiments, the method is an in situ method. In some embodiments, the cysteine-reactive probe is not 4-hydroxynonenal or 15-deoxy-.DELTA.12,14-prostaglandin J2.
[0006] Disclosed herein, in certain embodiments, is a method of screening a small molecule fragment for interaction with a cysteine containing protein, comprising: (a) harvesting a set of cysteine-reactive probe-protein complexes from a sample treated with a cysteine-reactive probe wherein the cysteine-reactive probe comprises a reactive moiety capable of forming a covalent bond with a cysteine residue located on the cysteine containing protein; (b) analyzing the set of cysteine-reactive probe-protein complexes by a proteomic analysis means; and (c) based on step b), identifying the small molecule fragment as interacting with the cysteine containing protein. In some embodiments, the method further comprises assigning a value to each of the cysteine containing protein from the set of cysteine-reactive probe-protein complexes prior to identifying the small molecule fragment as interacting with the cysteine containing protein, wherein the value is determined based on the proteomic analysis means of step b). In some embodiments, the sample comprises a first cell solution and a second cell solution. In some embodiments, the method further comprises contacting the first cell solution with a small molecule fragment for an extended period of time prior to incubating the first cell solution with a first cysteine-reactive probe to generate a first group of cysteine-reactive probe-protein complexes. In some embodiments, the extended period of time is about 5, 10, 15, 20, 30, 60, 90, 120 minutes or longer. In some embodiments, the method further comprises contacting the second cell solution with a second cysteine-reactive probe to generate a second group of cysteine-reactive probe-protein complexes. In some embodiments, the first cysteine-reactive probe and the second cysteine-reactive probe are the same. In some embodiments, the first group and the second group of cysteine-reactive probe-protein complexes comprise the set of cysteine-reactive probe-protein complexes. In some embodiments, cells from the second cell solution are grown in a media (e.g., an isotopically enriched media). In some embodiments, cells from the first cell solution are grown in a media (e.g., an isotopically enriched media). In some embodiments, cells from both the first cell solution and the second cell solution are grown in two different isotopically enriched media so that cells from the first cell solution is distinguishable from cells obtained from the second cell solution. In other embodiments, cells from only one of the cell solutions (e.g., either the first cell solution or the second cell solution) are grown in an isotopically enriched media. In some embodiments, the method further comprises contacting the first cell solution with a first set of small molecule fragments and a complementing set of cysteine-reactive probes wherein each small molecule fragment competes with its complementing cysteine-reactive probe for binding with a cysteine residue, and wherein each small molecule fragment and each complementing cysteine-reactive probe are different within each respective set. In some embodiments, the method further comprises contacting the second cell solution with a second set of cysteine-reactive probes wherein the second set of cysteine-reactive probes is the same as the complementing set of cysteine-reactive probes, and wherein each cysteine-reactive probe is different within the set. In some embodiments, the first set of cysteine-reactive probes generates a third group of cysteine-reactive probe-protein complexes and the second set of cysteine-reactive probes generates a fourth group of cysteine-reactive probe-protein complexes. In some embodiments, the cysteine containing protein comprises a biologically active cysteine residue. In some embodiments, the biologically active cysteine site is a cysteine residue that is located about 10 .ANG. or less to an active-site ligand or residue. In some embodiments, the cysteine residue that is located about 10 .ANG. or less to the active-site ligand or residue is an active site cysteine. In some embodiments, the biologically active cysteine site is an active site cysteine. In some embodiments, the biologically active cysteine site is a cysteine residue that is located greater than 10 .ANG. from an active-site ligand or residue. In some embodiments, the cysteine residue that is located greater than 10 .ANG. from the active-site ligand or residue is a non-active site cysteine. In some embodiments, the biologically active cysteine site is a non-active site cysteine. In some embodiments, the small molecule fragment that covalently interacts with the biologically active cysteine impairs and/or inhibits activity of the cysteine containing protein. In some embodiments, the cysteine containing protein exists in an active form. In some embodiments, the small molecule fragment and/or the cysteine-reactive probe interact with the active form of the cysteine containing protein. In some embodiments, the cysteine containing protein exists in a pro-active form. In some embodiments, the small molecule fragment and/or the cysteine-reactive probe interact with the pro-active form of the cysteine containing protein. In some embodiments, the structural environment of the biologically active cysteine residue modulates the reactivity of the cysteine residue. In some embodiments, the structural environment is a hydrophobic environment or a hydrophilic environment. In some embodiments, the structural environment is a charged environment. In some embodiments, the structural environment is a nucleophilic environment. In some embodiments, the cysteine containing protein is selected from an enzyme, a transporter, a receptor, a channel protein, an adaptor protein, a chaperone, a signaling protein, a plasma protein, transcription related protein, translation related protein, mitochondrial protein, or cytoskeleton related protein. In some embodiments, the cysteine containing protein is selected from an enzyme, a transporter, a receptor, a channel protein, an adaptor protein, a chaperone, a signaling protein, transcription related protein, or translation related protein. In some embodiments, the enzyme comprises kinases, proteases, or deubiquitinating enzymes. In some embodiments, the protease is a cysteine protease. In some embodiments, the cysteine protease comprises caspases. In some embodiments, the signaling protein comprises vascular endothelial growth factor. In some embodiments, the signaling protein comprises a redox signaling protein. In some embodiments, the cysteine containing protein is selected from Table 1. In some embodiments, the cysteine containing protein is a protein illustrated in Table 2. In some embodiments, the cysteine containing protein is a protein illustrated in Table 3. In some embodiments, the cysteine containing protein comprises a cysteine residue denoted in Table 3. In some embodiments, the cysteine containing protein is a protein illustrated in Table 8. In some embodiments, the cysteine containing protein is a protein illustrated in Table 9. In some embodiments, the cysteine containing protein is a protein illustrated in Table 10A, Table 10B, Table 10C, Table 10D or Table 10E. In some embodiments, the cysteine containing protein is TIGAR. IMPDH2, IDH1, IDH2, BTK, ZAK, TGM2, Map2k7, XPO1, Casp5, Casp8, ERCC3, Park 7 (Toxoplasma DJ-1), GSTO1, ALDH2, CTSZ, STAT1, STAT3, SMAD2, RBPJ, FOXK1, IRF4, IRF8, GTF3C1, or TCERG1. In some embodiments, the small molecule fragment is a small molecule fragment of Formula (I):
##STR00003##
wherein: RM is a reactive moiety selected from a Michael acceptor moiety, a leaving group moiety, or a moiety capable of forming a covalent bond with the thiol group of a cysteine residue; and F is a small molecule fragment moiety. In some embodiments, the Michael acceptor moiety comprises an alkene or an alkyne moiety. In some embodiments, F is obtained from a compound library. In some embodiments, the compound library comprises ChemBridge fragment library, Pyramid Platform Fragment-Based Drug Discovery, Maybridge fragment library, FRGx from AnalytiCon, TCI-Frag from AnCoreX, Bio Building Blocks from ASINEX, BioFocus 3D from Charles River, Fragments of Life (FOL) from Emerald Bio, Enamine Fragment Library, IOTA Diverse 1500, BIONET fragments library, Life Chemicals Fragments Collection, OTAVA fragment library, Prestwick fragment library, Selcia fragment library, TimTec fragment-based library, Allium from Vitas-M Laboratory, or Zenobia fragment library. In some embodiments, F is a small molecule fragment moiety illustrated in FIG. 3. In some embodiments. F further comprises a linker moiety that connects F to the carbonyl moiety. In some embodiments, the small molecule fragment is a small molecule fragment illustrated in FIG. 3. In some embodiments, the small molecule fragment is a specific inhibitor or a pan inhibitor. In some embodiments, the cysteine-reactive probe is a cysteine-reactive probe of Formula (II):
##STR00004##
wherein: RM is a reactive moiety selected from a Michael acceptor moiety, a leaving group moiety, or a moiety capable of forming a covalent bond to the thiol group of a cysteine residue; and AHM is an affinity handle moiety. In some embodiments, the Michael acceptor moiety comprises an alkene or an alkyne moiety. In some embodiments, the affinity handle moiety comprises an affinity handle and a binding moiety that facilitates covalent interaction of the cysteine-reactive probe to a cysteine residue of a cysteine-containing protein. In some embodiments, the binding moiety is a small molecule fragment obtained from a compound library. In some embodiments, the compound library comprises ChemBridge fragment library, Pyramid Platform Fragment-Based Drug Discovery, Maybridge fragment library, FRGx from AnalytiCon, TCI-Frag from AnCoreX, Bio Building Blocks from ASINEX, BioFocus 3D from Charles River. Fragments of Life (FOL) from Emerald Bio, Enamine Fragment Library, IOTA Diverse 1500, BIONET fragments library, Life Chemicals Fragments Collection, OTAVA fragment library, Prestwick fragment library, Selcia fragment library, TimTec fragment-based library, Allium from Vitas-M Laboratory, or Zenobia fragment library. In some embodiments, the affinity handle is a bioorthogonal affinity handle. In some embodiments, the affinity handle comprises a carbodiimide, N-hydroxysuccinimide (NHS) ester, imidoester, pentafluorophenyl ester, hydroxymethyl phosphine, maleimide, haloacetyl, pyridyl disulfide, thiosulfonate, vinylsulfone, hydrazide, alkoxyamine, alkyne, azide, or isocyanate group. In some embodiments, the affinity handle comprises an alkyne or an azide group. In some embodiments, the affinity handle is further conjugated to an affinity ligand. In some embodiments, the affinity ligand comprises a chromophore, a labeling group, or a combination thereof. In some embodiments, the chromophore comprises fluorochrome, non-fluorochrome chromophore, quencher, an absorption chromophore, fluorophore, organic dye, inorganic dye, metal chelate, or a fluorescent enzyme substrate. In some embodiments, the fluorophore comprises rhodamine, rhodol, fluorescein, thiofluorescein, aminofluorescein, carboxyfluorescein, chlorofluorescein, methylfluorescein, sulfofluorescein, aminorhodol, carboxyrhodol, chlororhodol, methylrhodol, sulforhodol, aminorhodamine, carboxyrhodamine, chlororhodamine, methylrhodamine, sulforhodamine, thiorhodamine, cyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, merocyanine, cyanine 2, cyanine 3, cyanine 3.5, cyanine 5, cyanine 5.5, cyanine 7, oxadiazole derivatives, pyridyloxazole, nitrobenzoxadiazole, benzoxadiazole, pyren derivatives, cascade blue, oxazine derivatives, Nile red, Nile blue, cresyl violet, oxazine 170, acridine derivatives, proflavin, acridine orange, acridine yellow, arylmethine derivatives, auramine, crystal violet, malachite green, tetrapyrrole derivatives, porphin, phtalocyanine, bilirubin 1-dimethylaminonaphthyl-5-sulfonate, 1-anilino-8-naphthalene sulfonate, 2-p-touidinyl-6-naphthalene sulfonate, 3-phenyl-7-isocyanatocoumarin, N-(p-(2-benzoxazolyl)phenyl)maleimide, stilbenes, pyrenes, 6-FAM (Fluorescein), 6-FAM (NHS Ester), 5(6)-FAM, 5-FAM, Fluorescein dT, 5-TAMRA-cadavarine, 2-aminoacridone, HEX, JOE (NHS Ester), MAX, TET, ROX, TAMRA, TARMA.TM. (NHS Ester), TEX 615, ATTO.TM. 488, ATTO.TM. 532, ATTO.TM.550, ATTO.TM. 565, ATTO.TM. Rho101, ATTO.TM. 590, ATTO.TM. 633, ATTO.TM. 647N, TYE.TM. 563, TYE.TM. 665, or TYE.TM. 705. In some embodiments, the labeling group is biotin moiety, streptavidin moiety, bead, resin, a solid support, or a combination thereof. In some embodiments, the affinity handle moiety further comprises a chromophore. In some embodiments, the cysteine-reactive probe is a cysteine-reactive probe illustrated in FIG. 3. In some embodiments, the second cell solution further comprises a control. In some embodiments, the control is dimethyl sulfoxide (DMSO). In some embodiments, the proteomic analysis means comprises a mass spectroscopy method. In some embodiments, the mass spectroscopy method is a MALDI-TOF based method. In some embodiments, the mass spectroscopy method is a liquid-chromatography-mass spectrometry (LC-MS) method. In some embodiments, the method further comprises analyzing the results from the mass spectroscopy method by an algorithm for protein identification. In some embodiments, the algorithm combines the results from the mass spectroscopy method with a protein sequence database for protein identification. In some embodiments, the algorithm comprises ProLuCID algorithm, Probity, Scaffold, SEQUEST, or Mascot. In some embodiments, the value assigned to each of the cysteine containing protein is obtained from the mass spectroscopy analysis. In some embodiments, the value assigned to each of the cysteine containing protein is the area-under-the curve from a plot of signal intensity as a function of mass-to-charge ratio. In some embodiments, the identifying in step c) further comprises (i) locating a first value assigned to a cysteine containing protein from the first group of cysteine-reactive probe-protein complex and a second value of the same cysteine containing protein from the second group of cysteine-reactive probe-protein complex; and (ii) calculating a ratio between the two values assigned to the same cysteine containing protein. In some embodiments, the ratio of greater than 2 indicates that the cysteine containing protein is a candidate for interacting with the small molecule fragment. In some embodiments, the ratio of greater than 3 indicates that the cysteine containing protein is a candidate for interacting with the small molecule fragment. In some embodiments, the identifying in step c) further comprises calculating a percentage of inhibition of the cysteine-reactive probe to the cysteine containing protein. In some embodiments, the percentage of inhibition of greater than 50%, 60%, 70%, 80%, 90%, or at 100% indicates that the cysteine containing protein is a candidate for interacting with the small molecule fragment. In some embodiments, the cell is obtained from a tumor cell line. In some embodiments, the cell is obtained from a MDA-MB-231, Ramos, or Jurkat cell line. In some embodiments, the cell is obtained from a tumor sample. In some embodiments, the sample is a tissue sample. In some embodiments, the method is an in situ method.
[0007] Disclosed herein, in certain embodiments, is a method of mapping a biologically active cysteine site on a protein, comprising (a) harvesting a set of cysteine-reactive probe-protein complexes from a sample treated with a cysteine-reactive probe wherein the cysteine-reactive probe comprises a reactive moiety capable of forming a covalent bond with a cysteine residue located on the cysteine containing protein; (b) analyzing the set of cysteine-reactive probe-protein complexes by a proteomic analysis means; and (c) based on step b), mapping the biologically active cysteine site on the protein. In some embodiments, the sample comprises a first cell solution and a second cell solution. In some embodiments, the method further comprises contacting the first cell solution with a small molecule fragment for an extended period of time prior to incubating the first cell solution with a first cysteine-reactive probe to generate a first group of cysteine-reactive probe-protein complexes. In some embodiments, the extended period of time is about 5, 10, 15, 20, 30, 60, 90, 120 minutes or longer. In some embodiments, the method further comprises contacting the second cell solution with a second cysteine-reactive probe to generate a second group of cysteine-reactive probe-protein complexes. In some embodiments, the first cysteine-reactive probe and the second cysteine-reactive probe are the same. In some embodiments, the biologically active cysteine site is a cysteine residue that is located about 10 .ANG. or less to an active-site ligand or residue. In some embodiments, the cysteine residue that is located about 10 .ANG. or less to the active-site ligand or residue is an active site cysteine. In some embodiments, the biologically active cysteine site is an active site cysteine. In some embodiments, the biologically active cysteine site is a cysteine residue that is located greater than 10 .ANG. from an active-site ligand or residue. In some embodiments, the cysteine residue that is located greater than 10 .ANG. from the active-site ligand or residue is a non-active site cysteine. In some embodiments, the biologically active cysteine site is a non-active site cysteine. In some embodiments, the small molecule fragment that covalently interacts with the biologically active cysteine impairs and/or inhibits activity of the cysteine containing protein. In some embodiments, the cysteine containing protein exists in an active form. In some embodiments, the small molecule fragment and/or the cysteine-reactive probe interact with the active form of the cysteine containing protein. In some embodiments, the cysteine containing protein exists in a pro-active form. In some embodiments, the small molecule fragment and/or the cysteine-reactive probe interact with the pro-active form of the cysteine containing protein. In some embodiments, the structural environment of the biologically active cysteine residue modulates the reactivity of the cysteine residue. In some embodiments, the structural environment is a hydrophobic environment or a hydrophilic environment. In some embodiments, the structural environment is a charged environment. In some embodiments, the structural environment is a nucleophilic environment. In some embodiments, the protein is an enzyme, a transporter, a receptor, a channel protein, an adaptor protein, a chaperone, a signaling protein, a plasma protein, transcription related protein, translation related protein, mitochondrial protein, or cytoskeleton related protein. In some embodiments, the protein is an enzyme, a transporter, a receptor, a channel protein, an adaptor protein, a chaperone, a signaling protein, transcription related protein, or translation related protein. In some embodiments, the enzyme comprises kinases, proteases, or deubiquitinating enzymes. In some embodiments, the protease is a cysteine protease. In some embodiments, the cysteine protease comprises caspases. In some embodiments, the signaling protein comprises vascular endothelial growth factor. In some embodiments, the signaling protein comprises a redox signaling protein. In some embodiments, the protein is a protein illustrated in Table 1. In some embodiments, the cysteine containing protein is a protein illustrated in Table 2. In some embodiments, the cysteine containing protein is a protein illustrated in Table 3. In some embodiments, the cysteine containing protein comprises a cysteine residue denoted in Table 3. In some embodiments, the cysteine containing protein is a protein illustrated in Table 8. In some embodiments, the cysteine containing protein is a protein illustrated in Table 9. In some embodiments, the cysteine containing protein is a protein illustrated in Table 10A. Table 10B. Table 10C, Table 10D or Table 10E. In some embodiments, the small molecule fragment is a small molecule fragment of Formula (I):
##STR00005##
wherein: RM is a reactive moiety selected from a Michael acceptor moiety, a leaving group moiety, or a moiety capable of forming a covalent bond with the thiol group of a cysteine residue, and F is a small molecule fragment moiety. In some embodiments, the Michael acceptor moiety comprises an alkene or an alkyne moiety. In some embodiments, F is obtained from a compound library. In some embodiments, the compound library comprises ChemBridge fragment library, Pyramid Platform Fragment-Based Drug Discovery, Maybridge fragment library, FRGx from AnalytiCon, TCI-Frag from AnCoreX, Bio Building Blocks from ASINEX, BioFocus 3D from Charles River. Fragments of Life (FOL) from Emerald Bio, Enamine Fragment Library, IOTA Diverse 1500, BIONET fragments library, Life Chemicals Fragments Collection, OTAVA fragment library, Prestwick fragment library, Selcia fragment library, TimTec fragment-based library, Allium from Vitas-M Laboratory. or Zenobia fragment library. In some embodiments, F is a small molecule fragment moiety illustrated in FIG. 3. In some embodiments. F further comprises a linker moiety that connects F to the carbonyl moiety. In some embodiments, the small molecule fragment is a small molecule fragment illustrated in FIG. 3. In some embodiments, the small molecule fragment is a specific inhibitor or a pan inhibitor. In some embodiments, the cysteine-reactive probe is a cysteine-reactive probe of Formula (II):
##STR00006##
wherein: RM is a reactive moiety selected from a Michael acceptor moiety, a leaving group moiety, or a moiety capable of forming a covalent bond to the thiol group of a cysteine residue; and AHM is an affinity handle moiety. In some embodiments, the Michael acceptor moiety comprises an alkene or an alkyne moiety. In some embodiments, the affinity handle moiety comprises an affinity handle and a binding moiety that facilitates covalent interaction of the cysteine-reactive probe to a cysteine residue of a cysteine-containing protein. In some embodiments, the binding moiety is a small molecule fragment obtained from a compound library. In some embodiments, the compound library comprises ChemBridge fragment library, Pyramid Platform Fragment-Based Drug Discovery, Maybridge fragment library, FRGx from AnalytiCon. TCI-Frag from AnCoreX, Bio Building Blocks from ASINEX, BioFocus 3D from Charles River. Fragments of Life (FOL) from Emerald Bio, Enamine Fragment Library, IOTA Diverse 1500, BIONET fragments library, Life Chemicals Fragments Collection, OTAVA fragment library, Prestwick fragment library, Selcia fragment library, TimTec fragment-based library, Allium from Vitas-M Laboratory, or Zenobia fragment library. In some embodiments, the affinity handle is a bioorthogonal affinity handle. In some embodiments, the affinity handle comprises a carbodiimide, N-hydroxysuccinimide (NHS) ester, imidoester, pentafluorophenyl ester, hydroxymethyl phosphine, maleimide, haloacetyl, pyridyl disulfide, thiosulfonate, vinylsulfone, hydrazide, alkoxyamine, alkyne, azide, or isocyanate group. In some embodiments, the affinity handle comprises an alkyne or an azide group. In some embodiments, the affinity handle is further conjugated to an affinity ligand. In some embodiments, the affinity ligand comprises a chromophore, a labeling group, or a combination thereof. In some embodiments, the chromophore comprises fluorochrome, non-fluorochrome chromophore, quencher, an absorption chromophore, fluorophore, organic dye, inorganic dye, metal chelate, or a fluorescent enzyme substrate. In some embodiments, the fluorophore comprises rhodamine, rhodol, fluorescein, thiofluorescein, aminofluorescein, carboxyfluorescein, chlorofluorescein, methylfluorescein, sulfofluorescein, aminorhodol, carboxyrhodol, chlororhodol, methylrhodol, sulforhodol, aminorhodamine, carboxyrhodamine, chlororhodamine, methylrhodamine, sulforhodamine, thiorhodamine, cyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, merocyanine, cyanine 2, cyanine 3, cyanine 3.5, cyanine 5, cyanine 5.5, cvanine 7, oxadiazole derivatives, pyridyloxazole, nitrobenzoxadiazole, benzoxadiazole, pyren derivatives, cascade blue, oxazine derivatives, Nile red, Nile blue, cresyl violet, oxazine 170, acridine derivatives, proflavin, acridine orange, acridine yellow, arylmethine derivatives, auramine, crystal violet, malachite green, tetrapyrrole derivatives, porphin, phtalocyanine, bilirubin 1-dimethylaminonaphthyl-5-sulfonate, 1-anilino-8-naphthalene sulfonate, 2-p-touidinyl-6-naphthalene sulfonate, 3-phenyl-7-isocyanatocoumarin, N-(p-(2-benzoxazolyl)phenyl)maleimide, stilbenes, pyrenes, 6-FAM (Fluorescein), 6-FAM (NHS Ester), 5(6)-FAM, 5-FAM, Fluorescein dT, 5-TAMRA-cadavarine, 2-aminoacridone, HEX, JOE (NHS Ester), MAX, TET, ROX, TAMRA, TARMA.TM. (NHS Ester), TEX 615, ATTO.TM. 488, ATTO.TM. 532, ATTO.TM. 550, ATTO.TM. 565, ATTO.TM. Rho101 ATTO.TM. 590, ATTO.TM. 633, ATTO.TM. 647N, TYE.TM. 563, TYE.TM. 665, or TYE.TM. 705. In some embodiments, the labeling group is biotin moiety, streptavidin moiety, bead, resin, a solid support, or a combination thereof. In some embodiments, the affinity handle moiety further comprises a chromophore. In some embodiments, the cysteine-reactive probe is a cysteine-reactive probe illustrated in FIG. 3. In some embodiments, the second cell solution further comprises a control. In some embodiments, the control is dimethyl sulfoxide (DMSO). In some embodiments, the proteomic analysis means comprises a mass spectroscopy method. In some embodiments, the mass spectroscopy method is a liquid-chromatography-mass spectrometry (LC-MS) method. In some embodiments, the method further comprises analyzing the results from the mass spectroscopy method by an algorithm for protein identification. In some embodiments, the algorithm combines the results from the mass spectroscopy method with a protein sequence database for protein identification. In some embodiments, the algorithm comprises ProLuCID algorithm, Probity, Scaffold, SEQUEST, or Mascot. In some embodiments, the mass spectroscopy method is a MALDI-TOF based method. In some embodiments, the cell is obtained from a tumor cell line. In some embodiments, the cell is obtained from a MDA-MB-231, Ramos, or Jurkat cell line. In some embodiments, the cell is obtained from a tumor sample. In some embodiments, the sample is a tissue sample. In some embodiments, the method is an in situ method.
[0008] Disclosed herein, in certain embodiments, is a composition comprising: a small molecule fragment of Formula (I):
##STR00007##
wherein: RM is a reactive moiety selected from a Michael acceptor moiety, a leaving group moiety, or a moiety capable of forming a covalent bond with the thiol group of a cysteine residue; and F is a small molecule fragment moiety; and a cysteine containing protein wherein the cysteine containing protein is covalently bond to the small molecule fragment. In some embodiments, the Michael acceptor moiety comprises an alkene or an alkyne moiety. In some embodiments, F is obtained from a compound library. In some embodiments, F is a small molecule fragment moiety illustrated in FIG. 3. In some embodiments, F further comprises a linker moiety that connects F to the carbonyl moiety.
[0009] Disclosed herein, in certain embodiments, is a composition comprising: a cysteine-reactive probe of Formula (II):
##STR00008##
wherein: RM is a reactive moiety selected from a Michael acceptor moiety, a leaving group moiety, or a moiety capable of forming a covalent bond to the thiol group of a cysteine residue; and AHM is an affinity handle moiety; and a cysteine containing protein wherein the cysteine containing protein is covalently bond to the cysteine-reactive probe. In some embodiments, the Michael acceptor moiety comprises an alkene or an alkyne moiety. In some embodiments, the affinity handle moiety comprises an affinity handle and a binding moiety that facilitates covalent interaction of the cysteine-reactive probe to a cysteine residue of a cysteine-containing protein. In some embodiments, the binding moiety is a small molecule fragment obtained from a compound library. In some embodiments, the affinity handle is a bioorthogonal affinity handle. In some embodiments, the affinity handle comprises a carbodiimide, N-hydroxysuccinimide (NHS) ester, imidoester, pentafluorophenyl ester, hydroxymethyl phosphine, maleimide, haloacetyl, pyridyl disulfide, thiosulfonate, vinylsulfone, hydrazide, alkoxyamine, alkyne, azide, or isocyanate group. In some embodiments, the affinity handle is further conjugated to an affinity ligand. In some embodiments, the affinity handle moiety further comprises a chromophore. In some embodiments, the cysteine-reactive probe is a cysteine-reactive probe illustrated in FIG. 3.
[0010] Disclosed herein, in certain embodiments, is a composition comprising: an isolated sample wherein the isolated sample is an isolated cell or a tissue sample; and a cysteine-reactive probe to be assayed for its ability to interact with a cysteine containing protein expressed in the isolated sample. In some embodiments, the composition further comprises contacting the isolated sample with a small molecule fragment for an extended period of time prior to incubating the isolated sample with the cysteine-reactive probe to generate a cysteine-reactive probe-protein complex. In some embodiments, the extended period of time is about 5, 10, 15, 20, 30, 60, 90, 120 minutes or longer.
[0011] Disclosed herein, in certain embodiments, is an isolated treated cell comprising a cysteine-reactive probe covalently attached to a cysteine containing protein. In some embodiments, the isolated treated cell further comprises a set of cysteine-reactive probes wherein each of the cysteine-reactive probes is covalently attached to a cysteine containing protein.
[0012] Disclosed herein, in certain embodiments, is an isolated treated cell comprising a small molecule fragment covalently attached to a cysteine containing protein. In some embodiments, the isolated treated cell further comprises a set of small molecule fragments wherein each of the small molecule fragments is covalently attached to a cysteine containing protein. In some embodiments, the isolated treated cell further comprises a cysteine-reactive probe. In some embodiments, the isolated treated cell further comprises a set of cysteine-reactive probes.
[0013] Disclosed herein, in certain embodiments, is an isolated treated population of cells comprising a set of cysteine-reactive probes covalently attached to cysteine containing proteins. Also disclosed herein, in certain embodiments, is an isolated treated population of cells comprising a set of small molecule fragments covalently attached to cysteine containing proteins. In some embodiments, the isolated treated population of cells further comprises a set of cysteine-reactive probes.
[0014] Disclosed herein, in certain embodiments, is an isolated and purified polypeptide comprising at least 90% sequence identity to at least seven contiguous amino acids of an amino acid sequence selected from Tables 1-3 or 8-9. In some embodiments, the isolated and purified polypeptide comprising at least 95% sequence identity to at least seven contiguous amino acids of an amino acid sequence selected from Tables 1-3 or 8-9. In some embodiments, the isolated and purified polypeptide comprising 100% sequence identity to at least seven contiguous amino acids of an amino acid sequence selected from Tables 1-3 or 8-9. In some embodiments, the isolated and purified polypeptide consisting 100% sequence identity to the full length of an amino acid sequence selected from Tables 1-3 or 8-9. In some embodiments, the isolated and purified polypeptide is at most 50 amino acids in length. A polypeptide probe for screening a small molecule fragment comprising an isolated and purified polypeptide described herein.
[0015] Further disclosed herein, in certain embodiments, is a nucleic acid encoding a polypeptide comprising at least 90% sequence identity at least seven contiguous amino acids of an amino acid sequence selected from Tables 1-3 or 8-9. In some embodiments, the nucleic acid encoding a polypeptide comprising at least 95% sequence identity at least seven contiguous amino acids of an amino acid sequence selected from Tables 1-3 or 8-9. In some embodiments, the nucleic acid encoding a polypeptide comprising 100% sequence identity at least seven contiguous amino acids of an amino acid sequence selected from Tables 1-3 or 8-9. In some embodiments, the nucleic acid encoding a polypeptide consisting 100% sequence identity to the full length of an amino acid sequence selected from Tables 1-3 or 8-9.
[0016] Disclosed herein, in certain embodiments, is a modified cysteine containing protein comprising a small molecule fragment having a covalent bond to a cysteine residue of a cysteine containing protein, wherein the small molecule fragment is a small molecule fragment of Formula (I):
##STR00009##
wherein: RM is a reactive moiety selected from a Michael acceptor moiety, a leaving group moiety, or a moiety capable of forming a covalent bond with the thiol group of a cysteine residue; and F is a small molecule fragment moiety. In some embodiments, the cysteine containing protein is a protein illustrated in Table 1. In some embodiments, the cysteine containing protein is a protein illustrated in Table 2. In some embodiments, the cysteine containing protein is a protein illustrated in Table 3. In some embodiments, the cysteine containing protein comprises a cysteine residue denoted in Table 3. In some embodiments, the cysteine containing protein is a protein illustrated in Table 8. In some embodiments, the cysteine containing protein is a protein illustrated in Table 9. In some embodiments, the Michael acceptor moiety comprises an alkene or an alkyne moiety. In some embodiments, F is obtained from a compound library. In some embodiments, F is a small molecule fragment moiety illustrated in FIG. 3. In some embodiments, F further comprises a linker moiety that connects F to the carbonyl moiety. In some embodiments, the small molecule fragment binds irreversibly to the cysteine containing protein. In some embodiments, the small molecule fragment binds reversibly to the cysteine containing protein.
[0017] Disclosed herein, in certain embodiments, is a modified cysteine containing protein comprising a small molecule fragment having a covalent bond to a cysteine residue of a cysteine containing protein, wherein the small molecule fragment has a molecular weight of about 150 Dalton or higher. In some embodiments, the small molecule fragment has a molecular weight of about 175, 200, 225, 250, 275, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 Dalton, or higher. In some embodiments, the molecular weight of the small molecule fragment is prior to enrichment with a halogen, a nonmetal, or a transition metal. In some embodiments, the molecular weight of the small molecule fragment is calculated based on carbon and hydrogen atoms and optionally further based on nitrogen, oxygen and/or sulfur atoms. In some embodiments, the molecular weight of the small molecule fragment does not include the molecular weight of a halogen, a transition metal or a combination thereof. In some embodiments, the cysteine containing protein is about 20, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000 amino acid residues in length or more. In some embodiments, the cysteine containing protein is a protein illustrated in Table 1. In some embodiments, the cysteine containing protein is a protein illustrated in Table 2. In some embodiments, the cysteine containing protein is a protein illustrated in Table 3. In some embodiments, the cysteine containing protein comprises a cysteine residue denoted in Table 3. In some embodiments, the cysteine containing protein is a protein illustrated in Table 8. In some embodiments, the cysteine containing protein is a protein illustrated in Table 9. In some embodiments, the small molecule fragment is a small molecule fragment of Formula (I):
##STR00010##
wherein: RM is a reactive moiety selected from a Michael acceptor moiety, a leaving group moiety, or a moiety capable of forming a covalent bond with the thiol group of a cysteine residue; and F is a small molecule fragment moiety. In some embodiments, the small molecule fragment of Formula (I) has a molecular weight of about 150, 175, 200, 225, 250, 275, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 Dalton, or higher. In some embodiments, the Michael acceptor moiety comprises an alkene or an alkyne moiety. In some embodiments, F is obtained from a compound library. In some embodiments, F is a small molecule fragment moiety illustrated in FIG. 3. In some embodiments, F further comprises a linker moiety that connects F to the carbonyl moiety. In some embodiments, the small molecule fragment bond irreversibly to the cysteine containing protein. In some embodiments, the small molecule fragment bond reversibly to the cysteine containing protein.
[0018] Disclosed herein, in certain embodiments, is a cysteine containing protein-small molecule fragment complex produced by a process comprising contacting a cell solution with a small molecule fragment of Formula (I):
##STR00011##
wherein: RM is a reactive moiety selected from a Michael acceptor moiety, a leaving group moiety, or a moiety capable of forming a covalent bond with the thiol group of a cysteine residue, and F is a small molecule fragment moiety; and wherein the contacting time is between about 5 minutes and about 2 hours. In some embodiments, the Michael acceptor moiety comprises an alkene or an alkyne moiety. In some embodiments, F is obtained from a compound library. In some embodiments, F is a small molecule fragment moiety illustrated in FIG. 3. In some embodiments, F further comprises a linker moiety that connects F to the carbonyl moiety. In some embodiments, the small molecule fragment of Formula (I) has a molecular weight of about 150, 175, 200, 225, 250, 275, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 Dalton, or higher. In some embodiments, the cysteine containing protein is a protein illustrated in Table 1. In some embodiments, the cysteine containing protein is a protein illustrated in Table 2. In some embodiments, the cysteine containing protein is a protein illustrated in Table 3. In some embodiments, the cysteine containing protein comprises a cysteine residue denoted in Table 3. In some embodiments, the cysteine containing protein is a protein illustrated in Table 8. In some embodiments, the cysteine containing protein is a protein illustrated in Table 9. In some embodiments, the cysteine containing protein is a protein illustrated in Table 10A, Table 10B, Table 10C, Table 10D or Table 10E. In some embodiments, the small molecule fragment binds irreversibly to the cysteine containing protein. In some embodiments, the small molecule fragment binds reversibly to the cysteine containing protein.
[0019] Disclosed herein, in certain embodiments, is a modified cysteine containing protein comprising a cysteine-reactive probe having a covalent bond to a cysteine residue of a cysteine containing protein, wherein the cysteine-reactive probe is a cysteine-reactive probe of Formula (II):
##STR00012##
wherein: RM is a reactive moiety selected from a Michael acceptor moiety, a leaving group moiety, or a moiety capable of forming a covalent bond to the thiol group of a cysteine residue; and AHM is an affinity handle moiety. In some embodiments, the cysteine containing protein is a protein illustrated in Table 1. In some embodiments, the cysteine containing protein is a protein illustrated in Table 2. In some embodiments, the cysteine containing protein is a protein illustrated in Table 8. In some embodiments, the cysteine containing protein is a protein illustrated in Table 9. In some embodiments, the cysteine containing protein is a protein illustrated in Table 10A, Table 10B, Table 10C, Table 10D or Table 10E. In some embodiments, the Michael acceptor moiety comprises an alkene or an alkyne moiety. In some embodiments, the affinity handle moiety comprises an affinity handle and a binding moiety that facilitates covalent interaction of the cysteine-reactive probe to a cysteine residue of a cysteine-containing protein. In some embodiments, the binding moiety is a small molecule fragment obtained from a compound library. In some embodiments, the affinity handle is a bioorthogonal affinity handle. In some embodiments, the affinity handle comprises a carbodiimide, N-hydroxysuccinimide (NHS) ester, imidoester, pentafluorophenyl ester, hydroxymethyl phosphine, maleimide, haloacetyl, pyridyl disulfide, thiosulfonate, vinylsulfone, hydrazide, alkoxyamine, alkyne, azide, or isocyanate group. In some embodiments, the affinity handle is further conjugated to an affinity ligand. In some embodiments, the affinity handle moiety further comprises a chromophore. In some embodiments, the cysteine-reactive probe is a cysteine-reactive probe illustrated in FIG. 3. In some embodiments, the cysteine-reactive probe binds irreversibly to the cysteine containing protein. In some embodiments, the cysteine-reactive probe binds reversibly to the cysteine containing protein.
[0020] Disclosed herein, in certain embodiments, is a cysteine-reactive probe of Formula (II):
##STR00013##
wherein: RM is a reactive moiety selected from a Michael acceptor moiety, a leaving group moiety, or a moiety capable of forming a covalent bond to the thiol group of a cysteine residue, and AHM is an affinity handle moiety. In some embodiments, the cysteine-reactive probe covalently binds to a cysteine residue on a cysteine containing protein. In some embodiments, cysteine containing protein is a protein illustrated in Table 1. In some embodiments, the cysteine containing protein is a protein illustrated in Table 2. In some embodiments, the cysteine containing protein is a protein illustrated in Table 3. In some embodiments, the cysteine containing protein comprises a cysteine residue denoted in Table 3. In some embodiments, the cysteine containing protein is a protein illustrated in Table 8. In some embodiments, the cysteine containing protein is a protein illustrated in Table 9. In some embodiments, the cysteine containing protein is a protein illustrated in Table 10A, Table 10B, Table 10C, Table 10D or Table 10E. In some embodiments, the cysteine-reactive probe binds irreversibly to the cysteine containing protein. In some embodiments, the cysteine-reactive probe binds reversibly to the cysteine containing protein.
[0021] Disclosed herein, in certain embodiments, is a compound capable of covalently binding to a cysteine containing protein identified, using the method comprising: (a) obtaining a set of cysteine-reactive probe-protein complexes from a sample wherein the cysteine-reactive probe comprises a reactive moiety capable of forming a covalent bond with a cysteine residue located on the cysteine containing protein; (b) analyzing the set of cysteine-reactive probe-protein complexes by a proteomic analysis means; (c) based on step b), identifying a cysteine containing protein as the binding target for the compound. In some embodiments, the compound is a small molecule fragment. In some embodiments, the small molecule fragment is a small molecule fragment of Formula (I):
##STR00014##
wherein: RM is a reactive moiety selected from a Michael acceptor moiety, a leaving group moiety, or a moiety capable of forming a covalent bond with the thiol group of a cysteine residue; and F is a small molecule fragment moiety. In some embodiments, the Michael acceptor moiety comprises an alkene or an alkyne moiety. In some embodiments, F is obtained from a compound library. In some embodiments, the compound library comprises ChemBridge fragment library, Pyramid Platform Fragment-Based Drug Discovery, Maybridge fragment library, FRGx from AnalytiCon, TCI-Frag from AnCoreX, Bio Building Blocks from ASINEX, BioFocus 3D from Charles River, Fragments of Life (FOL) from Emerald Bio, Enamine Fragment Library, IOTA Diverse 1500, BIONET fragments library, Life Chemicals Fragments Collection, OTAVA fragment library, Prestwick fragment library, Selcia fragment library, TimTec fragment-based library, Allium from Vitas-M Laboratory. or Zenobia fragment library. In some embodiments, F is a small molecule fragment moiety illustrated in FIG. 3. In some embodiments, F further comprises a linker moiety that connects F to the carbonyl moiety. In some embodiments, the small molecule fragment is a small molecule fragment illustrated in FIG. 3. In some embodiments, the small molecule fragment is a specific inhibitor or a pan inhibitor. In some embodiments, the cysteine containing protein comprises a biologically active cysteine residue. In some embodiments, the biologically active cysteine site is a cysteine residue that is located about 10 .ANG. or less to an active-site ligand or residue. In some embodiments, the cysteine residue that is located about 10 .ANG. or less to the active-site ligand or residue is an active site cysteine. In some embodiments, the biologically active cysteine site is an active site cysteine. In some embodiments, the biologically active cysteine site is a cysteine residue that is located greater than 10 .ANG. from an active-site ligand or residue. In some embodiments, the cysteine residue that is located greater than 10 .ANG. from the active-site ligand or residue is a non-active site cysteine. In some embodiments, the biologically active cysteine site is a non-active site cysteine. In some embodiments, the small molecule fragment that covalently interacts with the biologically active cysteine impairs and/or inhibits activity of the cysteine containing protein. In some embodiments, the cysteine containing protein exists in an active form. In some embodiments, the small molecule fragment and/or the cysteine-reactive probe interact with the active form of the cysteine containing protein. In some embodiments, the cysteine containing protein exists in a pro-active form. In some embodiments, the small molecule fragment and/or the cysteine-reactive probe interact with the pro-active form of the cysteine containing protein. In some embodiments, the structural environment of the biologically active cysteine residue modulates the reactivity of the cysteine residue. In some embodiments, the structural environment is a hydrophobic environment or a hydrophilic environment. In some embodiments, the structural environment is a charged environment. In some embodiments, the structural environment is a nucleophilic environment. In some embodiments, the cysteine containing protein is an enzyme, a transporter, a receptor, a channel protein, an adaptor protein, a chaperone, a signaling protein, a plasma protein, transcription related protein, translation related protein, mitochondrial protein, or cytoskeleton related protein. In some embodiments, the cysteine containing protein is an enzyme, a transporter, a receptor, a channel protein, an adaptor protein, a chaperone, a signaling protein, transcription related protein, or translation related protein. In some embodiments, the enzyme comprises kinases, proteases, or deubiquitinating enzymes. In some embodiments, the protease is a cysteine protease. In some embodiments, the cysteine protease comprises caspases. In some embodiments, the signaling protein comprises vascular endothelial growth factor. In some embodiments, the signaling protein comprises a redox signaling protein. In some embodiments, the cysteine containing protein is a protein illustrated in Table 1. In some embodiments, the cysteine containing protein is a protein illustrated in Table 2. In some embodiments, the cysteine containing protein is a protein illustrated in Table 3. In some embodiments, the cysteine containing protein comprises a cysteine residue denoted in Table 3. In some embodiments, the cysteine containing protein is a protein illustrated in Table 8. In some embodiments, the cysteine containing protein is a protein illustrated in Table 9. In some embodiments, the cysteine containing protein is a protein illustrated in Table 10A. Table 10B, Table 10C, Table 10D or Table 10E.
[0022] Disclosed herein, in certain embodiments, is a derivative of a cysteine-containing protein having the structure of Formula (I),
##STR00015##
wherein, the derivation occurs at a cysteine residue; R is selected from:
##STR00016##
wherein R.sup.1 is H, C1-C3 alkyl, or aryl; and F' is a small molecule fragment moiety. In some embodiments, F' has a molecular weight of about 175, 200, 225, 250, 275, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 Dalton, or higher. In some embodiments, the molecular weight of F' is prior to enrichment with a halogen, a nonmetal, or a transition metal. In some embodiments, the molecular weight of the small molecule fragment is calculated based on carbon and hydrogen atoms and optionally further based on nitrogen, oxygen and/or sulfur atoms. In some embodiments, the molecular weight of the small molecule fragment does not include the molecular weight of a halogen, a transition metal or a combination thereof. In some embodiments, F' is a small molecule fragment moiety illustrated in FIG. 3. In some embodiments, the cysteine containing protein is a cysteine containing protein described herein. In some embodiments, the cysteine containing protein is a protein illustrated in Tables 1, 2, 3, 8 or 9. In some embodiments, the cysteine containing protein is a protein illustrated in Table 1. In some embodiments, the cysteine containing protein is a protein illustrated in Table 2. In some embodiments, the cysteine containing protein is a protein illustrated in Table 3. In some embodiments, the cysteine containing protein is a protein illustrated in Table 8. In some embodiments, the cysteine containing protein is a protein illustrated in Table 9.
[0023] Disclosed herein, in certain embodiments, is a derivative of IDH1 protein having the structure of Formula (I),
##STR00017##
wherein, the derivation occurs at IDH1 cysteine residue position 269 based on SEQ ID NO: 1; R is selected from:
##STR00018##
wherein R.sup.1 is H, C1-C3 alkyl, or aryl, and F' is a small molecule fragment moiety. In some embodiments, F' has a molecular weight of about 175, 200, 225, 250, 275, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 Dalton, or higher. In some embodiments, the molecular weight of F' is prior to enrichment with a halogen, a nonmetal, or a transition metal. In some embodiments, the molecular weight of the small molecule fragment is calculated based on carbon and hydrogen atoms and optionally further based on nitrogen, oxygen and/or sulfur atoms. In some embodiments, the molecular weight of the small molecule fragment does not include the molecular weight of a halogen, a transition metal or a combination thereof. In some embodiments, F' is a small molecule fragment moiety illustrated in FIG. 3.
[0024] Disclosed herein, in certain embodiments, is a derivative of IDH2 protein having the structure of Formula (I),
##STR00019##
wherein the derivation occurs at IDH2 cysteine residue position 308 based on SEQ ID NO: 2; R is selected from:
##STR00020##
wherein R.sup.1 is H, C1-C3 alkyl, or aryl; and F' is a small molecule fragment moiety. In some embodiments, F' has a molecular weight of about 175, 200, 225, 250, 275, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 Dalton, or higher. In some embodiments, the molecular weight of F' is prior to enrichment with a halogen, a nonmetal, or a transition metal. In some embodiments, the molecular weight of the small molecule fragment is calculated based on carbon and hydrogen atoms and optionally further based on nitrogen, oxygen and/or sulfur atoms. In some embodiments, the molecular weight of the small molecule fragment does not include the molecular weight of a halogen, a transition metal or a combination thereof. In some embodiments, F' is a small molecule fragment moiety illustrated in FIG. 3.
[0025] Disclosed herein, in certain embodiments, is a derivative of caspase-8 protein having the structure of Formula (I)
##STR00021##
wherein the derivation occurs at caspase-8 cysteine residue position 360 based on SEQ ID NO: 3; R is selected from:
##STR00022##
wherein R.sup.1 is H, C1-C3 alkyl, or aryl; and F' is a small molecule fragment moiety. In some embodiments, F' has a molecular weight of about 175, 200, 225, 250, 275, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 Dalton, or higher. In some embodiments, the molecular weight of F' is prior to enrichment with a halogen, a nonmetal, or a transition metal. In some embodiments, the molecular weight of the small molecule fragment is calculated based on carbon and hydrogen atoms and optionally further based on nitrogen, oxygen and/or sulfur atoms. In some embodiments, the molecular weight of the small molecule fragment does not include the molecular weight of a halogen, a transition metal or a combination thereof. In some embodiments, F' is a small molecule fragment moiety illustrated in FIG. 3.
[0026] Disclosed herein, in certain embodiments, is a derivative of caspase-10 protein having the structure of Formula (I),
##STR00023##
wherein the derivation occurs at caspase-10 cysteine residue position 401 based on SEQ ID NO: 4; R is selected from:
##STR00024##
wherein R.sup.1 is H, C1-C3 alkyl, or aryl; and F' is a small molecule fragment moiety. In some embodiments, F' has a molecular weight of about 175, 200, 225, 250, 275, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 Dalton, or higher. In some embodiments, the molecular weight of F' is prior to enrichment with a halogen, a nonmetal, or a transition metal. In some embodiments, the molecular weight of the small molecule fragment is calculated based on carbon and hydrogen atoms and optionally further based on nitrogen, oxygen and/or sulfur atoms. In some embodiments, the molecular weight of the small molecule fragment does not include the molecular weight of a halogen, a transition metal or a combination thereof. In some embodiments, F' is a small molecule fragment moiety illustrated in FIG. 3.
[0027] Disclosed herein, in certain embodiments, is a derivative of PRMT-1 protein having the structure of Formula (I),
##STR00025##
wherein the derivation occurs at PRMT-1 cysteine residue position 109 based on SEQ ID NO: 5; R is selected from:
##STR00026##
wherein R.sup.1 is H, C1-C3 alkyl, or aryl; and F' is a small molecule fragment moiety. In some embodiments, F' has a molecular weight of about 175, 200, 225, 250, 275, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 Dalton, or higher. In some embodiments, the molecular weight of F' is prior to enrichment with a halogen, a nonmetal, or a transition metal. In some embodiments, the molecular weight of the small molecule fragment is calculated based on carbon and hydrogen atoms and optionally further based on nitrogen, oxygen and/or sulfur atoms. In some embodiments, the molecular weight of the small molecule fragment does not include the molecular weight of a halogen, a transition metal or a combination thereof. In some embodiments, F' is a small molecule fragment moiety illustrated in FIG. 3.
[0028] Disclosed herein, in certain embodiments, is a derivative of ZAK protein having the structure of Formula (I),
##STR00027##
wherein the derivation occurs at ZAK cysteine residue position 22 based on SEQ ID NO: 6; R is selected from:
##STR00028##
wherein R.sup.1 is H, C1-C3 alkyl, or aryl; and F' is a small molecule fragment moiety. In some embodiments, F' has a molecular weight of about 175, 200, 225, 250, 275, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 Dalton, or higher. In some embodiments, the molecular weight of F' is prior to enrichment with a halogen, a nonmetal, or a transition metal. In some embodiments, the molecular weight of the small molecule fragment is calculated based on carbon and hydrogen atoms and optionally further based on nitrogen, oxygen and/or sulfur atoms. In some embodiments, the molecular weight of the small molecule fragment does not include the molecular weight of a halogen, a transition metal or a combination thereof. In some embodiments, F' is a small molecule fragment moiety illustrated in FIG. 3.
[0029] Disclosed herein, in certain embodiments, is a derivative of IMPDH2 protein having the structure of Formula (I),
##STR00029##
wherein the derivation occurs at IMPDH2 cysteine residue position 140 based on SEQ ID NO: 7; R is selected from:
##STR00030##
wherein R.sup.1 is H, C1-C3 alkyl, or aryl; and F' is a small molecule fragment moiety. In some embodiments, F' has a molecular weight of about 175, 200, 225, 250, 275, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 Dalton, or higher. In some embodiments, the molecular weight of F' is prior to enrichment with a halogen, a nonmetal, or a transition metal. In some embodiments, the molecular weight of the small molecule fragment is calculated based on carbon and hydrogen atoms and optionally further based on nitrogen, oxygen and/or sulfur atoms. In some embodiments, the molecular weight of the small molecule fragment does not include the molecular weight of a halogen, a transition metal or a combination thereof. In some embodiments, F' is a small molecule fragment moiety illustrated in FIG. 3.
[0030] Disclosed herein, in certain embodiments, is a derivative of IMPDH2 protein having the structure of Formula (I),
##STR00031##
wherein the derivation occurs at IMPDH2 cysteine residue position 331 based on SEQ ID NO: 7; R is selected from:
##STR00032##
wherein R.sup.1 is H, C1-C3 alkyl, or aryl; and F' is a small molecule fragment moiety. In some embodiments, F' has a molecular weight of about 175, 200, 225, 250, 275, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 Dalton, or higher. In some embodiments, the molecular weight of F' is prior to enrichment with a halogen, a nonmetal, or a transition metal. In some embodiments, the molecular weight of the small molecule fragment is calculated based on carbon and hydrogen atoms and optionally further based on nitrogen, oxygen and/or sulfur atoms. In some embodiments, the molecular weight of the small molecule fragment does not include the molecular weight of a halogen, a transition metal or a combination thereof. In some embodiments, F' is a small molecule fragment moiety illustrated in FIG. 3.
[0031] Disclosed herein, in certain embodiments, is a derivative of TIGAR protein having the structure of Formula (I),
##STR00033##
wherein the derivation occurs at TIGAR cysteine residue position 114 based on SEQ ID NO: 8; R is selected from:
##STR00034##
wherein R.sup.1 is H, C1-C3 alkyl, or aryl; and F' is a small molecule fragment moiety. In some embodiments, F' has a molecular weight of about 175, 200, 225, 250, 275, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 Dalton. or higher. In some embodiments, the molecular weight of F' is prior to enrichment with a halogen, a nonmetal, or a transition metal. In some embodiments, the molecular weight of the small molecule fragment is calculated based on carbon and hydrogen atoms and optionally further based on nitrogen, oxygen and/or sulfur atoms. In some embodiments, the molecular weight of the small molecule fragment does not include the molecular weight of a halogen, a transition metal or a combination thereof. In some embodiments, F' is a small molecule fragment moiety illustrated in FIG. 3.
[0032] Disclosed herein, in certain embodiments, is a derivative of TIGAR protein having the structure of Formula (I),
##STR00035##
wherein the derivation occurs at TIGAR cysteine residue position 161 based on SEQ ID NO: 8; R is selected from:
##STR00036##
wherein R.sup.1 is H, C1-C3 alkyl, or aryl; and F' is a small molecule fragment moiety. In some embodiments, F' has a molecular weight of about 175, 200, 225, 250, 275, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 Dalton, or higher. In some embodiments, the molecular weight of F' is prior to enrichment with a halogen, a nonmetal, or a transition metal. In some embodiments, the molecular weight of the small molecule fragment is calculated based on carbon and hydrogen atoms and optionally further based on nitrogen, oxygen and/or sulfur atoms. In some embodiments, the molecular weight of the small molecule fragment does not include the molecular weight of a halogen, a transition metal or a combination thereof. In some embodiments, F' is a small molecule fragment moiety illustrated in FIG. 3.
[0033] Disclosed herein, in certain embodiments, is a derivative of PKC.theta. protein having the structure of Formula (I),
##STR00037##
wherein the derivation occurs at PKC.theta. cysteine residue position 14 based on SEQ ID NO: 9; R is selected from:
##STR00038##
wherein R.sup.1 is H, C1-C3 alkyl, or aryl; and F' is a small molecule fragment moiety. In some embodiments, F' has a molecular weight of about 175, 200, 225, 250, 275, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 Dalton, or higher. In some embodiments, the molecular weight of F' is prior to enrichment with a halogen, a nonmetal, or a transition metal. In some embodiments, the molecular weight of the small molecule fragment is calculated based on carbon and hydrogen atoms and optionally further based on nitrogen, oxygen and/or sulfur atoms. In some embodiments, the molecular weight of the small molecule fragment does not include the molecular weight of a halogen, a transition metal or a combination thereof. In some embodiments, F' is a small molecule fragment moiety illustrated in FIG. 3.
[0034] Disclosed herein, in certain embodiments, is a derivative of PKC.theta. protein having the structure of Formula (I),
##STR00039##
wherein the derivation occurs at PKC.theta. cysteine residue position 17 based on SEQ ID NO: 9; R is selected from:
##STR00040##
wherein R.sup.1 is H, C1-C3 alkyl, or aryl; and F' is a small molecule fragment moiety. In some embodiments, F' has a molecular weight of about 175, 200, 225, 250, 275, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 Dalton, or higher. In some embodiments, the molecular weight of F' is prior to enrichment with a halogen, a nonmetal, or a transition metal. In some embodiments, the molecular weight of the small molecule fragment is calculated based on carbon and hydrogen atoms and optionally further based on nitrogen, oxygen and/or sulfur atoms. In some embodiments, the molecular weight of the small molecule fragment does not include the molecular weight of a halogen, a transition metal or a combination thereof. In some embodiments, F' is a small molecule fragment moiety illustrated in FIG. 3.
[0035] Disclosed herein, in certain embodiments, is a method of identifying a cysteine containing protein as a binding target for a small molecule fragment, comprising: (a) obtaining a set of cysteine-reactive probe-protein complexes from a sample comprising a first cell solution treated with a small molecule fragment and a cysteine reactive probe wherein the cysteine-reactive probe comprises a reactive moiety capable of forming a covalent bond with a cysteine residue located on the cysteine containing protein; (b) analyzing the set of cysteine-reactive probe-protein complexes by a proteomic analysis means; and (c) based on step b), identifying a cysteine containing protein as the binding target for the small molecule fragment. In some embodiments, the method further comprises determining a value of each of the cysteine containing protein from the set of cysteine-reactive probe-protein complexes for identifying a cysteine containing protein as the binding target for the small molecule fragment, wherein the value is determined based on the proteomic analysis means of step b). In some embodiments, the sample further comprises a second cell solution. In some embodiments, the method further comprises contacting the first cell solution with a small molecule fragment for an extended period of time prior to incubating the first cell solution with a first cysteine-reactive probe to generate a first group of cysteine-reactive probe-protein complexes. In some embodiments, the extended period of time is about 5, 10, 15, 20, 30, 60, 90, 120 minutes or longer. In some embodiments, the method further comprises contacting the second cell solution with a second cysteine-reactive probe to generate a second group of cysteine-reactive probe-protein complexes. In some embodiments, the first cysteine-reactive probe and the second cysteine-reactive probe are the same. In some embodiments, the first group and the second group of cysteine-reactive probe-protein complexes comprise the set of cysteine-reactive probe-protein complexes. In some embodiments, the cysteine containing protein is an enzyme, a transporter, a receptor, a channel protein, an adaptor protein, a chaperone, a signaling protein, a plasma protein, transcription related protein, translation related protein, mitochondrial protein, or cytoskeleton related protein. In some embodiments, the cysteine containing protein is a protein illustrated in Table 3. In some embodiments, the cysteine containing protein is a protein illustrated in Table 1, Table 2, Table 8. Table 9, Table 10A, Table 10B, Table 10C, Table 10D or Table 10E. In some embodiments, the small molecule fragment is a small molecule fragment of Formula (I):
##STR00041##
wherein: RM is a reactive moiety selected from a Michael acceptor moiety, a leaving group moiety, or a moiety capable of forming a covalent bond with the thiol group of a cysteine residue; and F is a small molecule fragment moiety. In some embodiments, the Michael acceptor moiety comprises an alkene or an alkyne moiety. In some embodiments. F is obtained from a compound library. In some embodiments, the compound library comprises ChemBridge fragment library, Pyramid Platform Fragment-Based Drug Discovery, Maybridge fragment library, FRGx from AnalytiCon. TCI-Frag from AnCoreX, Bio Building Blocks from ASINEX, BioFocus 3D from Charles River, Fragments of Life (FOL) from Emerald Bio, Enamine Fragment Library, IOTA Diverse 1500, BIONET fragments library, Life Chemicals Fragments Collection, OTAVA fragment library, Prestwick fragment library, Selcia fragment library, TimTec fragment-based library, Allium from Vitas-M Laboratory, or Zenobia fragment library. In some embodiments, F is a small molecule fragment moiety illustrated in FIG. 3. In some embodiments, the cysteine-reactive probe is a cysteine-reactive probe of Formula (II):
##STR00042##
wherein: RM is a reactive moiety selected from a Michael acceptor moiety, a leaving group moiety, or a moiety capable of forming a covalent bond to the thiol group of a cysteine residue; and AHM is an affinity handle moiety. In some embodiments, the Michael acceptor moiety comprises an alkene or an alkyne moiety. In some embodiments, the affinity handle moiety comprises an affinity handle and a binding moiety that facilitates covalent interaction of the cysteine-reactive probe to a cysteine residue of a cysteine-containing protein. In some embodiments, the binding moiety is a small molecule fragment obtained from a compound library. In some embodiments, the affinity handle comprises a carbodiimide, N-hydroxysuccinimide (NHS) ester, imidoester, pentafluorophenyl ester, hydroxymethyl phosphine, maleimide, haloacetyl, pyridyl disulfide, thiosulfonate, vinylsulfone, hydrazide, alkoxyamine, alkyne, azide, or isocyanate group. In some embodiments, the affinity handle is further conjugated to an affinity ligand. In some embodiments, the affinity ligand comprises a chromophore, a labeling group, or a combination thereof. In some embodiments, the chromophore comprises non-fluorochrome chromophore, quencher, an absorption chromophore, fluorophore, organic dye, inorganic dye, metal chelate, or a fluorescent enzyme substrate. In some embodiments, the labeling group is a biotin moiety, a streptavidin moiety, bead, resin, a solid support, or a combination thereof. In some embodiments, the cysteine-reactive probe is a cysteine-reactive probe illustrated in FIG. 3. In some embodiments, the proteomic analysis means comprises a mass spectroscopy method. In some embodiments, the identifying in step c) further comprises (i) locating a first value assigned to a cysteine containing protein from the first group of cysteine-reactive probe-protein complex and a second value of the same cysteine containing protein from the second group of cysteine-reactive probe-protein complex; and (ii) calculating a ratio between the two values assigned to the same cysteine containing protein. In some embodiments, the ratio of greater than 2 indicates that the cysteine containing protein is a candidate for interacting with the small molecule fragment. In some embodiments, the identifying in step c) further comprises calculating a percentage of inhibition of the cysteine-reactive probe to the cysteine containing protein. In some embodiments, the percentage of inhibition of greater than 50%, 60%, 70%, 80%, 90%, or at 100% indicates that the cysteine containing protein is a candidate for interacting with the small molecule fragment. In some embodiments, the method is an in situ method. In some embodiments, the cysteine-reactive probe is not 4-hydroxynonenal or 15-deoxy-.DELTA.12,14-prostaglandin J2.
[0036] Disclosed herein, modified cysteine containing protein comprising a small molecule fragment having a covalent bond to a cysteine residue of a cysteine containing protein, wherein the small molecule fragment has a molecular weight of about 150 Dalton or higher. In some embodiments, the cysteine containing protein comprises a cysteine residue site denoted in Table 3. In some embodiments, the cysteine containing protein comprises a protein sequence illustrated in Table 1, Table 2. Table 8, Table 9, Table 10A, Table 10B, Table 10C, Table 10D or Table 10E. In some embodiments, the cysteine containing protein is about 20, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000 amino acid residues in length or more. In some embodiments, the cysteine residue of the modified cysteine containing protein has the structure SR, wherein R is selected from:
##STR00043##
wherein R.sup.1 is H, C1-C3 alkyl, or aryl; and F' is the small molecule fragment moiety. In some embodiments, the small molecule fragment has a molecular weight of about 175, 200, 225, 250, 275, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 Dalton, or higher. In some embodiments, the molecular weight of the small molecule fragment is calculated based on carbon and hydrogen atoms and optionally further based on nitrogen, oxygen and/or sulfur atoms. In some embodiments, the modified cysteine containing protein is selected from IDH2, caspase-8, caspase-10 or PRMT1. In some embodiments, IDH2 is modified at cysteine position 308. In some embodiments, caspase-8 is modified at cysteine position 360. In some embodiments, caspase-10 exist in the proform and is modified at cysteine position 401. In some embodiments, PRMT1 is modified at cysteine position 109. In some embodiments, the small molecule fragment is a small molecule fragment of Formula (I):
##STR00044##
wherein: RM is a reactive moiety selected from a Michael acceptor moiety, a leaving group moiety, or a moiety capable of forming a covalent bond with the thiol group of a cysteine residue; and F is a small molecule fragment moiety. In some embodiments, the Michael acceptor moiety comprises an alkene or an alkyne moiety. In some embodiments. F is obtained from a compound library. In some embodiments, F is a small molecule fragment moiety illustrated in FIG. 3. In some embodiments. F further comprises a linker moiety that connects F to the carbonyl moiety. In some embodiments, the small molecule fragment binds irreversibly to the cysteine containing protein. In some embodiments, the small molecule fragment binds reversibly to the cysteine containing protein.
[0037] Disclosed herein, in certain embodiments, is a method of screening a small molecule fragment for interaction with a cysteine containing protein, comprising: (a) harvesting a set of cysteine-reactive probe-protein complexes from a sample comprising a first cell solution treated with a small molecule fragment and a cysteine reactive probe wherein the cysteine-reactive probe comprises a reactive moiety capable of forming a covalent bond with a cysteine residue located on the cysteine containing protein; (b) analyzing the set of cysteine-reactive probe-protein complexes by a proteomic analysis means; and (c) based on step b), identifying the small molecule fragment as interacting with the cysteine containing protein. In some embodiments, the method further comprises determining a value of each of the cysteine containing protein from the set of cysteine-reactive probe-protein complexes prior to identifying the small molecule fragment as interacting with the cysteine containing protein, wherein the value is determined based on the proteomic analysis means of step b). In some embodiments, the cysteine containing protein is a protein illustrated in Table 3. In some embodiments, the cysteine containing protein is a protein illustrated in Table 1. Table 2, Table 8, Table 9, Table 10A, Table 10B, Table 10C, Table 10D or Table 10E.
BRIEF DESCRIPTION OF THE DRAWINGS
[0038] Various aspects of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:
[0039] FIG. 1 illustrates proteome-wide screening of covalent fragments. A. General protocol for competitive isoTOP-ABPP. Cell lysate or intact cells are pre-treated with a fragment electrophile or DMSO and then reacted with an IA-alkyne probe 1. The fragment- and DMSO-treated samples are then conjugated to isotopically-differentiated TEV protease-cleavable biotin tags [light (red) and heavy (blue), respectively] by copper-mediated azide-alkyne cycloaddition (CuAAC or click) chemistry, mixed, and IA-labeled proteins enriched by streptavidin-conjugated beads and digested stepwise on-bead with trypsin and TEV to yield IA-labeled peptides for MS analysis. Competition ratios, or R values, are measured by dividing the MS1 ion peaks for IA-labeled peptides in DMSO-treated (heavy or blue) versus fragment-treated (light or red) samples. B, Representative members of the electrophilic fragment library, where the reactive (electrophilic) and binding groups are colored green and black, respectively. C, Initial analysis of the proteomic reactivity of fragments using an IA-rhodamine probe 16. Soluble proteome from Ramos cells was treated with the indicated fragments (500 .mu.M each) for 1 h, followed by labeling with IA-rhodamine (1 .mu.M, 1 h) and analysis by SDS-PAGE and in-gel fluorescence scanning. Several proteins were identified that show impaired reactivity with IA-rhodamine in the presence of one or more fragments (asterisks). Fluorescent gel shown in grayscale. D, Competitive isoTOP-ABPP analysis of fragment-cysteines interactions in the soluble proteome of MDA-MB-231 cells pre-treated with the following fragments (500 .mu.M each): 3,5-di(trifluoromethyl)aniline chloroacetamide 3, acrylamide 14, and acetamide 17. Proteomic reactivity values, or liganded cysteine rates, for fragments were calculated as the percentage of total cysteines with R values.gtoreq.4 in DMSO/fragment (heavy/light) comparisons. E, Concentration-dependent labeling of MDA-MB-231 soluble proteomes with acrylamide 18 and chloroacetamide 19 click probes detected by CuACC with a rhodamine-azide tag and analysis by SDS-PAGE and in-gel fluorescence scanning. F, Representative MS1 peptide ion chromatograms from competitive isoTOP-ABPP experiments performed with fragments 3, 4, and 23 marking liganded cysteines selectively targeted by one of three fragments (or, in the case of PHGDH C369, by all three fragments).
[0040] FIG. 2 illustrates a conceptual schematic of an exemplary computer server to be used for processing a method described herein.
[0041] FIG. 3 shows composition of fragment electrophile library and structures of additional tool compounds, click probes, and fragments.
[0042] FIG. 4 illustrates analysis of proteomic reactivities of fragment electrophiles determined by competitive isoTOP-ABPP in human cell lysates. A, Frequency of quantification of all cysteines across the complete set of competitive isoTOP-ABPP experiments performed with fragment electrophiles. Note that cysteines were required to have been quantified in at least three isoTOP-ABPP data sets for interpretation. B, Rank order of proteomic reactivity values (or liganded cysteine rates) of fragments calculated as the percentage of all quantified cysteines with R values.gtoreq.4 for each fragment. The majority of fragments were evaluated in 2-4 replicate experiments in MDA-MB-231 and/or Ramos cell lysates, and their proteomic reactivity values are reported as mean.+-.SEM values for the replicates. C, Comparison of the proteomic reactivities of representative fragments screened at 500 versus 25 .mu.M in cell lysates. D, Comparison of proteomic reactivity values for fragments tested in both Ramos and MDA-MB-231 lysates. E, Mean.+-.SEM data for proteomic reactivity values of representative fragments tested in at least three independent replicates. F, Relative GSH reactivity for representative fragment electrophiles. Consumption of GSH (125 .mu.M) was measured using Ellman's reagent (5 mM) after 1 h incubation with the indicated fragments (500 .mu.M). G, Proteomic reactivity values for fragments electrophiles (500 .mu.M) possessing different electrophilic groups attached to a common binding element.
[0043] FIG. 5 illustrates analysis of cysteines and proteins liganded by fragment electrophiles. A, Fraction of total quantified cysteines and proteins that were liganded by fragment electrophiles in competitive isoTOP-ABPP experiments. B, Fraction of liganded proteins found in DrugBank. C, Functional classes of DrugBank and non-DrugBank proteins containing liganded cysteines. D, Functional categorization of liganded and unliganded cysteines based on residue annotations from the Uniprot database. E, Comparison of the ligandability of cysteines as a function of their intrinsic reactivity with the IA-alkyne probe. Cysteine reactivity values were taken from Weerapana, et al. Nature 468, 790-795 (2010), where lower ratios correspond to higher cysteine reactivity. Individual cysteines are plotted on the x-axis and were sorted by reactivity, which is shown on the left y-axis. A moving average with a step-size of 50 is shown in blue for the percentage of liganded cysteines within each reactivity bin (percent values shown on the right y-axis). F, Number of liganded and quantified cysteines per protein measured by isoTOP-ABPP. Respective average values of one and three for liganded and quantified cysteines per protein were measured by isoTOP-ABPP. G, R values for six cysteines in XPO1 quantified by isoTOP-ABPP, identifying C528 as the most liganded cysteine in this protein. Each point represents a distinct fragment-cysteine interaction quantified by isoTOP-ABPP.
[0044] FIG. 6 illustrates analysis of fragment-cysteine interactions. A, Heatmap showing R values for representative cysteines and fragments organized by proteomic reactivity values (high to low, left to right) and percentage of fragment hits for individual cysteines (high to low, top to bottom). R values.gtoreq.4 designate fragment hits (colored medium and dark blue). White color designates fragment-cysteine interactions that were not detected (ND). B, C, Histograms depicting the percentage of fragments that are hits (R.gtoreq.4) for all 768 liganded cysteines (B) or for liganded cysteines found in enzymes for which X-ray and/or NMR structures have been reported (or reported for a close homologue of the enzyme) (C). D, Percentage of liganded cysteines targeted only by group A (red) or B (blue) fragments or both group A and B fragments (black). Shown for all liganded cysteines, liganded cysteines in enzyme active and non-active sites, and liganded cysteines in transcription factors/regulators. For C, D, active-site cysteines were defined as those that reside <10 .ANG. from established active-site residues and/or bound substrates/inhibitors in enzyme structures. E, Representative example of reactive docking predictions shown for XPO1 (PDB ID: 3GB8). All accessible cysteines were identified and reactive docking was conducted with all fragments from the library within a 25 .ANG. docking cube centered on each accessible cysteine. Categories of XPO1 cysteines based on combined docking and isoTOP-ABPP results are shown. F, Success rate of reactive docking predictions for liganded cysteines identified by isoTOP-ABPP in 29 representative proteins.
[0045] FIG. 7 illustrates analysis of cysteines liganded by fragment electrophiles in competitive isoTOP-ABPP experiments. A, Representative MS1 ion chromatograms for peptides containing C481 of BTK and C131 of MAP2K7, two cysteines known to be targeted by the anti-cancer drug ibrutinib. Ramos cells were treated with ibrutinib (1 .mu.M, 1 h, red trace) or DMSO (blue trace) and evaluated by isoTOP-ABPP. B, Total number of liganded cysteines found in the active sites and non-active sites of enzymes for which X-ray and/or NMR structures have been reported (or reported for a close homologue of the enzyme). C, R values for eight cysteines in PHGDH quantified by isoTOP-ABPP, identifying a single liganded cysteine C369 that is targeted by several fragment electrophiles. Each point represents a distinct fragment-cysteine interaction quantified by isoTOP-ABPP. D, Heatmap showing representative fragment interactions for liganded cysteines found in the active sites and non-active sites of kinases. E, Histogram showing the fragment hit rate for active- and non-active site cysteines in kinases. F, The percentage of liganded cysteines in kinases that were targeted by only group A, only group B, or both group A and B compounds. G, Heatmap showing representative fragment interactions for liganded cysteines found in transcription factors/regulators. H, The fraction of cysteines predicted to be ligandable or not ligandable by reactive docking that were quantified in isoTOP-ABPP experiments.
[0046] FIG. 8 illustrates confirmation and functional analysis of fragment-cysteine interactions. A, Representative MS1 chromatograms for the indicated Cys-containing peptides from PRMT1 quantified in competitive isoTOP-ABPP experiments of MDA-MB-231 cell lysates, showing blockade of IA-alkyne 1 labeling of C109 by fragment 11, but not control fragment 3. B, 11, but not 3 blocked IA-rhodamine (2 .mu.M) labeling of recombinant, purified WT-PRMT1 (1 .mu.M protein doped into HEK293T cell lysates). Note that a C109S-PRMT1 mutant did not react with IA-rhodaminc. C, IC.sub.50 curve for blockade of 16 labeling of PRMT1 by 11. CI, 95% confidence intervals. D, Effect of 11 and control fragment 3 on methylation of recombinant histone 4 by recombinant PRMT1. Shown is one representative experiment of three independent experiments that yielded similar results. E, 60, but not control fragment 3 (50 .mu.M of each fragment) blocked labeling of recombinant MLTK (or ZAK) kinase by a previously reported ibrutinib-derived activity probe 59 (upper panel). A C22A-MLTK mutant did not react with the ibrutinib probe. Anti-FLAG blotting confirmed similar expression of WT- and C22A-MLTK proteins, which were expressed as FLAG-fusion proteins in HEK293T cells (lower panel). F, IC.sub.50 curve for blockade of ibrutinib probe-labeling of MLTK by 60. G, 60, but not control fragment 3 (100 .mu.M of each fragment) inhibited the kinase activity of WT-, but not C22A-MLTK. H, Click probe 18 (25 .mu.M) labeled WT-IMPDH2 and C331S-IMPDH2, but not C140S-IMPDH2 (or C140S/C331S-IMPDH2). Labeling was detected by CuAAC conjugation to a rhodamine-azide reporter tag and analysis by SDS-PAGE and in-gel fluorescence scanning. Recombinant IMPDH2 WT and mutants were expressed and purified from E. coli and added to Jurkat lysates to a final concentration of 1 .mu.M protein. I, Nucleotide competition profile for 18-labeling of recombinant WT-IMPDH2 (500 .mu.M of each nucleotide). J, IC.sub.50 curve for blockade of 18 labeling of IMPDH2 by ATP. K, 5, but not control fragment 3 blocked IA-rhodamine (2 .mu.M) labeling of recombinant, purified C161S-TIGAR (2 .mu.M protein doped into Ramos cell lysates). L, IC.sub.50 curve for blockade of IA-rhodamine labeling of C161S-TIGAR by 5. M, 5, but not control fragment 3 (100 .mu.M of each fragment) inhibited the catalytic activity of WT-TIGAR, C161S-TIGAR, but not C114S-TIGAR or C114S/C161S-TIGAR. For panels C, F, C, I, J, L, and M, data represent mean values.+-.SEM for at least three independent experiments. Statistical significance was calculated with unpaired students t-tests comparing DMSO- to fragment-treated samples; **, p<0.01, ****, p<0.0001.
[0047] FIG. 9 illustrates confirmation and functional analysis of fragment-cysteine interactions. A, Representative MS1 ion chromatograms for the MLTK tryptic peptide containing liganded cysteine C22 quantified by isoTOP-ABPP in MDA-MB-231 lysates treated with fragment 4 or control fragment 3 (500 .mu.M each). B, Lysates from HEK293T cells expressing WT- or C22A-MLTK treated with the indicated fragments and then an ibrutinib-derived activity probe 59 at 10 .mu.M. MLTK labeling by 59 was detected by CuAAC conjugation to a rhodamine-azide tag and analysis by SDS-PAGE and in-gel fluorescence scanning. C, Representative MS1 ion chromatograms for IMPDH2 tryptic peptides containing the catalytic cysteine. C331, and Bateman domain cysteine. C140, quantified by isoTOP-ABPP in cell lysates treated with the indicated fragments (500 .mu.M each). D, Structure of human IMPDH2 (PDB ID: INF7) (light grey) and its structurally unresolved Bateman domain modeled by ITASSER (dark grey) showing the positions of C331 (red spheres), Ribavirin Monophosphate and C2-Mycophenolic Adenine Dinucleotide (blue), and C140 (yellow spheres). E, Fragment reactivity with recombinant, purified IMPDH2 added to Jurkat lysates to a final concentration of 1 .mu.M protein, where reactivity was detected in competition assays using the click probe 18 (25 .mu.M; see FIG. 8H for structure of 18). Note that 18 reacted with WT- and C331S-IMPDH2, but not C140S or C140S/C331S-IMPDH2. F. Nucleotide competition of 18 (25 .mu.M) labeling of WT-IMPDH2 added to cell lysates to a final concentration of 1 .mu.M protein. G, Representative MS1 chromatograms for TIGAR tryptic peptides containing C114 and C161 quantified by isoTOP-ABPP in cell lysates treated with the indicated fragments (500 .mu.M each). H, Crystal structure of TIGAR (PDB ID: 3DCY) showing C114 (red spheres), C161 (yellow spheres), and inorganic phosphate (blue). I, Labeling of recombinant, purified TIGAR and mutant proteins by the IA-rhodamine (2 .mu.M) probe. TIGAR proteins were added to cell lysates, to a final concentration of 2 .mu.M protein. J, Concentration-dependent inhibition of WT-TIGAR by 5. Note that the C140S-TIGAR mutant was not inhibited by 5. Data represent mean values.+-.SEM for 4 replicate experiments at each concentration.
[0048] FIG. 10 illustrates in situ activity of fragment electrophiles. A. X-ray crystal structure of IDH1 (PDB ID: 3MAS) showing the position of C269 and the frequently mutated residue in cancer, R132, B, C, Reactivity of 20 and control fragment 2 with recombinant, purified WT-IDH1 (B) or R132H-IDH1 (C) added to cell lysates to a final concentration of 2 or 4 .mu.M protein, respectively. Fragment reactivity was detected in competition assays using the IA-rhodamine probe (2 .mu.M); note that the C269S-IDH1 mutant did not react with IA-rhodamine. D, Representative MS1 ion chromatograms for the IDH1 tryptic peptides containing liganded cysteine C269 and an unliganded cysteine C379 quantified by isoTOP-ABPP in MDA-MB-231 lysates treated with fragment 20 (25 .mu.M). E, Western blot of MUM2C cells stably overexpressing GFP (mock) or R132H-IDH1 proteins. F, Representative MS1 chromatograms for the IDH1 tryptic peptides containing liganded cysteine C269 and an unliganded cysteine C379 quantified by isoTOP-ABPP in R132H-IDH-expressing MUM2C lysates treated with 20 or control fragment 2 (5 .mu.M, 2 h, in situ).
[0049] FIG. 11 illustrates in situ activity of fragment electrophiles. A, Blockade of 16 labeling of WT-IDH1 by representative fragment electrophiles. Recombinant, purified WT-IDH1 was added to MDA-MB-231 lysates at a final concentration of 2 .mu.M, treated with fragments at the indicated concentrations, followed by IA-rhodamine probe 16 (2 .mu.M) and analysis by SDS-PAGE and in-gel fluorescence scanning. Note that a C269S mutant of IDH1 did not label with IA-rhodamine 16. B. IC.sub.50 curve for blockade of IA-rhodamine-labeling of IDH1 by 20. Note that the control fragment 2 showed much lower activity. C, 20, but not 2, inhibited IDH1-catalyzed oxidation of isocitrate to .alpha.-ketoglutarate (.alpha.-KG) as measured by an increase in NADPH production (340 nm absorbance). 20 did not inhibit the C269S-IDH1 mutant. D, 20 inhibited oncometabolite 2-hydroxyglutarate (2-HG) production by R132H-IDH1. MUM2C cells stably overexpressing the oncogenic R132H-IDH1 mutant or control GFP-expressing MUM2C cells were treated with the indicated fragments (2 h, in situ). Cells were harvested, lysed and IDH I-dependent production of 2-HG from .alpha.-KG and NADPH was measured by LC-MS and from which 2-HG production of GFP-expressing MUM2C cells was subtracted (GFP-expressing MUM2C cells produced <10% of the 2-HG generated by R132H-IDH1-expressing MUM2C cells). E, Proteomic reactivity values for individual fragments are comparable in vitro and in situ. One fragment (11) marked in red showed notably lower reactivity in situ versus in vitro. Reactivity values were calculated as in FIG. 1D. Dashed line mark 90% prediction intervals for the comparison of in vitro and in situ proteomic reactivity values for fragment electrophiles. Blue and red circles mark fragments that fall above (or just at) or below these prediction intervals, respectively. F. Fraction of cysteines liganded in vitro that is also liganded in situ. Shown are liganded cysteine numbers for individual fragments determined in vitro and the fraction of these cysteines that were liganded by the corresponding fragments in situ. G, Representative cysteines that were selectively targeted by fragments in situ, but not in vitro. For in situ-restricted fragment-cysteine interactions, a second cysteine in the parent protein was detected with an unchanging ratio (R.about.1), thus controlling for potential fragment-induced changes in protein expression. For panels B-D, data represent mean values.+-.SEM for at least three independent experiments. Statistical significance was calculated with unpaired students t-tests comparing DMSO- to fragment-treated samples; ****, p<0.0001.
[0050] FIG. 12 illustrates fragment electrophiles that target pro-CASP8. A, Representative MS1 chromatograms for CASP8 tryptic peptide containing the catalytic cysteine C360 quantified by isoTOP-ABPP in cell lysates or cells treated with fragment 4 (250 .mu.M, in vitro; 100 .mu.M, in situ) and control fragment 21 (500 .mu.M, in vitro; 200 .mu.M, in situ). B, Fragment reactivity with recombinant, purified active CASP8 added to cell lysates, where reactivity was detected in competition assays using the caspase activity probe Rho-DEVD-AOMK probe ("DEVD" disclosed as SEQ ID NO: 857) (2 .mu.M, 1 h). C, Western blot of proteomes from MDA-MB-231, Jurkat, and CASP8-null Jurkat proteomes showing that CASP8 was only found in the pro-enzyme form in these cells. D, Fragment reactivity with recombinant, purified pro-CASP8 (D374A, D384A, C409S) added to cell lysates to a final concentration of 1 .mu.M protein, where reactivity was detected in competition assays with the IA-rhodamine probe (2 .mu.M). Note that mutation of both cysteine-360 and cysteine-409 to serine prevented labeling of pro-CASP8 by IA-rhodamine. E, Concentration-dependent reactivity of click probe 61, with recombinant, purified pro-CASP8 (D374A, D384A) added to cell lysates to a final concentration of 1 .mu.M protein. Note that pre-treatment with 7 blocked 61 reactivity with pro-CASP8 and mutation of C360 to serine prevented labeling of pro-CASP8 by 61 (25 .mu.M). F, Fragments 7 and 62 did not block labeling by Rho-DEVD-AOMK ("DEVD" disclosed as SEQ ID NO: 857) (2 .mu.M) of recombinant, purified active-CASP8 and active-CASP3 added to MDA-MB-231 cell lysates to a final concentration of 1 .mu.M protein. G, Representative MS1 chromatograms for tryptic peptides containing the catalytic cysteines of CASP8 (C360), CASP2 (C320), and CASP7 (C186) quantified by isoTOP-ABPP in Jurkat cell lysates treated with 7 or 62 (50 .mu.M, 1 h). H, Representative MS1 chromatograms for CASP8 trvptic peptide containing C360 quantified by isoTOP-ABPP in cell lysates treated with 10 versus 100 .mu.M of 61. Structure of CASP8 C360 tryptic peptide adduct (blue) modified by 61 (black) and conjugated to TEV cleavable tag (red), where underline indicates site of isotopic modification. Figure discloses SEQ ID NO: 864.
[0051] FIG. 13 illustrates fragment electrophiles that target pro-CASP8. A, 7 blocked IA-rhodamine 16 labeling of pro-CASP8. Experiments were performed with recombinant, purified pro-CASP8 (bearing a C409S mutation to eliminate IA-rhodamine labeling at this site) added to Ramos cell lysate at a final concentration of 1 .mu.M and treated with the indicated concentrations of 7 followed by IA-rhodamine (2 .mu.M). Note that a C360S/C409S-mutant of pro-CASP8 did not label with IA-rhodamine. B, IC.sub.50 curve for blockade of IA-rhodamine labeling of pro-CASP8 (C409S) by 7. C, 7 (50 .mu.M) fully competed IA-alkyne-labeling of C360 of endogenous CASP8 in cell lysates as measured by isoTOP-ABPP. Representative MS1 chromatograms are shown for the C360-containing peptide of CASP8. D, 7 selectively blocked probe labeling of pro-CASP8 compared to active CASP8. Recombinant pro- and active-CASP8 (added to Ramos cell lysates at a final concentration of 1 .mu.M each) were treated with 7 (50 .mu.M) or the established caspase inhibitor. Ac-DEVD-CHO ("DEVD" disclosed as SEQ ID NO: 857) (20 .mu.M), for 1 h followed by labeling with the click probe 61 (25 .mu.M) for pro-CASP8 and the Rho-DEVD-AOMK probe ("DEVD" disclosed as SEQ ID NO: 857) (2 .mu.M) for active-CASP8. SDS-PAGE and in-gel fluorescence scanning revealed that 7 competes 61-labeling of pro-CASP8, but not Rho-DEVD-AOMK ("DEVD" disclosed as SEQ ID NO: 857) of active-CASP8, and, conversely, DEVD-CHO ("DEVD" disclosed as SEQ ID NO: 857) competed Rho-DEVD-AOMK ("DEVD" disclosed as SEQ ID NO: 857) labeling of active-CASP8, but not 61-labeling of pro-CASP8. E, Neither 7 nor control fragment 62 (100 .mu.M each) inhibited the activity of recombinant, purified active CASP8 and CASP3, which were assayed following addition to Ramos cell lysate using DEVD-AMC and IETD-AFC substrates, respectively. DEVD-CHO ("DEVD" disclosed as SEQ ID NO: 857) (20 .mu.M) inhibited the activity of both CASP8 and CASP3. F, 7 (30 .mu.M) blocked IA-alkyne labeling of C360 of pro-CASP8, but not active-CASP8 as measured by isoTOP-ABPP. Recombinant pro- and active-CASP8 were added to Ramos lysates at 1 .mu.M and then treated with 7 (30 .mu.M) followed by isoTOP-ABPP. G, Substitution of a naphthylamine for the aniline portion of 7 furnishes a control fragment 62 that did not compete with IA-rhodamine labeling of C360 of pro-CASP8. H, 7, but not control fragment 62, blocked extrinsic, but not intrinsic apoptosis. Jurkat cells (1.5 million cells/mL) were incubated with 7 or 62 (30 .mu.M) or the pan-caspase inhibitor VAD-FMK (100 .mu.M) for 30 min prior to addition of staurosporine (2 .mu.M) or SuperFasLigand.TM. (100 ng/mL). Cells were incubated for 6 hours and viability was quantified with CellTiter-Glo.RTM.. RLU--relative light unit. I, For cells treated as described in H, cleavage of PARP (89 kDa), CASP8 (p43/p41), and CASP3 (p19/p17) was visualized by western blot. For panels B, E, and H, data represent mean values.+-.SEM for at least three independent experiments.
[0052] FIG. 14 shows electrophile compounds that target pro-CASP8 and pro-CASP10. Heatmap showing R values for caspases measured by quantitative proteomics in Jurkat cells treated with 7, 63-R, or 62 followed by probe 61 (10 .mu.M, 1 h) (A). Comparison of effects of 7 and 63-R on FasL-induced apoptosis in Jurkat cells or anti-CD3, anti-CD28-activated primary human T cells (B). For B, data represent mean values.+-.SEM for at least three independent experiments, and results are representative of multiple experiments performed with T cells from different human subjects. Statistical significance was calculated with unpaired students t-tests comparing DMSO- to fragment treated samples; ****, p<0.0001 and comparing Jurkat to T cells ####, p<0.0001.
[0053] FIG. 15 illustrates a fraction of liganded (62%; 341 of 553 quantified cysteines) and unliganded (20%; 561 of 2870 quantified cysteines) cysteines that are sensitive to heat denaturation measured by IA-alkyne labeling (R>3 native/heat denatured).
[0054] FIG. 16 shows a percentage of proteins identified by isoTOP-ABPP as liganded by fragments 3 and 14 and enriched by their corresponding click probes 19 and 18 that are sensitive to heat denaturation (64% (85 of 133 quantified protein targets) and 73% (19 of 26 quantified protein targets), respectively). Protein enrichment by 18 and 19 was measured by whole protein capture of isotopically-SILAC labeled MDA-MB-231 cells using quantitative (SILAC) proteomics.
[0055] FIG. 17A-FIG. 17B illustrate exemplary fractions of cysteines predicted based on isoTOP-ABPP method or IA-alkyne probe. FIG. 17A shows the fraction of cysteines predicted to be ligandable or unligandable by reactive docking that were quantified in isoTOP-ABPP experiments. FIG. 17B shows the fraction of cysteines predicted to be ligandable or unligandable by reactive docking that show heat-sensitive labeling by the IA-alkyne probe (R>3 native/heat denatured).
[0056] FIG. 18 shows lysates from HEK293T cells expressing WT or C22A-MLTK treated with the indicated fragments and then an ibrutinib-derived activity probe 59 at 10 .mu.M. MLTK labeling by 59 was detected by CuAAC conjugation to a rhodamine-azide tag and analysis by SDS-PAGE and in-gel fluorescence scanning.
[0057] FIG. 19 shows click probe 18 (2 .mu.M) labeled WT-IMPDH2 and C331S-IMPDH2, but not C140S-IMPDH2 (or C140S/C331S-IMPDH2). Labeling was detected by CuAAC conjugation to a rhodamine-azide reporter tag and analysis by SDS-PAGE and in-gel fluorescence scanning. Recombinant IMPDH2 WT and mutants were expressed and purified from E. coli and added to Jurkat lysates to a final concentration of 1 .mu.M protein.
[0058] FIG. 20 shows the apparent IC.sub.50 curve for blockade of IA rhodamine-labeling of R132H-IDH1 by 20.
[0059] FIG. 21A-FIG. 21C show the activity of compounds 7 and 62 with respect to different recombinant caspases. FIG. 21A shows that 7 does not inhibit active caspases. Recombinant, active caspases were added to MDA-MD-231 lysate to a final concentration of 200 nM (CASP2, 3, 6, 7) or 1 gtM (CASP8, 10), treated with z-VAD-FMK (25 .mu.M) or 7 (50 .mu.M), followed by labeling with the Rho-DEVD-AOMK probe ("DEVD" disclosed as SEQ ID NO: 857) (2 .mu.M). FIG. 21B shows a western blot of the cleavage of PARP (96 kDa), CASP8 (p43/p41, p18), and CASP3 (p17). FIG. 21C shows that 7 protects Jurkat cells from extrinsic, but not intrinsic apoptosis. Cleavage of PARP, CASP8, and CASP3 detected by western blotting as shown in FIG. 21B was quantified for three (STS) or two (FasL) independent experiments. Cleavage products (PARP (96 kDa), CASP8 (p43/p4), CASP3 (p17)) were quantified for compound treatment and the % cleavage relative to DMSO treated samples was calculated. For FIG. 21C, STS data represent mean values.+-.SEM for three independent experiments, and FasL data represent mean values.+-.SD for two independent experiments. Statistical significance was calculated with unpaired students t-tests comparing active compounds (VAD-FMK and 7) to control compound 62; **, p<0.01, ***, p<0.001, ****, p<0.0001.
[0060] FIG. 22 shows that CASP10 is involved in intrinsic apoptosis in primary human T cells. A, Representative MS1 peptide signals showing R values for caspases detected by quantitative proteomics using probe 61. ABPP-SILAC experiments. Jurkat cells (10 million cells) were treated with either DMSO (heavy cells) or the indicated compounds (light cells) for 2 h followed by probe 61 (10 .mu.M, 1 h). B, 7 competed 61-labeling of pro-CASP8 and CASP10, whereas 63-R selectively blocked probe labeling of pro-CASP8. C, 7, but not 63-R block probe labeling of pro-CASP10. Recombinant pro-CASP10 was added to MDA-MB-231 lysates to a final concentration of 300 nM, treated with the indicated compounds, and labeled with probe 61. Mutation of the catalytic cysteine C401A fully prevented labeling by 61. D, Apparent IC50 curve for blockade of 61-labeling of pro-CASP10 by 7, 63-R or 63-S. E. Neither 7 nor 63 (25 .mu.M each) inhibited the activity of recombinant, purified active CASP10 (500 nM), which was assayed following addition of the protein to MDA-MB-231 lysate using fluorometric AEVD-AMC substrate ("AEVD" disclosed as SEQ ID NO: 859). DEVD-CHO ("DEVD" disclosed as SEQ ID NO: 857) (20 .mu.M) inhibited the activity of CASP10. F, Apparent IC.sub.50 curve for blockade of 61 labeling of pro-CASP8 and pro-CASP0 by 63-R. G, 63-R shows increased potency against pro-CASP8. Recombinant pro-CASP8 was added to MDA-MB-231 lysates to a final concentration of 300 nM, treated with the indicated compounds and labeled with probe 61. H, Apparent IC50 curve for blockade of 61 labeling of pro-CASP8 by 63-R compared with 63-S. The structure of 63-S is shown. I. CASP10 is more highly expressed in primary human T cells compared to Jurkat cells. Western blot analysis of full-length CASP10, CASP8 and GAPDH expression levels in Jurkat and T-cell lysates (2 mg/mL). J, Jurkat cells (150,000 cells/well) were incubated with 7 or 63-R at the indicated concentrations for 30 min prior to addition of staurosporine (2 .mu.M) or SuperFasLigand.TM. (100 ng/mL). Cells were incubated for 4 h and viability was quantified with CellTiter-Glo.RTM.. K, Jurkat cells treated as in J, but with 63-R or 63-S. L, HeLa cells (20,000 cells/well) were seeded and 24 h later treated with the indicated compounds for 30 minutes prior to addition of SuperFasLigandr (100 ng/mL) and cycloheximide (CHX, 2.5 ng/mL). Cells were incubated for 6 h and viability quantified with CTG. M, For T cells treated as in FIG. 14B cleavage of CASP10 (p22), CASP8 (p18). CASP3 (p17) and RIPK (33 kDa) was visualized by western blotting. For panels D-F, H, and J-K data represent mean values.+-.SEM for at least three independent experiments. Statistical significance was calculated with unpaired students t-tests comparing DMSO- to fragment-treated samples; **, p<0.01, ****, p<0.0001.
[0061] FIG. 23A-FIG. 23F exemplify DMF inhibits the activation of primary human T cells. FIG. 23A illustrates the chemical structures of DMF, MMF, and DMS. FIG. 23B-FIG. 23E illustrate bar graphs that exemplify IL-2 release (FIG. 23B), CD25 expression (FIG. 23C and FIG. 23D), and CD69 expression (FIG. 23E) in primary human T cells, either unstimulated (Unstim) or stimulated (Stim) with anti-CD3+anti-CD28 in the presence of DMSO or the indicated concentrations of DMF, MMF, and DMS for 8 hours. FIG. 23F illustrates a bar graph that exemplifies time course of DMF effects. T cells were stimulated with anti-CD3+anti-CD28 for the indicated periods of time before beginning DMF treatment. Cells were harvested 24 h after beginning T cell stimulation. Shown are data gated on CD4+ cells. Data represent mean.+-.SE, n=4-6 experiments/group. *p<0.05, **p<0.01, ***p<0.001 in comparison to DMSO group.
[0062] FIG. 24 illustrates a bar graph that exemplifies DMF does not affect T cell viability. Primary human T cells were stimulated with anti-CD3 and anti-CD28 antibodies as indicated and treated concomitantly with compound for 8 h. Cells were then stained with LIVE/DEAD fixable blue stain, and analyzed by flow cytometry. Shown are data gated on CD4+ cells. Data represent mean.+-.SE for four experiments per group.
[0063] FIG. 25A-FIG. 25B illustrate bar graphs that exemplify DMF, but not MMF, inhibits the activation of primary mouse T cells. Splenic T cells were harvested from C57BL/6 mice and left either unstimulated (Unstim) or stimulated (Stim) with anti-CD3+anti-CD28 in the presence of DMSO or the indicated concentrations of DMF. MMF, and DMS for 8 h. Activation was assessed by measuring CD25 (FIG. 25A) and CD69 (FIG. 25B) expression. Data represent mean.+-.SE for four experiments per group. ***p<0.001 in comparison to DMSO group.
[0064] FIG. 26A-FIG. 26D illustrate bar graphs that exemplify inhibitory effects of DMF are equivalent in Nrf2(+/+) and (-/-) T cells and not caused by reductions in cellular GSH. FIG. 26A exemplifies CD25 expression in anti-CD3+anti-CD28-stimulated Nrf2(+/+) and (-/-) T cells. Splenic T cells were harvested from Nrf2(+/+) and (-/-) mice, then stimulated in the presence of indicated compounds for 24 h. FIG. 26B and FIG. 26C exemplify treatment with DMF or BSO causes significant reductions in GSH content of human T cells. Primary human T cells were stimulated with anti-CD3+anti-CD28 antibodies and treated with DMF (50 .mu.M, 2 hours) or BSO (2.5 mM, 4 hours), after which intracellular GSH levels were measured. FIG. 26D exemplifies that BSO does not alter T cell activation. Primary human T cells were treated with DMSO, DMF (50 .mu.M), or BSO (2.5 mM) and stimulated as indicated for 8 h, after which CD25 expression was measured. Data represent mean.+-.SE for two biological replicates, with 3-4 technical replicates per biological replicate. *p<0.05, **p<0.01, ***p<0.001 in comparison to DMSO groups.
[0065] FIG. 27A-FIG. 27F exemplify isoTOP-ABPP of DMF-treated primary human T cells. FIG. 27A illustrates a graph that exemplifies isoTOP-ABPP ratios, or R values, for >2400 Cys residues in primary human T cells treated with DMSO or DMF or MMF (50 .mu.M, 4 h). FIG. 27B illustrates a graph that exemplifies expanded profile for DMF-sensitive Cys residues (R values>4 for DMSO/DMF). For FIG. 27A and FIG. 27B, data represent aggregate quantified Cys residues from five biological replicates. For Cys residues quantified in more than one replicate, average ratios are reported. Dashed line designates R values>4, which was used to define DMF-sensitive Cys residues (>4-fold reductions in IA-alkyne reactivity in DMF-treated T cells). FIG. 27C and FIG. 27D illustrate graphs that exemplify concentration- and time-dependent profiles for DMF-sensitive Cys residues in T cells, respectively. For additional concentrations (10 and 25 .mu.M) and time points (1 and 2 h), data represent aggregate quantified Cys residues from one-three isoTOP-ABPP experiments per group. FIG. 27E illustrates a chart which exemplifies fraction of proteins for which both a DMF-sensitive Cys residue and at least one additional Cys residue was quantified (Left) and, fraction of these proteins where additional Cys residue was clearly unchanged (Right) (R value<2.0 for DMSO/DMF). Unclear calls mark proteins with DMF-sensitive Cys residues where the R value for second Cys showed marginal evidence of potential change (R values between 2.0 and 3.9). FIG. 27F illustrates representative MS profiles for quantified Cys residues in PRKDC, one of which (C4045) shows sensitivity to DMF.
[0066] FIG. 28A-FIG. 28B illustrate bar graphs that exemplify the total number of unique quantified peptides (FIG. 28A) and proteins (FIG. 28B) begin to plateau after five biological replicates of the isoTOP-ABPP experiment in primary human T cells (treated with 50 uM DMF for 4 h).
[0067] FIG. 29 illustrates a graph that exemplifies isoTOP-ABPP of BSO-treated primary human T cells. Cells were treated with 2.5 mM BSO for 4 hours. Data represent aggregate quantified Cys residues from two isoTOP-ABPP experiments per group.
[0068] FIG. 30A-FIG. 30C exemplify conservation and functional analysis of DMF-sensitive cysteines. FIG. 30A exemplifies fraction of DMF-sensitive cysteines in the human T cell proteome that are conserved in mice. FIG. 30B exemplifies fraction of conserved DMF-sensitive Cys residues in human T cells that were quantified and also sensitive to DMF in mouse T cells. FIG. 30C exemplifies distribution of proteins harboring DMF-sensitive Cys residues by functional class.
[0069] FIG. 31A-FIG. 31C exemplify DMF inhibits p65 translocation to the nucleus in primary human T cells. FIG. 31A exemplify Human T cells were either left unstimulated or stimulated with anti-CD3 and anti-CD28 antibodies and treated with DMSO or DMF (50 uM) for 1 h. FIG. 31B illustrates a bar graph that exemplifies ratio of nuclear to cytoplasmic localization of p65 for samples shown in FIG. 31A, as well as samples treated with MMF (50 uM) or DMS (50 uM). FIG. 31C exemplifies p65 levels in whole cell lysate.
[0070] FIG. 32A-FIG. 32G exemplify DMF-sensitive C14/C17 residues in PKC.theta. are important for CD28 interactions and T cell activation. FIG. 32A illustrates representative MS1 profiles for DMF-sensitive (C14/C17) and -insensitive (C322) Cys residues in PKC.theta.. FIG. 32B exemplifies sequence conservation analysis of human and mouse PKC.theta., human PKC.delta., and human PKC.epsilon. (SEQ ID NOS 865-868, respectively, in order of appearance). Shown in red are C14 and C17. FIG. 32C illustrates location of DMF-sensitive C14 and C17 residues in the C2 domain of PKC.theta. (PDB accession number 2ENJ). FIG. 32D exemplifies DMF, but not MMF, treatment blocks the association of PKC.theta. with CD28. Peripheral CD4+ T cells from C57BL/6 mice were pre-incubated with DMSO, DMF (50 .mu.M), or MMF (50 .mu.M), either left unstimulated or stimulated with anti-CD3+anti-CD28 for 5 min, then washed and lysed. Immunoprecipitations (IPs) were performed in the cell lysates with anti-CD28 or control IgG antibodies and IPs blotted for CD28 or PKC.theta.. FIG. 32E illustrates Co-IP of WT PKC.theta. and the C14S/C17S (2CS) PKC.theta. mutant with CD28. PKC.theta.(-/-) T cells were reconstituted with empty vector (EV), WT PKC.theta., or the 2CS PKC.theta. mutant. FIG. 32F and FIG. 32G illustrate PKC.theta.(-/-) T cells reconstituted with WT or 2CS PKC.theta. were assayed for activation potential by measuring CD25 expression (FIG. 32F) and IL-2 (FIG. 32G). For FIG. 32E-FIG. 32G, PKC.theta.(-/-) T cell cultures were pre-activated with plate-coated anti-CD3+anti-CD28 for 24 h before retroviral transduction with empty vector, WT PKC.theta., or the 2CS PKC.theta. mutant. Cells were rested in culture medium without stimulation for 48 h. then re-stimulated with or without 1 .mu.g/mL plate-coated anti-CD3(+28) overnight (FIG. 32F), for 48 h (FIG. 32G), or with soluble 10 .mu.g/mL anti-CD3+anti-CD28 for 5 min prior to IP (FIG. 32D). For FIG. 32D and FIG. 32E, data are from a single experiment representative of three biological replicates. For FIG. 32F and FIG. 32G, data represent mean.+-.SE for three biological replicates. ***p<0.001 in comparison to WT PKC.theta. group.
[0071] FIG. 33A-FIG. 33D exemplify DMF sensitivity of C14/C17 in PKC.theta.. FIG. 33A illustrates representative MS1 profile of C14/C17 of mouse PKC.theta. shows sensitivity to DMF (50 .mu.M, 4 h) in isoTOP-ABPP experiments. FIG. 33B and FIG. 33C exemplify Time- and concentration-dependence of DMF sensitivity of C14/C17 in human PKC.theta., respectively, as determined by isoTOP-ABPP experiments. FIG. 33D exemplifies C14/C17 of human PKC.theta. are insensitive to MMF treatment (50 .mu.M MMF, 4 h).
[0072] FIG. 34A-FIG. 34B exemplify DMF-sensitive Cys residue in ADA. FIG. 34A illustrates the DMF-sensitive Cys, C75 (magenta), is .about.25 angstroms from the ADA active site (orange). FIG. 34B illustrates mutations in both residues neighboring C75 (G74 and R76 (blue)) have been associated with the severe combined immunodeficiency known as ADASCID (OMIM: 608958). PDB accession number: 3IAR.
DETAILED DESCRIPTION OF THE INVENTION
[0073] Cysteine containing proteins encompass a large repertoire of proteins that participate in numerous cellular functions such as mitogenesis, proliferation, apoptosis, gene regulation, and proteolysis. These proteins include enzymes, transporters, receptors, channel proteins, adaptor proteins, chaperones, signaling proteins, plasma proteins, transcription related proteins, translation related proteins, mitochondrial proteins, or cytoskeleton related proteins. Dysregulated expression of a cysteine containing protein, in many cases, is associated with or modulates a disease, such as an inflammatory related disease, a neurodegenerative disease, or cancer. As such, identification of a potential agonist/antagonist to a cysteine containing protein aids in improving the disease condition in a patient.
[0074] In some instances, potential constrains exist in drug screening due to the structurally complex compound and the inability of some of the structurally complexed compound to interact with the protein. As such, small molecule fragments are employed in some instances to serve as launching point for structure-guided elaboration of an initial interaction into a high-affinity drug. In some instances, one method of identifying a small molecule fragment that interacts with a cysteine containing protein is through monitoring their interaction under an in vitro environment. However in some cases, the in vitro environment does not mimic the native condition of the cysteine containing protein. In other cases, the in vitro environment lacks additional helper proteins to facilitate interaction with the small molecule fragment. Further still, in some instances, difficulties arise during the expression and/or purification stage of the cysteine-containing protein.
[0075] Described herein is another method of identifying small molecule fragments for interaction with a cysteine containing protein. In some instances, this method allows for mapping of small molecule fragments for interaction with a cysteine containing protein under native conditions, thereby allows for an accurate mapping of interaction with potential small molecule fragments. In some instances, this method also allows for identification of novel cysteine containing protein targets as this method eliminates the need of recombinant expression and purification.
[0076] In some embodiments, also described herein are compositions, cells, cell populations, assays, probes, and service related to the method of identifying a small molecule fragment for interaction with a cysteine containing protein.
General Methodology
[0077] In some embodiments, the methods described herein utilize a small molecule fragment and a cysteine-reactive probe for competitive interaction with a cysteine-containing protein. In some embodiments, the method is as described in FIG. 1A. FIG. 1A illustrates contacting a first cell solution with a small molecule fragment for an extended period of time prior to incubating the first cell solution with a first cysteine-reactive probe to generate a first group of cysteine-reactive probe-protein complexes. In some embodiments, the extended period of time is about 5, 10, 15, 20, 30, 60, 90, 120 minutes or longer. In some instances, the small molecule fragment competes with the first cysteine-reactive probe for interaction with a protein target. In some instances, the small molecule fragment or the cysteine-reactive probe form a covalent bond via a Michael's reaction with a cysteine residue of the cysteine containing protein. FIG. 1A further illustrates contacting a second cell solution with a second cysteine-reactive probe to generate a second group of cysteine-reactive probe-protein complexes. In some instances, the first cysteine-reactive probe and the second cysteine-reactive probe are the same.
[0078] In some embodiments, cells from the second cell solution are grown in an enriched media (e.g., an isotopically enriched media). In some cases, cells from the first cell solution are grown in an enriched media (e.g., an isotopically enriched media). In some instances, cells from both the first cell solution and the second cell solution are grown in two different enriched media (e.g., two different isotopically enriched media) so that a protein obtained from cells grown in the first cell solution is distinguishable from a protein obtained from cells grown in the second cell solution. In other embodiments, cells from only one of the cell solutions (e.g., either the first cell solution or the second cell solution) are grown in an enriched media (e.g., isotopically enriched media). In such cases, a protein obtained from the enriched cells (e.g., isotopically enriched cells) is distinguishable from a protein obtained from cells that have not been enriched (e.g., isotopically enriched).
[0079] As illustrated in FIG. 1A, in some instances the second cell solution is not treated with a small molecule fragment. In such cases, the second cell solution acts as a control.
[0080] In some instants, cells from the second cell solution are ae-further treated with a buffer. In some cases, the buffer is DMSO. In some cases, cells from the second cell solution are not treated with a small molecule fragment and the second cell solution acts as a control.
[0081] In some instances, a first group of cysteine-reactive probe-protein complexes and a second group of cysteine-reactive probe-protein complexes are harvested separately and combined to generate a set of cysteine-reactive probe-protein complexes which is further processed by a proteomic analysis means. In some cases, either the first group of cysteine-reactive probe-protein complexes or the second group of cysteine-reactive probe-protein complexes contain labeled proteins obtained from cells grown in an enriched media (e.g., isotopically enriched media). In some cases, both groups of cysteine-reactive probe-protein complexes contain labeled proteins obtained from cells grown in two different enriched media (e.g., two different isotopically enriched media). In other cases, either the first group of cysteine-reactive probe-protein complexes, the second group of cysteine-reactive probe-protein complexes, or both groups of cysteine-reactive probe-protein complexes contain labeled proteins in which the proteins have been labeled after harvesting from a cell.
[0082] In some instances, a first group of cysteine-reactive probe-protein complexes and a second group of cysteine-reactive probe-protein complexes are harvested separately and the proteins from one of the two groups of cysteine-reactive probe-protein complexes are subsequently labeled (e.g., by methylation). In some cases, first group of cysteine-reactive probe-protein complexes and a second group of cysteine-reactive probe-protein complexes are then combined and subjected to proteomic analysis means.
[0083] In other instances, a first group of cysteine-reactive probe-protein complexes and a second group of cysteine-reactive probe-protein complexes are harvested separately and both groups are subjected to proteomic analysis means. In some cases, data obtained from a proteomic analysis means is then combined for further analysis.
[0084] In some embodiments, the proteomic analysis means comprises a mass spectroscopy method. In some instances, the mass spectroscopy method is a liquid-chromatography-mass spectrometry (LC-MS) method. In some cases, the proteomic analysis means further comprise analyzing the results from the mass spectroscopy method by an algorithm for protein identification. In some cases, the algorithm combines the results from the mass spectroscopy method with a protein sequence database for protein identification. In some cases, the algorithm comprises ProLuCID algorithm, Probity, Scaffold, SEQUEST, or Mascot. In some cases, the mass spectroscopy method is a MALDI-TOF based method.
[0085] In some embodiments, a value is assigned to each of the cysteine binding protein from the cysteine-reactive probe-protein complexes after proteomic analysis, in which the value is determined from the proteomic analysis. In some cases, the value assigned to each of the cysteine containing protein is obtained from a mass spectroscopy analysis. In some instances, the value is an area-under-the curve from a plot of signal intensity as a function of mass-to-charge ratio. In some embodiments, a first value is assigned to a cysteine binding protein from the first group of cysteine-reactive probe-protein complex of the first cell solution and a second value of the same cysteine binding protein from the second group of cysteine-reactive probe-protein complex of the second cell solution. In some instances, a ratio is then calculated between the two values, the first value and the second value, and assigned to the same cysteine binding protein. In some instances, a ratio of greater than 2 indicates that the cysteine binding protein is a candidate for interacting with the small molecule fragment. In some instances, the ratio is greater than 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, or 10. In some cases, the ratio is at most 20. In some instances, the same small molecule fragment interacts with a number of cysteine binding proteins in the presence of a cysteine-reactive probe. In some instances, the small molecule modulates the interaction of a cysteine-reactive probe with its cysteine binding protein partners. In some instances, the spectrum of ratios for a small molecule fragment with its interacting protein partners in the presence of a cysteine-reactive probe indicates the specificity of the small molecule fragment toward the protein. In some instances, the spectrum of ratio indicates whether the small molecule fragment is a specific inhibitor to a protein or a pan inhibitor.
[0086] In some embodiments, the cysteine containing protein identified by the above method comprises a biologically active cysteine residue. In some instances, the biologically active cysteine site is a cysteine residue that is located about 10 .ANG. or less to an active-site ligand or residue. In some cases, the cysteine residue that is located about 10 .ANG. or less to the active-site ligand or residue is an active site cysteine. In some cases, the biologically active cysteine site is an active site cysteine. In some embodiments, the biologically active cysteine site is a cysteine residue that is located greater than 10 .ANG. from an active-site ligand or residue. In some cases, the cysteine residue that is located greater than 10 .ANG. from the active-site ligand or residue is a non-active site cysteine. In some instances, the biologically active cysteine site is a non-active site cysteine.
[0087] In some embodiments, the small molecule fragment that covalently interacts with the biologically active cysteine impairs and/or inhibits activity of the cysteine containing protein. In some instances, the cysteine containing protein exists in an active form. In some embodiments, the small molecule fragment and/or the cysteine-reactive probe interact with the active form of the cysteine containing protein. In some instances, the cysteine containing protein exists in a pro-active form. In some embodiments, the small molecule fragment and/or the cysteine-reactive probe interact with the pro-active form of the cysteine containing protein.
[0088] In some embodiments, the structural environment of the biologically active cysteine residue modulates the reactivity of the cysteine residue. In some embodiments, the structural environment is a hydrophobic environment or a hydrophilic environment. In some embodiments, the structural environment is a charged environment. In some embodiments, the structural environment is a nucleophilic environment.
[0089] In some embodiments, the cysteine containing protein is an enzyme, a transporter, a receptor, a channel protein, an adaptor protein, a chaperone, a signaling protein, a plasma protein, transcription related protein, translation related protein, mitochondrial protein, or cytoskeleton related protein. In some instances, the cysteine containing protein is an enzyme, a transporter, a receptor, a channel protein, an adaptor protein, a chaperone, a signaling protein, transcription related protein, or translation related protein. In some embodiments, the cysteine containing protein is a protein illustrated in Tables 1, 2, 3, 8 or 9. In some instances, the cysteine residue of the cysteine-containing proteins illustrated in Tables 1, 2, 3, 8 or 9 is denoted by (*) in Tables 1, 2, 3, 8 or 9.
[0090] In some instances, a set of cysteine-reactive probes are added to the cell solutions. For example, a first set of cysteine-reactive probes are added to the first cell solution and a second set of cysteine-reactive probes are added to the second cell solution. In some cases, each cysteine-reactive probe is different within the set. In some instances, the first set of cysteine-reactive probes is the same as the second set of cysteine-reactive probes. In some cases, the first set of cysteine-reactive probes generate a third group of cysteine-reactive probe-protein complexes and the second set of cysteine-reactive probes generate a fourth group of cysteine-reactive probe-protein complexes. In some instances, the set of cysteine-reactive probes further facilitates identification of cysteine containing proteins.
[0091] In some embodiments, the sample is a cell sample. In other instances, the sample is a tissue sample.
[0092] In some instances, the method is an in-situ method.
Small Molecule Fragments
[0093] In some embodiments, the small molecule fragments described herein comprise non-naturally occurring molecules. In some instances, the non-naturally occurring molecules do not include natural and/or non-natural peptide fragments, or small molecules that are produced naturally within the body of a mammal.
[0094] In some embodiments, the small molecule fragments described herein comprise a molecule weight of about 100 Dalton or higher. In some embodiments, the small molecule fragments comprise a molecule weight of about 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 Dalton, or higher. In some instances, the molecule weight of the small molecule fragments are between about 150 and about 500, about 150 and about 450, abut 150 and about 440, about 150 and about 430, about 150 and about 400, about 150 and about 350, about 150 and about 300, about 150 and about 250, about 170 and about 500, about 180 and about 450, about 190 and about 400, about 200 and about 350, about 130 and about 300, or about 120 and about 250 Dalton.
[0095] In some embodiments, the molecule weight of the small molecule fragments described herein is the molecule weight prior to enrichment with one or more elements selected from a halogen, a nonmetal, a transition metal, or a combination thereof. In some embodiments, the molecule weight of the small molecule fragments described herein is the molecule weight prior to enrichment with a halogen. In some embodiments, the molecule weight of the small molecule fragments described herein is the molecule weight prior to enrichment with a nonmetal. In some embodiments, the molecule weight of the small molecule fragments described herein is the molecule weight prior to enrichment with a transition metal. In some embodiments, the molecular weight of the small molecule fragment is calculated based on carbon and hydrogen atoms and optionally further based on nitrogen, oxygen and/or sulfur atoms.
[0096] In some embodiments, the molecular weight of the small molecule fragment does not include the molecular weight of a halogen, a transition metal or a combination thereof. In some cases, the molecular weight of the small molecule fragment does not include the molecular weight of a halogen, the molecular weight of the small molecule fragment does not include the molecular weight of a transition metal.
[0097] In some embodiments, the small molecule fragments described herein comprise micromolar or millimolar binding affinity. In some instances, the small molecule fragments comprise a binding affinity of about 1 .mu.M, 10 .mu.M, 100 .mu.M, 500 .mu.M, 1 mM, 10 mM, or higher.
[0098] In some embodiments, the small molecule fragments described herein has a high ligand efficiency (LE). Ligand efficiency is the measurement of the binding energy per atom of a ligand to its binding partner. In some instances, the ligand efficiency is defined as the ratio of the Gibbs free energy (.DELTA.G) to the number of non-hydrogen atoms of the compound (N):
LE=(.DELTA.G)/N.
[0099] In some cases, LE is also arranged as:
LE=1.4(-log IC.sub.50)/N.
[0100] In some instances, the LE score is about 0.3 kcal mol.sup.-1 HA.sup.-1, about 0.35 kcal mol.sup.-1 HA.sup.-1, about 0.4 kcal mol.sup.-1 HA.sup.-1, or higher.
[0101] In some embodiments, the small molecule fragments described herein are designed based on the Rule of 3. In some embodiments, the Rule of 3 comprises a non-polar solvent-polar solvent (e.g. octanol-water) partition coefficient log P of about 3 or less, a molecular mass of about 300 Daltons or less, about 3 hydrogen bond donors or less, about 3 hydrogen bond acceptors or less, and about 3 rotatable bonds or less.
[0102] In some embodiments, the small molecule fragments described herein comprises three cyclic rings or less.
[0103] In some embodiments, the small molecule fragments described herein binds to a cysteine residue of a polypeptide that is about 20 amino acid residues in length or more. In some instances, the small molecule fragments described herein binds to a cysteine residue of a polypeptide that is about 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000 amino acid residues in length or more.
[0104] In some embodiments, the small molecule fragments described herein further comprise pharmacokinctic parameters that are unsuitable as a therapeutic agent for administration without further optimization of the small molecule fragments. In some instances, the pharmacokinetic parameters that are suitable as a therapeutic agent comprise parameters in accordance with FDA guideline, or in accordance with a guideline from an equivalent Food and Drug Administration outside of the United States. In some instances, the pharmacokinetic parameters comprise the peak plasma concentration (C max), the lowest concentration of a therapeutic agent (C min), volume of distribution, time to reach C max, elimination half-life, clearance, and the life. In some embodiments, the pharmacokinetic parameters of the small molecule fragments are outside of the parameters set by the FDA guideline, or by an equivalent Food and Drug Administration outside of the United States. In some instances, a skilled artisan understands in view of the pharmacokinetic parameters of the small molecule fragments described herein that these small molecule fragments are unsuited as therapeutic agents without further optimization.
[0105] In some embodiments, the small molecule fragments described herein comprise a reactive moiety which forms a covalent interaction with the thiol group of a cysteine residue of a cysteine containing protein, and an affinity handle moiety.
[0106] In some instances, a small molecule fragment described herein is a small molecule fragment of Formula (I):
##STR00045##
[0107] wherein:
[0108] RM is a reactive moiety selected from a Michael acceptor moiety, a leaving group moiety, or a moiety capable of forming a covalent bond with the thiol group of a cysteine residue; and F is a small molecule fragment moiety.
[0109] In some instances, the Michael acceptor moiety comprises an alkene or an alkyne moiety. In some cases, F is obtained from a compound library. In some cases, the compound library comprises ChemBridge fragment library, Pyramid Platform Fragment-Based Drug Discovery, Maybridge fragment library, FRGx from AnalytiCon, TCI-Frag from AnCoreX, Bio Building Blocks from ASINEX, BioFocus 3D from Charles River, Fragments of Life (FOL) from Emerald Bio. Enamine Fragment Library, IOTA Diverse 1500, BIONET fragments library, Life Chemicals Fragments Collection, OTAVA fragment library, Prestwick fragment library, Selcia fragment library, TimTec fragment-based library, Allium from Vitas-M Laboratory, or Zenobia fragment library.
[0110] In some embodiments, the small molecule fragment of Formula (I) does not contain a second binding site. In some instances, the small molecule fragment moiety does not bind to the protein. In some cases, the small molecule fragment moiety does not covalently bind to the protein. In some instances, the small molecule fragment moiety does not interact with a secondary binding site on the protein. In some instances, the secondary binding site is an active site such as an ATP binding site. In some cases, the active site is at least about 10, 15, 20, 25, 35, 40 .ANG., or more away from the biologically active cysteine residue. In some instances, the small molecule fragment moiety does not interact with an active site such as an ATP binding site.
[0111] In some instances, F is a small molecule fragment moiety illustrated in FIG. 3. In some cases, F further comprises a linker moiety that connects F to the carbonyl moiety. In some cases, the small molecule fragment is a small molecule fragment illustrated in FIG. 3.
[0112] In some instances, F is a small molecule fragment moiety selected from: N-(4-bromophenyl)-N-phenylacrylamide, N-(1-benzoylpiperidin-4-yl)-2-chloro-N-phenylacetamide, 1-(4-benzylpiperidin-1-yl)-2-chloroethan-1-one, N-(2-(1H-indol-3-yl)ethyl)-2-chloroacetamide, N-(3,5-bis(trifluoromethyl)phenyl)acrylamide, N-(4-phenoxy-3-(trifluoromethyl)phenyl)-N-(pyridin-3-ylmethyl)acrylamide, N-(3,5-bis(trifluoromethyl)phenyl)acetamide, 2-chloro-1-(4-(hydroxydiphenylmethyl)piperidin-1-yl)ethan-1-one, (E)-3-(3,5-bis(trifluoromethyl)phenyl)-2-cyanoacrylamide, N-(3,5-bis(trifluoromethyl)phenyl)-2-bromopropanamide, N-(3,5-bis(trifluoromethyl)phenyl)-2-chloropropanamide, N-(3,5-bis(trifluoromethyl)phenyl)-N-(pyridin-3-ylmethyl)acrylamide, 3-(2-chloroacetamido)-5-(trifluoromethyl)benzoic acid, 1-(4-(5-fluorobenzisoxazol-3-yl)piperidin-1-yl)prop-2-en-1-one, tert-butyl 4-(4-acrylamido-2,6-difluorophenyl)piperazine-1-carboxylate, N-(4-bromo-2,5-dimethylphenyl)acrylamide, 2-Chloroacetamido-2-deoxy-.alpha./.beta.-D-glucopyranose, 2-chloro-1-(2-methyl-3,4-dihydroquinolin-1(2H)-yl)ethan-1-one, N-cyclohexyl-N-phenylacrylamide, 1-(5-bromoindolin-1-yl)prop-2-en-1-one, N-(1-benzylpiperidin-4-yl)-N-phenylacrylamide, 2-chloro-N-(2-methyl-5-(trifluoromethyl)phenyl)acetamide, 1-(5-bromoindolin-1-yl)-2-chloroethan-1-one, 2-chloro-N-(quinolin-5-yl)acetamide, 1-(4-benzylpiperidin-1-yl)prop-2-en-1-one, 2-chloro-N-((3-hydroxy-5-(hydroxymethyl)-2-methylpyridin-4-yl)methyl)acet- amide, or 1-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)prop-2-en-1-one- .
[0113] In some embodiments, the small molecule fragment of Formula (I) comprise a molecule weight of about 100, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 Dalton, or higher. In some instances, the molecule weight of the small molecule fragment of Formula (I) is between about 150 and about 500, about 150 and about 450, abut 150 and about 440, about 150 and about 430, about 150 and about 400, about 150 and about 350, about 150 and about 300, about 150 and about 250, about 170 and about 500, about 180 and about 450, about 190 and about 400, about 200 and about 350, about 130 and about 300, or about 120 and about 250 Dalton.
[0114] In some embodiments, the molecule weight of the small molecule fragment of Formula (I) is the molecule weight prior to enrichment with one or more elements selected from a halogen, a nonmetal, a transition metal, or a combination thereof. In some embodiments, the molecule weight of the small molecule fragment of Formula (I) is the molecule weight prior to enrichment with a halogen. In some embodiments, the molecule weight of the small molecule fragment of Formula (I) is the molecule weight prior to enrichment with a nonmetal. In some embodiments, the molecule weight of the small molecule fragment of Formula (I) is the molecule weight prior to enrichment with a transition metal.
[0115] In some embodiments, the molecular weight of the small molecule fragment of Formula (I) does not include the molecular weight of a halogen, a transition metal or a combination thereof. In some embodiments, the molecular weight of the small molecule fragment of Formula (I) does not include the molecular weight of a halogen. In some embodiments, the molecular weight of the small molecule fragment of Formula (I) does not include the molecular weight of a transition metal.
[0116] In some instances, the small molecule fragment of Formula (I) comprises micromolar or millimolar binding affinity. In some instances, the small molecule fragment of Formula (I) comprises a binding affinity of about 1 .mu.M, 10 .mu.M, 100 .mu.M, 500 .mu.M, 1 mM, 10 mM, or higher.
[0117] In some cases, the small molecule fragment of Formula (I) has a LE score about 0.3 kcal mol.sup.-1 HA.sup.-1, about 0.35 kcal mol.sup.-1 HA.sup.-1, about 0.4 kcal mol.sup.-1 HA.sup.-1, or higher
[0118] In some embodiments, the small molecule fragment of Formula (I) follows the design parameters of Rule of 3. In some instances, the small molecule fragment of Formula (I) has a non-polar solvent-polar solvent (e.g. octanol-water) partition coefficient log P of about 3 or less, a molecular mass of about 300 Daltons or less, about 3 hydrogen bond donors or less, about 3 hydrogen bond acceptors or less, and about 3 rotatable bonds or less.
[0119] In some embodiments, the small molecule fragment of Formula (I) comprises three cyclic rings or less.
[0120] In some embodiments, the small molecule fragment of Formula (I) binds to a cysteine residue of a polypeptide (e.g., a cysteine containing protein) that is about 20 amino acid residues in length or more. In some instances, the small molecule fragments described herein binds to a cysteine residue of a polypeptide (e.g., a cysteine containing protein) that is about 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000 amino acid residues in length or more.
[0121] In some instances, the small molecule fragment of Formula (I) has pharmacokinetic parameters outside of the parameters set by the FDA guideline, or by an equivalent Food and Drug Administration outside of the United States. In some instances, a skilled artisan understands in view of the pharmacokinetic parameters of the small molecule fragment of Formula (I) described herein that these small molecule fragment is unsuited as a therapeutic agent without further optimization.
[0122] In some embodiments, the small molecule fragment is a specific inhibitor or a pan inhibitor.
Cysteine-Reactive Probes
[0123] In some embodiments, a cysteine-reactive probe comprises a reactive moiety which forms a covalent interaction with the thiol group of a cysteine residue of a cysteine containing protein, and an affinity handle moiety.
[0124] In some embodiments, a cysteine-reactive probe is a cysteine-reactive probe of Formula (II):
##STR00046##
[0125] wherein:
[0126] RM is a reactive moiety selected from a Michael acceptor moiety, a leaving group moiety, or a moiety capable of forming a covalent bond to the thiol group of a cysteine residue; and AHM is an affinity handle moiety.
[0127] In some instances, the Michael acceptor moiety comprises an alkene or an alkyne moiety. In some cases, the affinity handle moiety comprises an affinity handle and a binding moiety that facilitates covalent interaction of the cysteine-reactive probe to a cysteine residue of a cysteine-containing protein. In some cases, the binding moiety is a small molecule fragment obtained from a compound library. In some instances, the compound library comprises ChemBridge fragment library, Pyramid Platform Fragment-Based Drug Discovery, Maybridge fragment library, FRGx from AnalytiCon. TCI-Frag from AnCoreX, Bio Building Blocks from ASINEX, BioFocus 3D from Charles River, Fragments of Life (FOL) from Emerald Bio, Enamine Fragment Library, IOTA Diverse 1500, BIONET fragments library, Life Chemicals Fragments Collection, OTAVA fragment library. Prestwick fragment library, Selcia fragment library, TimTec fragment-based library. Allium from Vitas-M Laboratory, or Zenobia fragment library.
[0128] In some embodiments, the affinity handle is a bioorthogonal affinity handle. In some embodiments, the affinity handle utilizes bioorthogonal chemistry. As used herein, bioorthogonal chemistry refers to any chemical reaction that occurs inside of a living system (e.g. a cell) without interfering with native biochemical processes.
[0129] In some cases, the affinity handle comprises a carbodiimide, N-hydroxysuccinimide (NHS) ester, imidoester, pentafluorophenyl ester, hydroxymethyl phosphine, maleimide, haloacetyl, pyridyl disulfide, thiosulfonate, vinylsulfone, hydrazide, alkoxyamine, alkyne, azide, or isocyanate group. In some cases, the affinity handle comprises an alkyne or an azide group.
[0130] In some instances, the affinity handle is an alkyne group. The term "alkyne group" as used in the context of an affinity handle refers to a group with a chemical formula of H--C.ident.C--R, HC.sub.2R, R.sub.1--C.ident.C--R.sub.2, or R.sub.1C.sub.2R.sub.2. In the context of the present chemical formula, R, R.sub.1, and R.sub.2 are independently a cysteine-reactive probe portion described herein, a linker, or a combination thereof. In some cases, the alkyne group is capable of being covalently linked in a chemical reaction with a molecule containing an azide. In some instances, the affinity handle is an azide group.
[0131] In some instances, the affinity handle (e.g. alkyne group or azide group) serve as nonnative and non-perturbed bioorthogonal chemical handles. In some instances, the affinity handle (e.g. alkyne group or azide group) is further derivatized through chemical reactions such as click chemistry. In some instances, the click chemistry is a copper(I)-catalyzed [3+2]-Huisgen 1,3-dipolar cyclo-addition of alkynes and azides leading to 1,2,3-triazoles. In other instances, the click chemistry is a copper free variant of the above reaction.
[0132] In some instances, the affinity handle further comprises a linker. In some instances, the linker bridges the affinity handle to the reactive moiety.
[0133] In some instances, the affinity handle is further conjugated to an affinity ligand. In some cases, the affinity ligand comprises a chromophore, a labeling group, or a combination thereof. In some embodiments, the chromophore comprises fluorochrome, non-fluorochrome chromophore, quencher, an absorption chromophore, fluorophore, organic dye, inorganic dye, metal chelate, or a fluorescent enzyme substrate. In some cases, the chromophore comprises non-fluorochrome chromophore, quencher, an absorption chromophore, fluorophore, organic dye, inorganic dye, metal chelate, or a fluorescent enzyme substrate. In other cases, the chromophore comprises a fluorophore.
[0134] In some embodiments, the fluorophore comprises rhodamine, rhodol, fluorescein, thiofluorescein, aminofluorescein, carboxyfluorescein, chlorofluorescein, methylfluorescein, sulfofluorescein, aminorhodol, carboxythodol, chlororhodol, methylrhodol, sulforhodol, aminorhodamine, carboxyrhodamine, chlororhodamine, mcthylrhodamine, sulforhodamine, thiorhodamine, cyanine, indocarbocyanine, oxacarbocyvanine, thiacarbocyanine, merocyanine, cyanine 2, cyanine 3, cyanine 3.5, cyanine 5, cyanine 5.5, cyanine 7, oxadiazole derivatives, pyridyloxazole, nitrobenzoxadiazole, benzoxadiazole, pyren derivatives, cascade blue, oxazine derivatives. Nile red, Nile blue, cresyl violet, oxazine 170, acridine derivatives, proflavin, acridine orange, acridine yellow, arylmethine derivatives, auramine, crystal violet, malachite green, tetrapyrrole derivatives, porphin, phtalocyanine, bilirubin 1-dimethylaminonaphthyl-5-sulfonate, 1-anilino-8-naphthalene sulfonate, 2-p-touidinyl-6-naphthalene sulfonate, 3-phenyl-7-isocyanatocoumarin, N-(p-(2-benzoxazolyl)phenyl)maleimide, stilbenes, pyrenes, 6-FAM (Fluorescein), 6-FAM (NHS Ester), 5(6)-FAM, 5-FAM, Fluorescein dT, 5-TAMRA-cadavarine, 2-aminoacridone, HEX, JOE (NHS Ester), MAX, TET, ROX, TAMRA, TARMA.TM. (NHS Ester), TEX 615, ATTO.TM. 488, ATTO.TM. 532, ATTO.TM. 550, ATTO.TM. 565. ATTO.TM. Rho101, ATTO.TM. 590, ATTO.TM. 633, ATTO.TM. 647N, TYE.TM. 563, TYE.TM. 665, or TYE.TM. 705.
[0135] In some embodiments, the labeling group is a biotin moiety, a streptavidin moiety, bead, resin, a solid support, or a combination thereof. As used herein, a biotin moiety described herein comprises biotin and biotin derivatives. Exemplary biotin derivatives include, but are not limited by, desthiobiotin, biotin alkyne or biotin azide. In some instances, a biotin moiety described herein is desthiobiotin. In some cases, a biotin moiety described herein is d-Desthiobiotin.
[0136] In some instances, the labeling group is a biotin moiety. In some instances, the biotin moiety further comprises a linker such as a 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more residues in length. In some instances, the linker further comprises a cleavage site, such as a protease cleavage site. In some cases, the biotin moiety interacts with a streptavidin moiety. In some instances, the biotin moiety is further attached to a bead, such as a streptavidin-coupled bead. In some instances, the biotin moiety is further attached to a resin or a solid support, such as a streptavidin-coupled resin or a streptavidin-coupled solid support. In some instances, the solid support is a plate, a platform, a cover slide, a microfluidic channel, and the like.
[0137] In some embodiments, the affinity handle moiety further comprises a chromophore.
[0138] In some embodiments, the cysteine-reactive probe is a cysteine-reactive probe illustrated in FIG. 3. In some embodiments, the cysteine-reactive probe is a cysteine-reactive probe selected from: N-(hex-5-yn-1-yl)-2-iodoacetamide, lodoacetamide-rhodamine, 3-acrylamido-N-(hex-5-yn-1-yl)-5-(trifluoromethyl)benzamide, 3-acrylamido-N-(hex-5-yn-1-yl)-5-(trifluoromethyl)benzamide, or 2-chloro-N-(1-(3-ethynylbenzoyl)piperidin-4-yl)-N-phenylacetamide.
Cysteine Containing Proteins
[0139] In some instances, the cysteine containing protein is a soluble protein or a membrane protein. In some instances, the cysteine containing protein is involved in one or more of a biological process such as protein transport, lipid metabolism, apoptosis, transcription, electron transport, mRNA processing, or host-virus interaction. In some instances, the cysteine containing protein is associated with one or more of diseases such as cancer or one or more disorders or conditions such as immune, metabolic, developmental, reproductive, neurological, psychiatric, renal, cardiovascular, or hematological disorders or conditions.
[0140] In some embodiments, the cysteine containing protein comprises a biologically active cysteine residue. In some embodiments, the cysteine containing protein comprises one or more cysteines in which at least one cysteine is a biologically active cysteine residue. In some cases, the biologically active cysteine site is a cysteine residue that is located about 10 .ANG. or less to an active-site ligand or residue. In some cases, the cysteine residue that is located about 10 .ANG. or less to the active-site ligand or residue is an active site cysteine. In other cases, the biologically active cysteine site is a cysteine residue that is located greater than 10 .ANG. from an active-site ligand or residue. In some instances, the cysteine residue is located greater than 12 .ANG., 15 .ANG., 20 .ANG., 25 .ANG., 30 .ANG., 35 .ANG., 40 .ANG., 45 .ANG., or greater than 50 .ANG. from an active-site ligand or residue. In some cases, the cysteine residue that is located greater than 10 .ANG. from the active-site ligand or residue is a non-active site cysteine. In additional cases, the cysteine containing protein exists in an active form, or in a pro-active form.
[0141] In some embodiments, the cysteine containing protein comprises one or more functions of an enzyme, a transporter, a receptor, a channel protein, an adaptor protein, a chaperone, a signaling protein, a plasma protein, transcription related protein, translation related protein, mitochondrial protein, or cvtoskeleton related protein. In some embodiments, the cysteine containing protein is an enzyme, a transporter, a receptor, a channel protein, an adaptor protein, a chaperone, a signaling protein, a plasma protein, transcription related protein, translation related protein, mitochondrial protein, or cytoskeleton related protein. In some instances, the cysteine containing protein has an uncategorized function.
[0142] In some embodiments, the cysteine containing protein is an enzyme. An enzyme is a protein molecule that accelerates or catalyzes chemical reaction. In some embodiments, non-limiting examples of enzymes include kinases, proteases, or deubiquitinating enzymes.
[0143] In some instances, exemplary kinases include tyrosine kinases such as the TEC family of kinases such as Tec, Bruton's tvrosine kinase (Btk), interleukin-2-indicible T-cell kinase (Itk) (or Emt/Tsk). Bmx. and Txk/Rlk; spleen tyrosine kinase (Syk) family such as SYK and Zeta-chain-associated protein kinase 70 (ZAP-70); Src kinases such as Src, Yes, Fyn, Fgr, Lck, Hck. Blk, Lyn, and Frk; JAK kinases such as Janus kinase 1 (JAK1), Janus kinase 2 (JAK2), Janus kinase 3 (JAK3), and Tyrosine kinase 2 (TYK2); or ErbB family of kinases such as Her1 (EGFR, ErbB1), Her2 (Neu. ErbB2), Her3 (ErbB3), and Her4 (ErbB4).
[0144] In some embodiments, the cysteine containing protein is a protease. In some embodiments, the protease is a cysteine protease. In some cases, the cysteine protease is a caspase. In some instances, the caspase is an initiator (apical) caspase. In some instances, the caspase is an effector (executioner) caspase. Exemplary caspase includes CASP2, CASP8, CASP9, CASP10, CASP3, CASP6, CASP7, CASP4, and CASP5. In some instances, the cysteine protease is a cathepsin. Exemplary cathepsin includes Cathepsin B, Cathepsin C, CathepsinF, Cathepsin H, Cathepsin K, Cathepsin L1, Cathepsin L2, Cathepsin O. Cathepsin S, Cathepsin W, or Cathepsin Z.
[0145] In some embodiments, the cysteine containing protein is a deubiquitinating enzyme (DUB). In some embodiments, exemplary deubiquitinating enzymes include cysteine proteases DUBs or metalloproteases. Exemplary cysteine protease DUBs include ubiquitin-specific protease (USP/UBP) such as USP1, USP2, USP3, USP4, USP5, USP6, USP7, USP8, USP9X, USP9Y, USP10, USP11, USP12, USP13, USP14, USP15, USP16, USP17, USP17L2, USP17L3, USP17L4, USP17L5, USP17L7, USP17L8, USP18, USP19, USP20, USP21. USP22, USP23, USP24, USP25, USP26, USP27X, USP28. USP29, USP30, USP31, USP32, USP33, USP34, USP35. USP36, USP37, USP38, USP39, USP40, USP41, USP42, USP43, USP44, USP45, or USP46; ovarian tumor (OTU) proteases such as OTUB1 and OTUB2; Machado-Josephin domain (MJD) proteases such as ATXN3 and ATXN3L; and ubiquitin C-terminal hydrolase (UCH) proteases such as BAP1, UCHL1, UCHL3, and UCHL5. Exemplary metalloproteases include the Jab1/Mov34/Mpr1 Pad1 N-terminal+(MPN+) (JAMM) domain proteases.
[0146] In some embodiments, exemplary cysteine containing proteins as enzymes include, but are not limited to, Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Protein arginine N-methyltransferase 1 (PRMT1), Peptidyl-prolyl cis-trans isomerase NIMA-interaction (PIN1), Acetyl-CoA acetyltransferase (mitochondrial) (ACAT1). Glutathione S-transferase P (GSTP1). Elongation factor 2 (EEF2). Glutathione S-transferase omega-1 (GSTO1), Acetyl-CoA acetyltransferase (mitochondrial) (ACAT1), Protein disulfide-isomerase A4 (PDIA4), Prostaglandin E synthase 3 (PTGES3), Adenosine kinase (ADK), Elongation factor 2 (EEF2), Isoamyl acetate-hydrolyzing esterase 1 homolog (IAHI), Peroxiredoxin-5 (mitochondrial) (PRDX5), Inosine-5-monophosphate dehydrogenase 2 (IMPDH2), 3-hydroxyacyl-CoA dehydrogenase type-2 (HSD17B10), Omega-amidase NIT2 (NIT2), Aldose reductase (AKR1B1), Monofunctional C1-tetrahydrofolate synthase (mitochondrial) (MTHFD1L), Protein disulfide-isomerase A6 (PDIA6), Pyruvate kinase isozymes M1/M2 (PKM), 6-phosphogluconolactonase (PGLS), Acetl-CoA acetyltransferase (mitochondrial) (ACAT1), ERO1-like protein alpha (EROIL), Thioredoxin domain-containing protein 17 (TXNDC17), Protein disulfide-isomerase A4 (PDIA4), Protein disulfide-isomerase A3 (PDIA3), 3-ketoacvl-CoA thiolase (mitochondrial) (ACAA2), Dynamin-2 (DNM2), DNA replication licensing factor MCM3 (MCM3), Serine-tRNA ligase (cytoplasmic) (SARS), Fatty acid synthase (FASN), Acetyl-CoA acetyltransferase (mitochondrial) (ACAT1), Protein disulfide-isomerase (P4HB), Deoxycytidine kinase (DCK), Eukaryotic translation initiation factor 3 subunit (EIF3F), Protein disulfide-isomerase A6 (PDIA6), UDP-N-acetylglucosamine-peptide N-acetylglucosamine (OGT), Ketosamine-3-kinase (FN3KRP), Protein DJ-1 (PARK7), Phosphoglycolate phosphatase (PGP), DNA replication licensing factor MCM6 (MCM6), Fructose-2,6-bisphosphatase TIGAR (TIGAR), Cleavage and polyadenylation specificity factor subunit (CPSF3), Ubiquitin-conjugating enzyme E2 L3 (UBE2L3), Alanine-tRNA ligase, cytoplasmic (AARS), Mannose-1-phosphate guanyltransferase alpha (GMPPA), C-1-tetrahydrofolate synthase (cytoplasmic) (MTHFD1), Dynamin-1-like protein (DNM1L), Protein disulfide-isomerase A3 (PDIA3), Aspartyl aminopeptidase (DNPEP), Acetyl-CoA acetyltransferase (cytosolic) (ACAT2), Thioredoxin domain-containing protein 5 (TXNDC5), Thymidine kinase (cytosolic) (TK1), Inosine-5-monophosphate dehydrogenase 2 (IMPDH2), Ubiquitin carboxyl-terminal hydrolase isozyme L3 (UCHL3), Integrin-linked protein kinase (ILK), Cyclin-dependent kinase 2 (CDK2), Histone acetyltransferase type B catalytic subunit (HAT1), Enoyl-CoA delta isomerase 2 (mitochondrial) (ECI2), C-1-tetrahydrofolate synthase (cytoplasmic) (MTHFD1), Deoxycytidine kinase (DCK), Ubiquitin-like modifier-activating enzyme 6 (UBA6), Protein-L-isoaspartate(D-aspartate)O-methyltransferase (PCMTI), Monofunctional C1-tetrahydrofolate synthase (mitochondrial) (MTHFD1L), Thymidylate kinase (DTYMK), Protein ETHE (mitochondrial) (ETHE 1), Arginine-tRNA ligase (cvtoplasmic) (RARS), NEDD8-activating enzyme E1 catalytic subunit (UBA3), Dual specificity mitogen-activated protein kinase (MAP2K3). Ubiquitin-conjugating enzyme E2S (UBE2S), Amidophosphoribosyltransferase (PPAT), Succinate-semialdehyde dehydrogenase (mitochondrial) (ALDH5A1), CAD, Phosphoenolpyruvate carboxykinase (PCK2), 6-phosphofructokinase type C (PFKP), Acyl-CoA synthetase family member 2 (mitochondrial) (ACSF2), Multifunctional protein ADE2 (PAICS), Desumoylating isopeptidase 1 (DESI1), 6-phosphofructokinase type C (PFKP), V-type proton ATPasc catalytic subunit A (ATP6V1A), 3-ketoacyl-CoA thiolase (peroxisomal) (ACAA1), Galactokinase (GALK1), Thymidine kinase (cytosolic) (TK1), ATPase WRNIP1 (WRNIP1), Phosphoribosylformylglycinamidine synthase (PFAS), V-type proton ATPase catalytic subunit A (ATP6V1A), Thioredoxin domain-containing protein 5 (TXNDC5), 4-trimethylaminobutyraldehyde dehydrogenase (ALDH9A1). Dual specificity mitogen-activated protein kinase (MAP2K4), Calcineurin-like phosphoesterase domain-containing (CPPED1), Dual specificity protein phosphatase 12 (DUSP12), Phosphoribosylformylglycinamidine synthase (PFAS), Diphosphomevalonate decarboxylase (MVD), D-3-phosphoglycerate dehydrogenase (PHGDH), Cell cycle checkpoint control protein RAD9A (RAD9A), Peroxiredoxin-1 (PRDX1), Sorbitol dehydrogenase (SORD), Pcroxiredoxin-4 (PRDX4), AMP deaminase 2 (AMPD2), Isocitrate dehydrogenase (IDH1), Pyruvate carboxvlase (mitochondrial) (PC), Integrin-linked kinase-associated serine/threonine (ILKAP), Methylmalonate-semialdehyde dehydrogenase (ALDH6A1), 26S proteasome non-ATPase regulatory subunit 14 (PSMD 14), Thymidylate kinase (DTYMK), 6-phosphofructo-2-kinase/fructose-2,6-bisphosphata (PFKFB2). Peroxiredoxin-5 (mitochondrial) (PRDX5), PDP1, Cathepsin B (CTSB), Transmembrane protease serine 12 (TMPRSS 12), UDP-glucose 6-dehydrogenase (UGDH), Histidine triad nucleotide-binding protein 1 (HINT1), E3 ubiquitin-protein ligase UBR5L (UBRL5), SAM domain and HD domain-containing protein 1 (SAMHD1), Probable tRNA threonylcarbamovyladenosine biosynthesis (OSGEP), Methylated-DNA-protein-cysteine methyltransferase (MGMT). Fatty acid synthase (FASN), Adenosine deaminase (ADA), Cyclin-dependent kinase 19 (CDK19), Serine/threonine-protein kinase 38 (STK38), Mitogen-activated protein kinase 9 (MAPK9), tRNA (adenine(58)-N(1))-methyltransferase catalytic (TRMT61A), Glyoxylate reductase/hydroxypyruvate reductase (GRHPR), Aldehyde dehydrogenase (mitochondrial) (ALDH2), Mitochondrial-processing peptidase subunit beta (PMPCB), 3-ketoacyl-CoA thiolase, peroxisomal (ACAA1), Lysophosphatidic acid phosphatase type 6 (ACP6), Ubiquitin/ISG15-conjugating enzyme E2 L6 (UBE2L6), Caspase-8 (CASP8), 2,5-phosphodiesterase 12 (PDE12), Thioredoxin domain-containing protein 12 (TXNDC12), Nitrilase homolog 1 (NIT1). ERO1-like protein alpha (ERO1L), SUMO-activating enzyme subunit 1 (SAE1), Leucine-tRNA ligase (cytoplasmic) (LARS), Protein-glutamine gamma-glutamyltransferase 2 (TGM2), Probable DNA dC-dU-editing enzyme APOBEC-3C (APOBEC3C), Double-stranded RNA-specific adenosine deaminase (ADAR), Isocitrate dehydrogenase (IDH2), Methylcrotonoyl-CoA carboxylase beta chain (mitochondrial) (MCCC2). Uridine phosphorylase 1 (UPP1). Glycogen phosphorylase (brain form) (PYGB), E3 ubiquitin-protein ligase UBR5 (UBR5). Procollagen-lysine,2-oxoglutarate 5-dioxygenase 1 (PLOD1), Ubiquitin carboxyl-terminal hydrolase 48 (USP48), Aconitate hydratase (mitochondrial) (ACO2), GMP reductase 2 (GMPR2), Pyrroline-5-carboxylate reductase 1 (mitochondrial) (PYCR1), Cathepsin Z (CTSZ), E3 ubiquitin-protein ligase UBR2 (UBR2), Cysteine protease ATG4B (ATG4B), Serine/threonine-protein kinase Nek9 (NEK9), Lysine-specific demethylase 4B (KDM4B), Insulin-degrading enzyme (IDE), Dipeptidyl peptidase 9 (DPP9), Decaprenyl-diphosphate synthase subunit 2 (PDSS2), TFIIH basal transcription factor complex helicase (ERCC3). Methionine-R-sulfoxide reductase B2 (mitochondrial) (MSRB2), E3 ubiquitin-protein ligase BREIB (RNF40), Thymidylate synthase (TYMS), Cyclin-dependent kinase 5 (CDK5), Bifunctional 3-phosphoadenosine 5-phosphosulfate (PAPSS2), Short/branched chain specific acyl-CoA dehydrogenase (ACADSB), Cathepsin D (CTSD), E3 ubiquitin-protein ligase HUWE1 (HUWE1), Calpain-2 catalytic subunit (CAPN2), Dual specificity mitogen-activated protein kinase (MAP2K7), Mitogen-activated protein kinase kinase kinase MLT (MLTK), Bleomycin hydrolase (BLMH), Probable ATP-dependent RNA helicase DDX59 (DDX59), Cystathionine gamma-lyase (CTH). S-adenosylmethionine synthase isoform type-2 (MAT2A), 6-phosphofructokinase type C (PFKP), Cvtidine deaminase (CDA). DNA-directed RNA polymerase II subunit RPB2 (POLR2B), Protein disulfide-isomerase (P4HB), Procollagen-lysine,2-oxoglutarate 5-dioxygenase 3 (PLOD3), Nucleoside diphosphate-linked moiety X motif 8 (mitochondrial) (NUDT8), E3 ubiquitin-protein ligase HUWE1 (HUWE1). Methylated-DNA-protein-cysteine methyltransferase (MGMT), Nitrilase homolog 1 (NIT1), Interferon regulatory factor 2-binding protein 1 (IRF2BP1), Ubiquitin carboxyl-terminal hydrolase 16 (USP16), Glycylpeptide N-tetradecanoyltransferase 2 (NMT2), Cyclin-dependent kinase inhibitor 3 (CDKN3). Hydroxysteroid dehydrogenase-like protein 2 (HSDL2), Serine/threonine-protein kinase VRK1 (VRK1). Serine/threonine-protein kinase A-Raf (ARAF), ATP-citrate synthase (ACLY), Probable ribonuclease ZC3H12D (ZC3H12D), Peripheral plasma membrane protein CASK (CASK), DNA polymerase epsilon subunit 3 (POLE3), Aldehyde dehydrogenase X (mitochondrial) (ALDHIB1), UDP-N-acetylglucosamine transferase subunit ALG13 (ALG13), Protein disulfide-isomerase A4 (PDIA4), DNA polymerase alpha catalytic subunit (POLA1), Ethylmalonyl-CoA decarboxylase (ECHDC1), Protein-tyrosine kinase 2-beta (PTK2B), E3 SUMO-protein ligase RanBP2 (RANBP2), Legumain (LGMN), Non-specific lipid-transfer protein (SCP2), Long-chain-fatty-acid-CoA ligase 4 (ACSL4), Dual specificity protein phosphatase 12 (DUSP12), Oxidoreductase HTATIP2 (HTATIP2), Serine/threonine-protein kinase MRCK beta (CDC42BPB). Histone-lysine N-methyltransferase EZH2 (EZH2), Non-specific lipid-transfer protein (SCP2), Dual specificity mitogen-activated protein kinase (MAP2K7), Ubiquitin carboxyl-terminal hydrolase 28 (USP28), 6-phosphofructokinase (liver type) (PFKL), SWI/SNF-related matrix-associated actin-dependent (SMARCAD1), Protein phosphatase methylesterase 1 (PPME1). DNA replication licensing factor MCM5 (MCM5), 6-phosphofructo-2-kinase/fructose-2,6-bisphosphata (PFKFB4), Dehydrogenase/reductase SDR family member 11 (DHRS11), Pyroglutamyl-peptidase 1 (PGPEP1), Probable E3 ubiquitin-protein ligase (MYCBP2), DNA fragmentation factor subunit beta (DFFB), Deubiquitinating protein VCIP135 (VCPIP1), Putative transferase CAF17 (mitochondrial) (IBA57), Calpain-7 (CAPN7), GDP-L-fucose synthase (TSTA3), Protein disulfide-isomerase A4 (PDIA4, Probable ATP-dependent RNA helicase (DDX59), RNA exonuclease 4 (REXO4), PDKI, E3 SUMO-protein ligase (PIAS4), DNA (cytosine-5)-methyltransferase 1 (DNMT1), Alpha-aminoadipic semialdehyde dehydrogenase (ALDH7A), Hydroxymethylglutaryl-CoA synthase (cytoplasmic) (HMGCS 1), E3 ubiquitin-protein ligase (SMURF2), Aldehyde dehydrogenase X (mitochondrial) (ALDHIB1), Tvrosine-protein kinase (BTK), DNA repair protein RAD50 (RAD50), ATP-binding domain-containing protein 4 (ATPBD4), Nucleoside diphosphate kinase 3 (NME3), Interleukin-1 receptor-associated kinase 1 (IRAK1), Ribonuclease P/MRP protein subunit POP5 (POP5), Peptide-N(4)-(N-acetyl-beta-glucosaminyl)asparagin (NGLY 1), Caspase-2 (CASP2), Ribosomal protein S6 kinase alpha-3 (RPS6KA3), E3 ubiquitin-protein ligase UBR1 (UBR1), Serine/threonine-protein kinase Chk2 (CHEK2), Phosphatidylinositol 3,4,5-trisphosphate 5-phospha (INPPL 1), Histone acetvltransferase p300 (EP300), Creatine kinase U-type (mitochondrial) (CKMTIB), E3 ubiquitin-protein ligase TRIM33 (TRIM33), Cancer-related nucleoside-triphosphatase (NTPCR), Aconitate hydratase (mitochondrial) (ACO2), Ubiquitin carboxyl-terminal hydrolase 34 (USP34), Probable E3 ubiquitin-protein ligase HERC4 (HERC4), E3 ubiquitin-protein ligase HECTD1 (HECTD1), Peroxisomal 2,4-dienoyl-CoA reductase (DECR2), Helicase ARIP4 (RAD54L2). Ubiquitin-like modifier-activating enzyme 7 (UBA7), ER degradation-enhancing alpha-mannosidase-like 3 (EDEM3). Ubiquitin-conjugating enzyme E20 (UBE2O). Dual specificity mitogen-activated protein kinase (MAP2K7), Myotubularin-related protein 1 (MTMR1), Calcium-dependent phospholipase A2 (PLA2G5), Mitotic checkpoint serine/threonine-protein kinase (BUB1B). Putative transferase CAF17 (mitochondrial) (IBA57), Tyrosine-protein kinase ZAP-70 (ZAP70). E3 ubiquitin-protein ligase pellino homolog 1 (PELI1), Neuropathy target esterase (PNPLA6), Ribosomal protein S6 kinase alpha-3 (RPS6KA3), N6-adenosine-methyltransferase 70 kDa subunit (METTL3), Fructosamine-3-kinase (FN3K), Ubiquitin carboxyl-terminal hydrolase 22 (USP22), Rab3 GTPase-activating protein catalytic subunit (RAB3GAP1), Caspase-5 (CASP5), L-2-hydroxyglutarate dehydrogenase (mitochondrial) (L2HGDH), Saccharopine dehydrogenase-like oxidoreductase (SCCPDH), FLAD1 FAD synthase, Lysine-specific demethylase 3A (KDM3A), or Ubiquitin carboxyl-terminal hydrolase 34 (USP34).
[0147] In some embodiments, the cysteine containing protein is a signaling protein. In some instances, exemplary signaling protein includes vascular endothelial growth factor (VEGF) proteins or proteins involved in redox signaling. Exemplary VEGF proteins include VEGF-A, VEGF-B, VEGF-C, VEGF-D. and PGF. Exemplary proteins involved in redox signaling include redox-regulatory protein FAM213A.
[0148] In some embodiments, the cysteine containing protein is a transcription factor or regulator. Exemplary cysteine containing proteins as transcription factors and regulators include, but are not limited to, 40S ribosomal protein S3 (RPS3). Basic leucine zipper and W2 domain-containing protein (BZW1). Poly(rC)-binding protein 1 (PCBP1), 40S ribosomal protein S11 (RPS11), 40S ribosomal protein S4, X isoform (RPS4X), Signal recognition particle 9 kDa protein (SRP9), Non-POU domain-containing octamer-binding protein (NONO), N-alpha-acetyltransferase 15, NatA auxiliary subunit (NAA15), Cleavage stimulation factor subunit 2 (CSTF2), Lamina-associated polypeptide 2, isoform alpha (TMPO), Heterogeneous nuclear ribonucleoprotein R (HNRNPR), MMS19 nucleotide excision repair protein homolog (MMS19), SWI/SNF complex subunit SMARCC2 (SMARCC2), Enhancer of mRNA-decapping protein 3 (EDC3), H/ACA ribonucleoprotein complex subunit 2 (NHP2), WW domain-containing adapter protein with coiled-c (WAC), N-alpha-acetyltransferase 15 NatA auxiliary subunit (NAA15), 40S ribosomal protein S11 (RPS11), Signal transducer and activator of transcription 1 (STAT1), Mediator of RNA polymerase II transcription subunit (MED15), Lamina-associated polypeptide 2 (isoform alpha) (TMPO), MMS19 nucleotide excision repair protein homolog (MMS19), DNA mismatch repair protein Msh2 (MSH2). Recombining binding protein suppressor of hairless (RBPJ). Mediator of RNA polymerase II transcription subunit (MED17), Heterogeneous nuclear ribonucleoprotein U (HNRNPU), Transcription initiation factor IIA subunit 2 (GTF2A2), Chromatin accessibility complex protein 1 (CHRAC1), CDKN2A-interacting protein (CDKN2AIP). Zinc finger protein 217 (ZNF217), Signal transducer and activator of transcription 3 (STAT3), WD repeat and HMG-box DNA-binding protein 1 (WDHDI), Lamina-associated polypeptide 2 (isoform alpha) (TMPO), Lamina-associated polypeptide 2 (isoforms beta/gam) (TMPO), Interferon regulatory factor 4 (IRF4), Protein flightless-1 homolog (FLII), Heterogeneous nuclear ribonucleoprotein F (HNRNPF). Nucleus accumbens-associated protein 1 (NACC1), Transcription elongation regulator 1 (TCERG1), Protein HEXIM1 (HEXIM1). Enhancer of mRNA-decapping protein (EDC3), Zinc finger protein Aiolos (IKZF3), Transcription elongation factor SPT5 (SUPT5H), Forkhead box protein K1 (FOXK1), LIM domain-containing protein 1 (LIMD1), MMS19 nucleotide excision repair protein homolog (MMS19), Elongator complex protein 4 (ELP4). Ankyrin repeat and KH domain-containing protein 1 (ANKHD1), PML, Nuclear factor NF-kappa-B p100 subunit (NFKB2), Heterogeneous nuclear ribonucleoprotein L-like (HNRPLL), CCR4-NOT transcription complex subunit 3 (CNOT3), Constitutive coactivator of PPAR-gamma-like protein (FAM120A), Mediator of RNA polymerase II transcription subunit (MED15), 60S ribosomal protein L7 (RPL7), Interferon regulatory factor 8 (IRF8), COUP transcription factor 2 (NR2F2), Mediator of RNA polymerase II transcription subunit (MED1), tRNA (uracil-5-)-mcthyltransferase homolog A (TRMT2A), Transcription factor p65 (RELA), Exosome complex component RRP42 (EXOSC7), General transcription factor 3C polypeptide 1 (GTF3C1), Mothers against decapentaplegic homolog 2 (SMAD2), Ankyrin repeat domain-containing protein 17 (ANKRDI7), MMSl9 nucleotide excision repair protein homolog (MMS19), Death domain-associated protein 6 (DAXX), Zinc finger protein 318 (ZNF318), Thioredoxin-interacting protein (TXNIP), Glucocorticoid receptor (NR3C1), Iron-responsive element-binding protein 2 (IREB2), Zinc finger protein 295 (ZNF295). Polycomb protein SUZ12 (SUZ12), Cleavage stimulation factor subunit 2 tau variant (CSTF2T), C-myc promoter-binding protein (DENND4A), Pinin (PNN), Mediator of RNA polymerase II transcription subunit (MED9), POU domain, class 2, transcription factor 2 (POU2F2), Enhancer of mRNA-decapping protein 3 (EDC3), A-kinase anchor protein 1 (mitochondrial) (AKAP), Transcription factor RelB (RELB), RNA polymerase II-associated protein 1 (RPAP 1), Zinc finger protein 346 (ZNF346), Chromosome-associated kinesin KIF4A (KIF4A), Mediator of RNA polymerase II transcription subunit (MED12), Protein NPAT (NPAT), Leucine-rich PPR motif-containing protein (mitochondrial) (LRPPRC), AT-hook DNA-binding motif-containing protein 1 (AHDC1), Mediator of RNA polymerase II transcription subunit (MED12), Bromodomain-containing protein 8 (BRD8), Trinucleotide repeat-containing gene 6B protein (TNRC6B), Aryl hydrocarbon receptor nuclear translocator (ARNT), Activating transcription factor 7-interacting protein (ATF71P), Glucocorticoid receptor (NR3C1), Chromosome transmission fidelity protein 18 homolog (CHTF18), or C-myc promoter-binding protein (DENND4A).
[0149] In some embodiments, the cysteine containing protein is a channel, transporter or receptor. Exemplary cysteine containing proteins as channels, transporters, or receptors include, but are not limited to, Chloride intracellular channel protein 4 (CLIC4), Exportin-1 (XPO1), Thioredoxin (TXN), Protein SEC13 homolog (SEC13), Chloride intracellular channel protein 1 (CLIC1), Guanine nucleotide-binding protein subunit beta-2 (GNB2L1), Sorting nexin-6 (SNX6), Conserved oligomeric Golgi complex subunit 3 (COG3), Nuclear cap-binding protein subunit 1 (NCBP1), Cytoplasmic dynein 1 light intermediate chain 1 (DYNCILI1), MOB-like protein phocein (MOB4). Programmed cell death 6-interacting protein (PDCD6IP), Glutaredoxin-1 (GLRX), ATP synthase subunit alpha (mitochondrial) (ATP5A1), Treacle protein (TCOF1), Dynactin subunit 1 (DCTN1), Importin-7 (IPO7), Exportin-2 (CSE1L), ATP synthase subunit gamma (mitochondrial) (ATP5C1), Trafficking protein particle complex subunit 5 (TRAPPC5), Thioredoxin mitochondrial (TXN2), THO complex subunit 6 homolog (THOC6), Exportin-1 (XPO), Nuclear pore complex protein Nup50 (NUP50), Treacle protein (TCOF1), Nuclear pore complex protein Nup93 (NUP93), Nuclear pore glycoprotein p62 (NUP62), Cytoplasmic dynein 1 heavy chain 1 (DYNC1H1), Thioredoxin-like protein 1 (TXNL1), Nuclear pore complex protein Nup214 (NUP214). Protein lin-7 homolog C (LIN7C), ADP-ribosylation factor-binding protein GGA2 (GGA2), Trafficking protein particle complex subunit 4 (TRAPPC4), Protein quaking (QKI), Perilipin-3 (PLIN3), Copper transport protein ATOX1 (ATOX1), Unconventional myosin-Ic (MYO IC), Nucleoporin NUP53 (NUP35), Vacuolar protein sorting-associated protein 18 homolog (VPS 18), Dedicator of cytokinesis protein 7 (DOCK7), Nucleoporin p54 (NUP54), Ras-related GTP-binding protein C (RRAGC), Arf-GAP with Rho-GAP domain (ANK repeat and PH domain) (ARAPI), Exportin-5 (XPO5), Kinectin (KTN1), Chloride intracellular channel protein 6 (CLIC6), Voltage-gated potassium channel subunit beta-2 (KCNAB2), Exportin-5 (XPO5), Ras-related GTP-binding protein C (RRAGC), Ribosome-binding protein 1 (RRBP1), Acyl-CoA-binding domain-containing protein 6 (ACBD6), Chloride intracellular channel protein 5 (CLIC5), Pleckstrin homology domain-containing family A member (PLEKHA2), ADP-ribosylation factor-like protein 3 (ARL3), Protein transport protein Sec24C (SEC24C), Voltage-dependent anion-selective channel protein (VDAC3), Programmed cell death 6-interacting protein (PDCD6IP), Chloride intracellular channel protein 3 (CLIC3), Multivesicular body subunit 12A (FAM125A), Eukaryotic translation initiation factor 4E transporter (EIF4ENIF 1), NmrA-like family domain-containing protein 1 (NMRAL1), Nuclear pore complex protein Nup98-Nup96 (NUP98), Conserved oligomeric Golgi complex subunit 1 (COG1), Importin-4 (1P04), Pleckstrin homology domain-containing family A member (PLEKHA2), Cytoplasmic dynein 1 heavy chain 1 (DYNC1H1), DENN domain-containing protein 1C (DENND1C), Cytoplasmic dynein 1 heavy chain 1 (DYNC1H1), Protein ELYS (AHCTF), Trafficking protein particle complex subunit 1 (TRAPPC1), Guanine nucleotide-binding protein-like 3 (GNL3), or Importin-13 (IPO13).
[0150] In some embodiments, the cysteine containing protein is a chaperone. Exemplary cysteine containing proteins as chaperones include, but are not limited to, 60 kDa heat shock protein (mitochondrial) (HSPD1), T-complex protein 1 subunit eta (CCT7), T-complex protein 1 subunit epsilon (CCT5). Heat shock 70 kDa protein 4 (HSPA4), GrpE protein homolog 1 (mitochondrial) (GRPEL1), Tubulin-specific chaperone E (TBCE), Protein unc-45 homolog A (UNC45A), Serpin H1 (SERPINH1), Tubulin-specific chaperone D (TBCD). Peroxisomal biogenesis factor 19 (PEX19), BAG family molecular chaperone regulator 5 (BAG5), T-complex protein 1 subunit theta (CCT8), Protein canopy homolog 3 (CNPY3), DnaJ homolog subfamily C member 10 (DNAJC10), ATP-dependent Clp protease ATP-binding subunit clp (CLPX), or Midasin (MDN 1).
[0151] In some embodiments, the cysteine containing protein is an adapter, scaffolding or modulator protein. Exemplary cysteine containing proteins as adapter, scaffolding, or modulator proteins include, but are not limited to, Proteasome activator complex subunit 1 (PSME1), TIP41-like protein (TIPRL), Crk-like protein (CRKL), Cofilin-1 (CFL), Condensin complex subunit 1 (NCAPD2), Translational activator GCN1 (GCN1L1), Serine/threonine-protein phosphatase 2A 56 kDa regulatory (PPP2R5D), UPF0539 protein C7orf59 (C7orf59), Protein diaphanous homolog 1 (DIAPH1), Protein asunder homolog (Asun), Ras GTPase-activating-like protein IQGAP1 (IQGAP1), Sister chromatid cohesion protein PDS5 homolog A (PDS5A), Reticulon-4 (RTN4), Proteasome activator complex subunit 4 (PSME4), Condensin complex subunit 2 (NCAPH), Sister chromatid cohesion protein PDS5 homolog A (PDS5A), cAMP-dependent protein kinase type I-alpha regulatory (PRKARIA), Host cell factor 1 (HCFC1), Serine/threonine-protein phosphatase 4 regulatory (PPP4R2), Apoptotic chromatin condensation inducer in the nucleus (ACIN1), BRISC and BRCA1-A complex member 1 (BABAM1), Interferon-induced protein with tetratricopeptide (IFIT3), Ras association domain-containing protein 2 (RASSF2), Hsp70-binding protein 1 (HSPBP1). TBC1 domain family member 15 (TBC1D15), Dynamin-binding protein (DNMBP), Condensin complex subunit 1 (NCAPD2), Beta-2-syntrophin (SNTB2), Disks large homolog 1 (DLG1), TBC1 domain family member 13 (TBC ID13), Formin-binding protein 1-like (FNBPIL), Translational activator GCN1 (GCN1L1), GRB2-related adapter protein (GRAP), G2/mitotic-specific cyclin-B1 (CCNB1), Myotubularin-related protein 12 (MTMR12), Protein FADD (FADD), Translational activator GCN1 (GCNIL1), Wings apart-like protein homolog (WAPAL), cAMP-dependent protein kinase type II-beta regulatory (PRKAR2B), Malcavemin (CCM2), MPP1 55 kDa erythrocyte membrane protein, Actin filament-associated protein 1 (AFAP1), Tensin-3 (TNS3), tRNA methyltransferase 112 homolog (TRMT112). Symplekin (SYMPK), TBC1 domain family member 2A (TBC1D2), ATR-interacting protein (ATRIP), Ataxin-10 (ATXN10), Succinate dehydrogenase assembly factor 2 (mitochondrial) (SDHAF2), Formin-binding protein 1 (FNBPI), Myotubularin-related protein 12 (MTMR12), Interferon-induced protein with tetratricopeptide (IFIT3), Protein CBFA2T2 (CBFA2T2), Neutrophil cytosol factor 1 (NCF1), or Protein syndesmos (NUDT16L1).
[0152] In some embodiments, a cysteine containing protein comprises a protein illustrated in Tables 1-5 or Tables 7-9. In some instances, a cysteine containing protein comprises a protein illustrated in Table 1. In some embodiments, the cysteine containing protein comprises a cysteine residue denoted in Table 1. In some instances, a cysteine containing protein comprises a protein illustrated in Table 2. In some embodiments, the cysteine containing protein comprises a cysteine residue denoted in Table 2. In some instances, a cysteine containing protein comprises a protein illustrated in Table 3. In some embodiments, the cysteine containing protein comprises a cysteine residue denoted in Table 3. In some instances, a cysteine containing protein comprises a protein illustrated in Table 4. In some embodiments, the cysteine containing protein comprises a cysteine residue denoted in Table 4. In some instances, a cysteine containing protein comprises a protein illustrated in Table 5. In some embodiments, the cysteine containing protein comprises a cysteine residue denoted in Table 5. In some instances, a cysteine containing protein comprises a protein illustrated in Table 7. In some embodiments, the cysteine containing protein comprises a cysteine residue denoted in Table 7. In some instances, a cysteine containing protein comprises a protein illustrated in Table 8. In some embodiments, the cysteine containing protein comprises a cysteine residue denoted in Table 8. In some instances, a cysteine containing protein comprises a protein illustrated in Table 9. In some embodiments, the cysteine containing protein comprises a cysteine residue denoted in Table 9. In some instances, the cysteine containing protein is a modified protein, in which the protein is modified at a cysteine residue site by a small molecule fragment described herein, such as for example, by a small molecule fragment of Formula (I) described herein, a cysteine-reactive probe of Formula (II) described herein, or by a small molecule fragment illustrated in FIG. 3.
[0153] In some embodiments, described herein is a modified cysteine containing protein comprising a small molecule fragment having a covalent bond to a cysteine residue of a cysteine containing protein. In some instances, the cysteine containing protein is selected from Table 3. In some cases, one or more cysteine residues of each respective cysteine containing protein are denoted in Table 3. In some cases, a cysteine containing protein selected from Table 3 is modified by a small molecule fragment at at least one cysteine site denoted in Table 3 to generate a modified cysteine containing protein. In some cases, the cysteine containing protein is selected from AIP, PES1, IKBKB, XPO1, KDM4B, NR3C1, GSTP1, TNFAIP3, ACAT1, IRAK1, GNB2L1, IRF4, USP34. ZC3HAV1, USP7, PELI1, DCUN1D1, USP28, UBE2O, RRAGC, MLTK, USP22, KDM3A, or USP16. In some cases, the cysteine containing protein is selected from AIP, PES1, IKBKB, XPO1, GSTP1, ACAT1, IRAK1, IRF4, ZC3HAV1, USP7, PELI1, USP28, UBE2O, RRAGC, MLTK, USP22, KDM3A, or USP16. In some cases, the cysteine containing protein is selected from KDM4B, NR3C, TNFAIP3, USP7 or USP22. In some cases, the cysteine containing protein is selected from GNB2L1 or USP34. In some cases, the cysteine containing protein is DCUN1D1. In some cases, the cysteine containing protein is selected from PES1, IKBKB, GSTP1, ACAT1, IRAK1, ZC3HAV1 or RRAGC. In some cases, the cysteine containing protein is selected from XPO1, GNB2L1, USP34, UBE2O, MLTK or USP22. In some cases, the cysteine containing protein is selected from KDM4B or NR3C1. In some cases, the cysteine containing protein is selected from TNFAIP3, USP7, USP28, KDM3A or USP16. In some cases, the cysteine containing protein is selected from IRF4, PELI1, DCUNID1 or USP22. In some cases, the cysteine containing protein is AIP. In some cases, the cysteine containing protein is an enzyme and the enzyme is selected from IKBKB, KDM4B, GSTP1, TNFAIP3, ACAT1, IRAK1, USP34, USP7, PELI1, USP28, UBE2O, MLTK, USP22, KDM3A. or USP16. In some cases, the cysteine containing protein is a transcription factor or regulator and the transcription factor or regulator is selected from NR3C1, IRF4 or ZC3HAV1. In some cases, the cysteine containing protein is a channel, a transporter, or a receptor and the channel, transporter, or receptor is selected from GNB2L1 or RRAGC. In some cases, the cysteine containing protein is selected from AIP. PES1, XPO1 or DCUN1D1. In some cases, the cysteine containing protein is selected from PES1, CYR61, UBE2L6, XPO1, ADA, NR3C1, POU2F2, UCHL3, MGMT, ERCC3, ACAT1, STAT3, UBA7, CASP2, IDH2, LRBA, UBE2L3, RELB, IRF8, CASP8, PDIA6, PCK2, PFKFB4, PDE12, USP34, USP48, SMARCC2 or SAMHD1. In some cases, the cysteine containing protein is selected from PES1, CYR61, NR3C1, UCHL3, ERCC3, ACAT1, STAT3, CASP2, LRBA, UBE2L3. RELB, PDIA6, PCK2, PFKFB4, USP48 or SMARCC2. In some cases, the cysteine containing protein is selected from UBE2L6, POU2F2, MGMT, ACAT1, UBA7, CASP8, PDE12 or USP34. In some cases, the cysteine containing protein is selected from CYR61 or XPO1. In some cases, the cysteine containing protein is selected from ADA, MGMT, IDH2, IRF8 or SAMHD1. In some cases, the cysteine containing protein is selected from PES1, CYR61, XPO1, NR3C1 or SMARCC2. In some cases, the cysteine containing protein is selected from CYR61, UBE2L6, MGMT, ERCC3, ACAT1 or USP48. In some cases, the cysteine containing protein is selected from ADA, RELB or USP34. In some cases, the cysteine containing protein is selected from UCHL3. CASP2, IDH2. LRBA, CASP8, PCK2 or PDE12. In some cases, the cysteine containing protein is selected from MGMT. ACAT1, UBA7, UBE2L3 or IRF8. In some cases, the cysteine containing protein is selected from PFKFB4, ACAT1 or STAT3. In some cases, the cysteine containing protein is selected from POU2F2, PDIA6 or SAMHD1. In some cases, the cysteine containing protein is an enzyme and the enzyme is selected from UBE2L6, ADA, UCHL3. MGMT, ERCC3, ACAT1, UBA7, CASP2, IDH2, UBE2L3, CASP8, PDIA6, PCK2, PFKFB4, PDE12, USP34. USP48 or SAMHD1. In some cases, the cysteine containing protein is a transcription factor or a regulator and the transcription factor or regulator is selected from NR3C1, POU2F2, STAT3, RELB, IRF8 or SMARCC2. In some cases, the cysteine containing protein is selected from ZAP70, PRKCQ or PRMT1. In some cases, the cysteine containing protein is selected from ZAP70 or PRKCQ. In some cases, the cysteine containing protein is selected from CYR61, ZNF217, NCF1, IREB2, LRBA, CDK5, EP300, EZH2, UBE2S, VCPIP1, RRAGC or IRAK4. In some cases, the cysteine containing protein is selected from CYR61, ZNF217, IREB2, EP300, UBE2S, VCPIP1, RRAGC or IRAK4. In some cases, the cysteine containing protein is selected from NCF1, LRBA or CDK5. In some cases, the cysteine containing protein is EZH2. In some cases, the cysteine containing protein is selected from ZNF217, NCF1, CDK5, EP300 or IRAK4. In some cases, the cysteine containing protein is selected from CYR61, IREB2, LRBA or UBE2S. In some cases, the cysteine containing protein is selected from EZH2, VCPIP1 or RRAGC. In some cases, the cysteine containing protein is an enzyme and the enzyme is selected from CDK5, EP300, EZH2, UBE2S, VCPIP1 or IRAK4. In some cases, the cysteine containing protein is a transcription factor or a regulator and the transcription factor or regulator is selected from ZNF217 or IREB2. In some cases, the cysteine containing protein is an adapter, a scaffolding protein or a modulator protein and the adapter, scaffolding protein or the modulator protein is selected from NCF1. In some cases, the cysteine containing protein is a channel, a transporter or a receptor and the channel, transporter, or receptor is selected from RRAGC. In some cases, the cysteine containing protein is selected from CYR61 or LRBA. In some cases, the cysteine containing protein is about 20, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000 amino acid residues in length or more. In some cases, the cysteine residue of the modified cysteine containing protein has the structure SR, wherein R is selected from:
##STR00047##
wherein R.sup.1 is H, C1-C3 alkyl, or aryl; and F' is the small molecule fragment moiety. In some cases, the small molecule fragment has a molecular weight of about 175, 200, 225, 250, 275, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 Dalton. or higher. In some cases, the molecular weight of the small molecule fragment is prior to enrichment with a halogen, a nonmetal, or a transition metal. In some embodiments, the molecular weight of the small molecule fragment is calculated based on carbon and hydrogen atoms and optionally further based on nitrogen, oxygen and/or sulfur atoms. In some embodiments, the molecular weight of the small molecule fragment does not include the molecular weight of a halogen, a transition metal or a combination thereof. In some cases, the small molecule fragment is a small molecule fragment of Formula (I):
##STR00048##
wherein RM is a reactive moiety selected from a Michael acceptor moiety, a leaving group moiety, or a moiety capable of forming a covalent bond with the thiol group of a cysteine residue, and F is a small molecule fragment moiety. In some cases, the Michael acceptor moiety comprises an alkene or an alkyne moiety. In some cases, F is obtained from a compound library. In some cases, F is a small molecule fragment moiety illustrated in FIG. 3. In some cases, F further comprises a linker moiety that connects F to the carbonyl moiety. In some cases, the small molecule fragment binds irreversibly to the cysteine containing protein. In some cases, the small molecule fragment binds reversibly to the cysteine containing protein.
[0154] In some embodiments, described herein is a modified cysteine containing protein comprising a small molecule fragment having a covalent bond to a cysteine residue of a cysteine containing protein, in which the cysteine containing protein is selected from Table 10A, enzymes. In some cases, one or more cysteine residues of each respective cysteine containing protein are denoted in Table 10A. In some cases, a cysteine containing protein selected from Table 10 A is modified by a small molecule fragment at at least one cysteine site denoted in Table 10A to generate a modified cysteine containing protein. In some cases, the cysteine containing protein is about 20, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000 amino acid residues in length or more. In some cases, the cysteine residue of the modified cysteine containing protein has the structure SR, wherein R is selected from:
##STR00049##
wherein R.sup.1 is H, C1-C3 alkyl, or aryl; and F' is the small molecule fragment moiety. In some cases, the small molecule fragment has a molecular weight of about 175, 200, 225, 250, 275, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 Dalton, or higher. In some cases, the molecular weight of the small molecule fragment is prior to enrichment with a halogen, a nonmetal, or a transition metal. In some embodiments, the molecular weight of the small molecule fragment is calculated based on carbon and hydrogen atoms and optionally further based on nitrogen, oxygen and/or sulfur atoms. In some embodiments, the molecular weight of the small molecule fragment does not include the molecular weight of a halogen, a transition metal or a combination thereof. In some cases, the small molecule fragment is a small molecule fragment of Formula (I):
##STR00050##
wherein RM is a reactive moiety selected from a Michael acceptor moiety, a leaving group moiety, or a moiety capable of forming a covalent bond with the thiol group of a cysteine residue, and F is a small molecule fragment moiety. In some cases, the Michael acceptor moiety comprises an alkene or an alkyne moiety. In some cases, F is obtained from a compound library. In some cases. F is a small molecule fragment moiety illustrated in FIG. 3. In some cases, F further comprises a linker moiety that connects F to the carbonyl moiety. In some cases, the small molecule fragment binds irreversibly to the cysteine containing protein. In some cases, the small molecule fragment binds reversibly to the cysteine containing protein.
[0155] In some embodiments, described herein is a modified cysteine containing protein comprising a small molecule fragment having a covalent bond to a cysteine residue of a cysteine containing protein, in which the cysteine containing protein is selected from Table 10B, transcription factors and regulators. In some cases, one or more cysteine residues of each respective cysteine containing protein are denoted in Table 10B. In some cases, a cysteine containing protein selected from Table 10B is modified by a small molecule fragment at at least one cysteine site denoted in Table 10B to generate a modified cysteine containing protein. In some cases, the cysteine containing protein is about 20, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000 amino acid residues in length or more. In some cases, the cysteine residue of the modified cysteine containing protein has the structure SRI wherein R is selected from:
##STR00051##
wherein R.sup.1 is H, C1-C3 alkyl, or aryl; and F' is the small molecule fragment moiety. In some cases, the small molecule fragment has a molecular weight of about 175, 200, 225, 250, 275, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 Dalton, or higher. In some cases, the molecular weight of the small molecule fragment is prior to enrichment with a halogen, a nonmetal, or a transition metal. In some embodiments, the molecular weight of the small molecule fragment is calculated based on carbon and hydrogen atoms and optionally further based on nitrogen, oxygen and/or sulfur atoms. In some embodiments, the molecular weight of the small molecule fragment does not include the molecular weight of a halogen, a transition metal or a combination thereof. In some cases, the small molecule fragment is a small molecule fragment of Formula (I):
##STR00052##
wherein RM is a reactive moiety selected from a Michael acceptor moiety, a leaving group moiety, or a moiety capable of forming a covalent bond with the thiol group of a cysteine residue; and F is a small molecule fragment moiety. In some cases, the Michael acceptor moiety comprises an alkene or an alkyne moiety. In some cases, F is obtained from a compound library. In some cases, F is a small molecule fragment moiety illustrated in FIG. 3. In some cases, F further comprises a linker moiety that connects F to the carbonyl moiety. In some cases, the small molecule fragment binds irreversibly to the cysteine containing protein. In some cases, the small molecule fragment binds reversibly to the cysteine containing protein.
[0156] In some embodiments, described herein is a modified cysteine containing protein comprising a small molecule fragment having a covalent bond to a cysteine residue of a cysteine containing protein, in which the cysteine containing protein is selected from Table 10C, channels, transporters or receptors. In some cases, one or more cysteine residues of each respective cysteine containing protein are denoted in Table 10C. In some cases, a cysteine containing protein selected from Table 10C is modified by a small molecule fragment at at least one cysteine site denoted in Table 10C to generate a modified cysteine containing protein. In some cases, the cysteine containing protein is about 20, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000 amino acid residues in length or more. In some cases, the cysteine residue of the modified cysteine containing protein has the structure SRI wherein R is selected from:
##STR00053##
wherein R.sup.1 is H, C1-C3 alkyl, or aryl; and F' is the small molecule fragment moiety. In some cases, the small molecule fragment has a molecular weight of about 175, 200, 225, 250, 275, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 Dalton, or higher. In some cases, the molecular weight of the small molecule fragment is prior to enrichment with a halogen, a nonmetal, or a transition metal. In some embodiments, the molecular weight of the small molecule fragment is calculated based on carbon and hydrogen atoms and optionally further based on nitrogen, oxygen and/or sulfur atoms. In some embodiments, the molecular weight of the small molecule fragment does not include the molecular weight of a halogen, a transition metal or a combination thereof. In some cases, the small molecule fragment is a small molecule fragment of Formula (I):
##STR00054##
wherein RM is a reactive moiety selected from a Michael acceptor moiety, a leaving group moiety, or a moiety capable of forming a covalent bond with the thiol group of a cysteine residue; and F is a small molecule fragment moiety. In some cases, the Michael acceptor moiety comprises an alkene or an alkyne moiety. In some cases, F is obtained from a compound library. In some cases, F is a small molecule fragment moiety illustrated in FIG. 3. In some cases, F further comprises a linker moiety that connects F to the carbonyl moiety. In some cases, the small molecule fragment binds irreversibly to the cysteine containing protein. In some cases, the small molecule fragment binds reversibly to the cysteine containing protein.
[0157] In some embodiments, described herein is a modified cysteine containing protein comprising a small molecule fragment having a covalent bond to a cysteine residue of a cysteine containing protein, in which the cysteine containing protein is selected from Table 10D, adapter, scaffolding, or modulator proteins. In some cases, one or more cysteine residues of each respective cysteine containing protein are denoted in Table 10D. In some cases, a cysteine containing protein selected from Table 10D is modified by a small molecule fragment at at least one cysteine site denoted in Table 10D to generate a modified cysteine containing protein. In some cases, the cysteine containing protein is about 20, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000 amino acid residues in length or more. In some cases, the cysteine residue of the modified cysteine containing protein has the structure SR, wherein R is selected from:
##STR00055##
wherein R.sup.1 is H. C1-C3 alkyl, or aryl; and F' is the small molecule fragment moiety. In some cases, the small molecule fragment has a molecular weight of about 175, 200, 225, 250, 275, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 Dalton, or higher. In some cases, the molecular weight of the small molecule fragment is prior to enrichment with a halogen, a nonmetal, or a transition metal. In some embodiments, the molecular weight of the small molecule fragment is calculated based on carbon and hydrogen atoms and optionally further based on nitrogen, oxygen and/or sulfur atoms. In some embodiments, the molecular weight of the small molecule fragment does not include the molecular weight of a halogen, a transition metal or a combination thereof. In some cases, the small molecule fragment is a small molecule fragment of Formula (I):
##STR00056##
wherein RM is a reactive moiety selected from a Michael acceptor moiety, a leaving group moiety, or a moiety capable of forming a covalent bond with the thiol group of a cysteine residue; and F is a small molecule fragment moiety. In some cases, the Michael acceptor moiety comprises an alkene or an alkyne moiety. In some cases, F is obtained from a compound library. In some cases, F is a small molecule fragment moiety illustrated in FIG. 3. In some cases, F further comprises a linker moiety that connects F to the carbonyl moiety. In some cases, the small molecule fragment binds irreversibly to the cysteine containing protein. In some cases, the small molecule fragment binds reversibly to the cysteine containing protein.
[0158] In some embodiments, described herein is a modified cysteine containing protein comprising a small molecule fragment having a covalent bond to a cysteine residue of a cysteine containing protein, in which the cysteine containing protein is selected from Table 10E. In some cases, one or more cysteine residues of each respective cysteine containing protein are denoted in Table 10E. In some cases, a cysteine containing protein selected from Table 10E is modified by a small molecule fragment at at least one cysteine site denoted in Table 10E to generate a modified cysteine containing protein. In some cases, the cysteine containing protein is about 20, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000 amino acid residues in length or more. In some cases, the cysteine residue of the modified cysteine containing protein has the structure SR, wherein R is selected from
##STR00057##
wherein R.sup.1 is H, C1-C3 alkyl, or aryl; and F' is the small molecule fragment moiety. In some cases, the small molecule fragment has a molecular weight of about 175, 200, 225, 250, 275, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 Dalton, or higher. In some cases, the molecular weight of the small molecule fragment is prior to enrichment with a halogen, a nonmetal, or a transition metal. In some embodiments, the molecular weight of the small molecule fragment is calculated based on carbon and hydrogen atoms and optionally further based on nitrogen, oxygen and/or sulfur atoms. In some embodiments, the molecular weight of the small molecule fragment does not include the molecular weight of a halogen, a transition metal or a combination thereof. In some cases, the small molecule fragment is a small molecule fragment of Formula (I):
##STR00058##
wherein RM is a reactive moiety selected from a Michael acceptor moiety, a leaving group moiety, or a moiety capable of forming a covalent bond with the thiol group of a cysteine residue, and F is a small molecule fragment moiety. In some cases, the Michael acceptor moiety comprises an alkene or an alkyne moiety. In some cases, F is obtained from a compound library. In some cases, F is a small molecule fragment moiety illustrated in FIG. 3. In some cases, F further comprises a linker moiety that connects F to the carbonyl moiety. In some cases, the small molecule fragment binds irreversibly to the cysteine containing protein. In some cases, the small molecule fragment binds reversibly to the cysteine containing protein.
[0159] In some embodiments, described herein is a modified cysteine containing protein comprising a small molecule fragment having a covalent bond to a cysteine residue of a cysteine containing protein, in which the cysteine containing protein comprises the motif X.sub.pC*Z, wherein X, is a polar residue, C* denotes the site of modification, and Z is any amino acid. In some cases, the cysteine containing protein is selected from Table 3. In some cases, the cysteine containing protein is selected from AIP, PES1, IKBKB, XPO1. KDM4B, NR3C1, GSTP1. TNFAIP3, ACAT1, IRAK1, GNB2L1, IRF4, USP34, ZC3HAV1, USP7, PELI1, DCUN1D1, USP28, UBE2O, RRAGC, MLTK, USP22, KDM3A, or USP16.
[0160] In some instances, described herein is a modified cysteine containing protein comprising a small molecule fragment having a covalent bond to a cysteine residue of a cysteine containing protein, in which the cysteine containing protein comprises the motif X.sub.pC*X.sub.n, wherein X.sub.p is a polar residue. C* denotes the site of modification, and X.sub.n is a nonpolar residue. In some cases, the cysteine containing protein is selected from Table 3. In some cases, the cysteine containing protein is selected from AIP, PES1, IKBKB, XPO1, GSTP1, ACAT1, IRAK1, IRF4, ZC3HAV1, USP7, PELI1, USP28, UBE2O, RRAGC, MLTK, USP22, KDM3A, or USP16.
[0161] In some instances, described herein is a modified cysteine containing protein comprising a small molecule fragment having a covalent bond to a cysteine residue of a cysteine containing protein, in which the cysteine containing protein comprises the motif X.sub.pC*X.sub.p, wherein X.sub.p is a polar residue and C* denotes the site of modification. In some cases, the cysteine containing protein is selected from Table 3. In some cases, the cysteine containing protein is selected from KDM4B, NR3C1, TNFAIP3, USP7 or USP22.
[0162] In some instances, described herein is a modified cysteine containing protein comprising a small molecule fragment having a covalent bond to a cysteine residue of a cysteine containing protein, in which the cysteine containing protein comprises the motif X.sub.pC*X.sub.b, wherein X.sub.p is a polar residue, C* denotes the site of modification, and X.sub.b is a basic residue. In some cases, the cysteine containing protein is selected from Table 3. In some cases, the cysteine containing protein is selected from GNB2L1 or USP34.
[0163] In some instances, described herein is a modified cysteine containing protein comprising a small molecule fragment having a covalent bond to a cysteine residue of a cysteine containing protein, in which the cysteine containing protein comprises the motif X.sub.pC*X.sub.a, wherein X.sub.p is a polar residue, C* denotes the site of modification, and X.sub.a is an acidic residue. In some cases, the cysteine containing protein is selected from Table 3. In some cases, the cysteine containing protein is DCUN1D1.
[0164] In some instances, described herein is a modified cysteine containing protein comprising a small molecule fragment having a covalent bond to a cysteine residue of a cysteine containing protein, in which the cysteine containing protein comprises the motif SC*Z, wherein C* denotes the site of modification, and Z is any amino acid. In some cases, the cysteine containing protein is selected from Table 3. In some cases, the cysteine containing protein is selected from PES1, IKBKB, GSTP1, ACAT1, IRAK1, ZC3HAV1 or RRAGC.
[0165] In some instances, described herein is a modified cysteine containing protein comprising a small molecule fragment having a covalent bond to a cysteine residue of a cysteine containing protein, in which the cysteine containing protein comprises the motif NC*Z, wherein C* denotes the site of modification, and Z is any amino acid. In some cases, the cysteine containing protein is selected from Table 3. In some cases, the cysteine containing protein is selected from XPO1, GNB2L1, USP34, UBE2O, MLTK or USP22.
[0166] In some instances, described herein is a modified cysteine containing protein comprising a small molecule fragment having a covalent bond to a cysteine residue of a cysteine containing protein, in which the cysteine containing protein comprises the motif YC*Z, wherein C* denotes the site of modification, and Z is any amino acid. In some cases, the cysteine containing protein is selected from Table 3. In some cases, the cysteine containing protein is selected from KDM4B or NR3C1.
[0167] In some instances, described herein is a modified cysteine containing protein comprising a small molecule fragment having a covalent bond to a cysteine residue of a cysteine containing protein, in which the cysteine containing protein comprises the motif TC*Z, wherein C* denotes the site of modification, and Z is any amino acid. In some cases, the cysteine containing protein is selected from Table 3. In some cases, the cysteine containing protein is selected from TNFAIP3, USP7, USP28, KDM3A or USP16.
[0168] In some instances, described herein is a modified cysteine containing protein comprising a small molecule fragment having a covalent bond to a cysteine residue of a cysteine containing protein, in which the cysteine containing protein comprises the motif QC*Z, wherein C* denotes the site of modification, and Z is any amino acid. In some cases, the cysteine containing protein is selected from Table 3. In some cases, the cysteine containing protein is selected from IRF4, PELI1, DCUN1D1 or USP22.
[0169] In some instances, described herein is a modified cysteine containing protein comprising a small molecule fragment having a covalent bond to a cysteine residue of a cysteine containing protein, in which the cysteine containing protein comprises the motif CC*Z, wherein C* denotes the site of modification, and Z is any amino acid. In some cases, the cysteine containing protein is selected from Table 3. In some cases, the cysteine containing protein is AIP.
[0170] In some instances, described herein is a modified cysteine containing protein comprising a small molecule fragment having a covalent bond to a cysteine residue of a cysteine containing protein, in which the cysteine containing protein is an enzyme and the enzyme comprises the motif X.sub.pC*Z, wherein X.sub.p is a polar residue, C* denotes the site of modification, and Z is any amino acid. In some cases, the cysteine containing protein is selected from Table 3. In some cases, the enzyme is selected from IKBKB, KDM4B, GSTP1, TNFAIP3, ACAT1, IRAK1, USP34, USP7, PELI1, USP28, UBE20O, MLTK, USP22, KDM3A, or USP16.
[0171] In some instances, described herein is a modified cysteine containing protein comprising a small molecule fragment having a covalent bond to a cysteine residue of a cysteine containing protein, in which the cysteine containing protein is a transcription factor or a regulator and the transcription factor or regulator comprises the motif X.sub.pC*Z, wherein X.sub.p is a polar residue, C* denotes the site of modification, and Z is any amino acid. In some cases, the cysteine containing protein is selected from Table 3. In some cases, the transcription factor or regulator is selected from NR3C1, IRF4 or ZC3HAV1.
[0172] In some instances, described herein is a modified cysteine containing protein comprising a small molecule fragment having a covalent bond to a cysteine residue of a cysteine containing protein, in which the cysteine containing protein is a channel, transporter or a receptor and the channel, transporter or receptor comprises the motif X.sub.pC*Z, wherein X.sub.p is a polar residue, C* denotes the site of modification, and Z is any amino acid. In some cases, the cysteine containing protein is selected from Table 3. In some cases, the channel, transporter, or receptor is selected from GNB2L1 or RRAGC.
[0173] In some instances, described herein is a modified cysteine containing protein comprising a small molecule fragment having a covalent bond to a cysteine residue of a cysteine containing protein, in which the cysteine containing protein comprises the motif X.sub.pC*Z, wherein X.sub.p is a polar residue. C* denotes the site of modification, and Z is any amino acid. In some cases, the cysteine containing protein is selected from Table 3. In some cases, the cysteine containing protein is selected from AIP, PES1, XPO1 or DCUN1D1.
[0174] In some instances, described herein is a modified cysteine containing protein comprising a small molecule fragment having a covalent bond to a cysteine residue of a cysteine containing protein, in which the cysteine containing protein comprises the motif X.sub.nC*Z, wherein X.sub.n is a nonpolar residue, C* denotes the site of modification, and Z is any amino acid. In some cases, the cysteine containing protein is selected from Table 3. In some cases, the cysteine containing protein is selected from PES1, CYR61, UBE2L6, XPO1, ADA, NR3C1, POU2F2, UCHL3, MGMT, ERCC3, ACAT1, STAT3, UBA7, CASP2, IDH2, LRBA, UBE2L3, RELB, IRF8, CASP8, PDIA6, PCK2, PFKFB4, PDE12, USP34, USP48, SMARCC2 or SAMHD1.
[0175] In some instances, described herein is a modified cysteine containing protein comprising a small molecule fragment having a covalent bond to a cysteine residue of a cysteine containing protein, in which the cysteine containing protein comprises the motif X.sub.nC*X.sub.n, wherein X.sub.n is a nonpolar residue and C* denotes the site of modification. In some cases, the cysteine containing protein is selected from Table 3. In some cases, the cysteine containing protein is selected from PES1, CYR61, NR3C1, UCHL3, ERCC3, ACAT1, STAT3, CASP2, LRBA, UBE2L3, RELB, PDIA6, PCK2, PFKFB4, USP48 or SMARCC2.
[0176] In some instances, described herein is a modified cysteine containing protein comprising a small molecule fragment having a covalent bond to a cysteine residue of a cysteine containing protein, in which the cysteine containing protein comprises the motif X.sub.nC*X.sub.p, wherein X.sub.n is a nonpolar residue, C* denotes the site of modification, and X.sub.p is a polar residue. In some cases, the cysteine containing protein is selected from Table 3. In some cases, the cysteine containing protein is selected from UBE2L6, POU2F2, MGMT, ACAT1, UBA7, CASP8, PDE12 or USP34.
[0177] In some instances, described herein is a modified cysteine containing protein comprising a small molecule fragment having a covalent bond to a cysteine residue of a cysteine containing protein, in which the cysteine containing protein comprises the motif X.sub.nC*X.sub.a, wherein X.sub.n is a nonpolar residue, C* denotes the site of modification, and X.sub.a is an acidic residue. In some cases, the cysteine containing protein is selected from Table 3. In some cases, the cysteine containing protein is selected from CYR61 or XPO1.
[0178] In some instances, described herein is a modified cysteine containing protein comprising a small molecule fragment having a covalent bond to a cysteine residue of a cysteine containing protein, in which the cysteine containing protein comprises the motif X.sub.aC*X.sub.b, wherein X.sub.n is a nonpolar residue, C* denotes the site of modification, and X.sub.b is a basic residue. In some cases, the cysteine containing protein is selected from Table 3. In some cases, the cysteine containing protein is selected from ADA, MGMT, IDH2, IRF8 or SAMHD1.
[0179] In some instances, described herein is a modified cysteine containing protein comprising a small molecule fragment having a covalent bond to a cysteine residue of a cysteine containing protein, in which the cysteine containing protein comprises the motif LC*Z, wherein C* denotes the site of modification, and Z is any amino acid. In some cases, the cysteine containing protein is selected from Table 3. In some cases, the cysteine containing protein is selected from PES1, CYR61, XPO1, NR3C1 or SMARCC2.
[0180] In some instances, described herein is a modified cysteine containing protein comprising a small molecule fragment having a covalent bond to a cysteine residue of a cysteine containing protein, in which the cysteine containing protein comprises the motif PC*Z, wherein C* denotes the site of modification, and Z is any amino acid. In some cases, the cysteine containing protein is selected from Table 3. In some cases, the cysteine containing protein is selected from CYR61, UBE2L6, MGMT, ERCC3, ACAT1 or USP48.
[0181] In some instances, described herein is a modified cysteine containing protein comprising a small molecule fragment having a covalent bond to a cysteine residue of a cysteine containing protein, in which the cysteine containing protein comprises the motif GC*Z, wherein C* denotes the site of modification, and Z is any amino acid. In some cases, the cysteine containing protein is selected from Table 3. In some cases, the cysteine containing protein is selected from ADA, RELB or USP34.
[0182] In some instances, described herein is a modified cysteine containing protein comprising a small molecule fragment having a covalent bond to a cysteine residue of a cysteine containing protein, in which the cysteine containing protein comprises the motif AC*Z, wherein C* denotes the site of modification, and Z is any amino acid. In some cases, the cysteine containing protein is selected from Table 3. In some cases, the cysteine containing protein is selected from UCHL3, CASP2. IDH2, LRBA. CASP8, PCK2 or PDE12.
[0183] In some instances, described herein is a modified cysteine containing protein comprising a small molecule fragment having a covalent bond to a cysteine residue of a cysteine containing protein, in which the cysteine containing protein comprises the motif VC*Z, wherein C* denotes the site of modification, and Z is any amino acid. In some cases, the cysteine containing protein is selected from Table 3. In some cases, the cysteine containing protein is selected from MGMT, ACAT1, UBA7, UBE2L3 or IRF8.
[0184] In some instances, described herein is a modified cysteine containing protein comprising a small molecule fragment having a covalent bond to a cysteine residue of a cysteine containing protein, in which the cysteine containing protein comprises the motif IC*Z, wherein C* denotes the site of modification, and Z is any amino acid. In some cases, the cysteine containing protein is selected from Table 3. In some cases, the cysteine containing protein is selected from PFKFB4, ACAT1 or STAT3.
[0185] In some instances, described herein is a modified cysteine containing protein comprising a small molecule fragment having a covalent bond to a cysteine residue of a cysteine containing protein, in which the cysteine containing protein comprises the motif X.sub.rC*Z, wherein X.sub.r denotes an aromatic residue, C* denotes the site of modification, and Z is any amino acid. In some cases, the cysteine containing protein is selected from Table 3. In some cases, the cysteine containing protein is selected from POU2F2, PDIA6 or SAMHD1.
[0186] In some instances, described herein is a modified cysteine containing protein comprising a small molecule fragment having a covalent bond to a cysteine residue of a cysteine containing protein, in which the cysteine containing protein is an enzyme and the enzyme comprises the motif X.sub.nC*Z, wherein X.sub.n is a nonpolar residue, C* denotes the site of modification, and Z is any amino acid. In some cases, the cysteine containing protein is selected from Table 3. In some cases, the enzyme is selected from UBE2L6, ADA, UCHL3, MGMT, ERCC3, ACAT1, UBA7, CASP2, IDH2, UBE2L3, CASP8, PDIA6, PCK2, PFKFB4, PDE12, USP34, USP48 or SAMHD1.
[0187] In some instances, described herein is a modified cysteine containing protein comprising a small molecule fragment having a covalent bond to a cysteine residue of a cysteine containing protein, in which the cysteine containing protein is a transcription factor or a regulator and the transcription factor or regulator comprises the motif X.sub.nC*Z, wherein X.sub.n is a nonpolar residue, C* denotes the site of modification, and Z is any amino acid. In some cases, the cysteine containing protein is selected from Table 3. In some cases, the transcription factor or regulator is selected from NR3C1, POU2F2, STAT3, RELB, IRF8 or SMARCC2.
[0188] In some instances, described herein is a modified cysteine containing protein comprising a small molecule fragment having a covalent bond to a cysteine residue of a cysteine containing protein, in which the cysteine containing protein comprises the motif X.sub.nC*Z, wherein X.sub.n is a nonpolar residue, C* denotes the site of modification, and Z is any amino acid. In some cases, the cysteine containing protein is selected from Table 3. In some cases, the cysteine containing protein is selected from PES1, CYR61, XPO1 or LRBA.
[0189] In some instances, described herein is a modified cysteine containing protein comprising a small molecule fragment having a covalent bond to a cysteine residue of a cysteine containing protein, in which the cysteine containing protein comprises the motif X.sub.aC*Z, wherein X.sub.a is an acidic residue, C* denotes the site of modification, and Z is any amino acid. In some cases, the cysteine containing protein is selected from Table 3. In some cases, the cysteine containing protein is selected from ZAP70, PRKCQ or PRMT1.
[0190] In some instances, described herein is a modified cysteine containing protein comprising a small molecule fragment having a covalent bond to a cysteine residue of a cysteine containing protein, in which the cysteine containing protein comprises the motif EC*Z, wherein C* denotes the site of modification, and Z is any amino acid. In some cases, the cysteine containing protein is selected from Table 3. In some cases, the cysteine containing protein is selected from ZAP70 or PRKCQ.
[0191] In some instances, described herein is a modified cysteine containing protein comprising a small molecule fragment having a covalent bond to a cysteine residue of a cysteine containing protein, in which the cysteine containing protein comprises the motif X.sub.bC*Z, wherein X.sub.b is a basic residue, C* denotes the site of modification, and Z is any amino acid. In some cases, the cysteine containing protein is selected from Table 3. In some cases, the cysteine containing protein is selected from CYR61, ZNF217, NCF1, IREB2, LRBA, CDK5, EP300, EZH2, UBE2S, VCPIP1, RRAGC or IRAK4.
[0192] In some instances, described herein is a modified cysteine containing protein comprising a small molecule fragment having a covalent bond to a cysteine residue of a cysteine containing protein, in which the cysteine containing protein comprises the motif X.sub.bC*X.sub.n, wherein X.sub.b is a basic residue, C* denotes the site of modification, and X.sub.n is a nonpolar residue. In some cases, the cysteine containing protein is selected from Table 3. In some cases, the cysteine containing protein is selected from CYR61, ZNF217, IREB2, EP300, UBE2S, VCPIP1, RRAGC or IRAK4.
[0193] In some instances, described herein is a modified cysteine containing protein comprising a small molecule fragment having a covalent bond to a cysteine residue of a cysteine containing protein, in which the cysteine containing protein comprises the motif X.sub.bC*X.sub.p, wherein X.sub.b is a basic residue, C* denotes the site of modification, and X.sub.p is a polar residue. In some cases, the cysteine containing protein is selected from Table 3. In some cases, the cysteine containing protein is selected from NCF1, LRBA or CDK5.
[0194] In some instances, described herein is a modified cysteine containing protein comprising a small molecule fragment having a covalent bond to a cysteine residue of a cysteine containing protein, in which the cysteine containing protein comprises the motif X.sub.bC*X.sub.b, wherein X.sub.b is a basic residue and C* denotes the site of modification. In some cases, the cysteine containing protein is selected from Table 3. In some cases, the cysteine containing protein is EZH2.
[0195] In some instances, described herein is a modified cysteine containing protein comprising a small molecule fragment having a covalent bond to a cysteine residue of a cysteine containing protein, in which the cysteine containing protein comprises the motif RC*Z, wherein C* denotes the site of modification, and Z is any amino acid. In some cases, the cysteine containing protein is selected from Table 3. In some cases, the cysteine containing protein is selected from ZNF217, NCF1, CDK5, EP300 or IRAK4.
[0196] In some instances, described herein is a modified cysteine containing protein comprising a small molecule fragment having a covalent bond to a cysteine residue of a cysteine containing protein, in which the cysteine containing protein comprises the motif KC*Z, wherein C* denotes the site of modification, and Z is any amino acid. In some cases, the cysteine containing protein is selected from Table 3. In some cases, the cysteine containing protein is selected from CYR61, IREB2, LRBA or UBE2S.
[0197] In some instances, described herein is a modified cysteine containing protein comprising a small molecule fragment having a covalent bond to a cysteine residue of a cysteine containing protein, in which the cysteine containing protein comprises the motif HC*Z, wherein C* denotes the site of modification, and Z is any amino acid. In some cases, the cysteine containing protein is selected from Table 3. In some cases, the cysteine containing protein is selected from EZH2, VCPIP1 or RRAGC.
[0198] In some instances, described herein is a modified cysteine containing protein comprising a small molecule fragment having a covalent bond to a cysteine residue of a cysteine containing protein, in which the cysteine containing protein is an enzyme and the enzyme comprises the motif X.sub.bC*Z, wherein X.sub.b is a basic residue, C* denotes the site of modification, and Z is any amino acid. In some cases, the cysteine containing protein is selected from Table 3. In some cases, the enzyme is selected from CDK5, EP300, EZH2, UBE2S, VCPIP1 or IRAK4.
[0199] In some instances, described herein is a modified cysteine containing protein comprising a small molecule fragment having a covalent bond to a cysteine residue of a cysteine containing protein, in which the cysteine containing protein is a transcription factor or a regulator and the transcription factor or regulator comprises the motif X.sub.bC*Z, wherein X.sub.b is a basic residue, C* denotes the site of modification, and Z is any amino acid. In some cases, the cysteine containing protein is selected from Table 3. In some cases, the transcription factor or regulator is selected from ZNF217 or IREB2.
[0200] In some instances, described herein is a modified cysteine containing protein comprising a small molecule fragment having a covalent bond to a cysteine residue of a cysteine containing protein, in which the cysteine containing protein is an adapter, a scaffolding protein, or a modulator protein and the adapter, scaffolding protein or the modulator protein comprises the motif X.sub.bC*Z, wherein X.sub.b is a basic residue. C* denotes the site of modification, and Z is any amino acid. In some cases, the cysteine containing protein is selected from Table 3. In some cases, the adapter, scaffolding protein or the modulator protein is selected from NCF1.
[0201] In some instances, described herein is a modified cysteine containing protein comprising a small molecule fragment having a covalent bond to a cysteine residue of a cysteine containing protein, in which the cysteine containing protein is a channel, a transporter, or a receptor and the channel, transporter, or receptor comprises the motif X.sub.bC*Z, wherein X.sub.b is a basic residue, C* denotes the site of modification, and Z is any amino acid. In some cases, the cysteine containing protein is selected from Table 3. In some cases, the channel, transporter, or receptor is selected from RRAGC.
[0202] In some instances, described herein is a modified cysteine containing protein comprising a small molecule fragment having a covalent bond to a cysteine residue of a cysteine containing protein, in which the cysteine containing protein comprises the motif X.sub.bC*Z, wherein X.sub.b is a basic residue, C* denotes the site of modification, and Z is any amino acid. In some cases, the cysteine containing protein is selected from Table 3. In some cases, the cysteine containing protein is selected from CYR61 or LRBA.
[0203] In some cases, a cysteine containing protein described above comprises about 20, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000 amino acid residues in length or more.
[0204] In some cases, the cysteine residue of a modified cysteine containing protein described above has the structure SR, wherein R is selected from:
##STR00059##
wherein R.sup.1 is H, C1-C3 alkyl, or aryl; and F' is the small molecule fragment moiety. In some cases, the small molecule fragment has a molecular weight of about 175, 200, 225, 250, 275, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 Dalton, or higher. In some cases, the molecular weight of the small molecule fragment is prior to enrichment with a halogen, a nonmetal, or a transition metal. In some embodiments, the molecular weight of the small molecule fragment is calculated based on carbon and hydrogen atoms and optionally further based on nitrogen, oxygen and/or sulfur atoms. In some embodiments, the molecular weight of the small molecule fragment does not include the molecular weight of a halogen, a transition metal or a combination thereof. In some cases, the small molecule fragment is a small molecule fragment of Formula (I):
##STR00060##
wherein RM is a reactive moiety selected from a Michael acceptor moiety, a leaving group moiety, or a moiety capable of forming a covalent bond with the thiol group of a cysteine residue; and F is a small molecule fragment moiety. In some cases, the Michael acceptor moiety comprises an alkene or an alkyne moiety. In some cases, F is obtained from a compound library. In some cases, F is a small molecule fragment moiety illustrated in FIG. 3. In some cases, F further comprises a linker moiety that connects F to the carbonyl moiety. In some cases, the small molecule fragment binds irreversibly to a cysteine containing protein described above. In some cases, the small molecule fragment binds reversibly to a cysteine containing protein described above.
Compositions, Cells, and Cell Populations
[0205] Disclosed herein also include compositions of a small molecule fragment conjugated to a cysteine containing protein, a cysteine-reactive probe conjugated to a cysteine containing protein, and treated sample compositions. In some embodiments, a composition described herein comprises a small molecule fragment of Formula (I):
##STR00061##
[0206] wherein:
[0207] RM is a reactive moiety selected from a Michael acceptor moiety, a leaving group moiety, or a moiety capable of forming a covalent bond with the thiol group of a cysteine residue; and
[0208] F is a small molecule fragment moiety; and
[0209] a cysteine containing protein wherein the cysteine containing protein is covalently bond to the small molecule fragment.
[0210] In some embodiments, also described herein is a composition that comprises a cysteine-reactive probe of Formula (II):
##STR00062##
[0211] wherein:
[0212] RM is a reactive moiety selected from a Michael acceptor moiety, a leaving group moiety, or a moiety capable of forming a covalent bond to the thiol group of a cysteine residue; and
[0213] AHM is an affinity handle moiety; and
[0214] a cysteine containing protein wherein the cysteine containing protein is covalently bond to the cysteine-reactive probe.
[0215] In some embodiments, also described herein is a composition that comprises an isolated sample wherein the isolated sample is an isolated cell or a tissue sample; and a cysteine-reactive probe to be assayed for its ability to interact with a cysteine containing protein expressed in the isolated sample.
[0216] Disclosed herein further include isolated treated cell and cell populations. In some embodiments, described herein is an isolated treated cell that comprises a cysteine-reactive probe covalently attached to a cysteine containing protein. In some instances, the isolated treated cell further comprises a set of cysteine-reactive probes wherein each of the cysteine-reactive probes is covalently attached to a cysteine containing protein.
[0217] In some embodiments, described herein is an isolated treated cell that comprises a small molecule fragment covalently attached to a cysteine containing protein. In some instances, the isolated treated cell further comprises a set of small molecule fragments wherein each of the small molecule fragment is covalently attached to a cysteine containing protein. In some instances, the isolated treated cell further comprises a cysteine-reactive probe. In some instances, the isolated treated cell further comprises a set of cysteine-reactive probes.
[0218] In some embodiments, also described herein is an isolated treated population of cells that comprises a set of cysteine-reactive probes covalently attached to cysteine containing proteins.
[0219] In some embodiments, further described herein is an isolated treated population of cells that comprises a set of small molecule fragments covalently attached to cysteine containing proteins. In some instances, the isolated treated population of cells further comprises a set of cysteine-reactive probes.
[0220] As disclosed elsewhere herein, the small molecule fragment is a small molecule fragment of Formula (I):
##STR00063##
[0221] wherein:
[0222] RM is a reactive moiety selected from a Michael acceptor moiety, a leaving group moiety, or a moiety capable of forming a covalent bond with the thiol group of a cysteine residue; and
[0223] F is a small molecule fragment moiety.
[0224] In some instances, the Michael acceptor moiety comprises an alkene or an alkyne moiety. In some cases, F is obtained from a compound library. In some embodiments, the compound library comprises ChemBridge fragment library, Pyramid Platform Fragment-Based Drug Discovery, Maybridge fragment library, FRGx from AnalytiCon, TCI-Frag from AnCoreX, Bio Building Blocks from ASINEX, BioFocus 3D from Charles River, Fragments of Life (FOL) from Emerald Bio. Enamine Fragment Library, IOTA Diverse 1500, BIONET fragments library, Life Chemicals Fragments Collection, OTAVA fragment library, Prestwick fragment library, Selcia fragment library, TimTec fragment-based library, Allium from Vitas-M Laboratory, or Zenobia fragment library. In some cases, F is a small molecule fragment moiety illustrated in FIG. 3. In some cases, F further comprises a linker moiety that connects F to the carbonyl moiety. In some embodiments, the small molecule fragment is a small molecule fragment illustrated in FIG. 3.
[0225] Also described elsewhere herein, the cysteine-reactive probe is a cysteine-reactive probe of Formula (II):
##STR00064##
[0226] wherein:
[0227] RM is a reactive moiety selected from a Michael acceptor moiety, a leaving group moiety, or a moiety capable of forming a covalent bond to the thiol group of a cysteine residue; and
[0228] AHM is an affinity handle moiety.
[0229] In some embodiments, the Michael acceptor moiety comprises an alkene or an alkyne moiety. In some instances, the affinity handle moiety comprises an affinity handle and a binding moiety that facilitates covalent interaction of the cysteine-reactive probe to a cysteine residue of a cysteine-containing protein. In some cases, the binding moiety is a small molecule fragment obtained from a compound library. In some cases, the compound library comprises ChemBridge fragment library, Pyramid Platform Fragment-Based Drug Discovery, Maybridge fragment library. FRGx from AnalytiCon, TCI-Frag from AnCoreX, Bio Building Blocks from ASINEX, BioFocus 3D from Charles River, Fragments of Life (FOL) from Emerald Bio, Enamine Fragment Library, IOTA Diverse 1500, BIONET fragments library, Life Chemicals Fragments Collection, OTAVA fragment library, Prestwick fragment library, Selcia fragment library, TimTec fragment-based library, Allium from Vitas-M Laboratory, or Zenobia fragment library.
[0230] In some instances, the affinity handle is a bioorthogonal affinity handle. In some cases, the affinity handle comprises a carbodiimide, N-hydroxysuccinimide (NHS) ester, imidoester, pentafluorophenyl ester, hydroxymethyl phosphine, maleimide, haloacetyl, pyridyl disulfide, thiosulfonate, vinylsulfone, hydrazide, alkoxyamine, alkyne, azide, or isocyanate group. In some cases, the affinity handle comprises an alkyne or an azide group. In some instances, the affinity handle is further conjugated to an affinity ligand. In some instances, the affinity ligand comprises a chromophore, a labeling group, or a combination thereof. In some cases, the chromophore comprises fluorochrome, non-fluorochrome chromophore, quencher, an absorption chromophore, fluorophore, organic dye, inorganic dye, metal chelate, or a fluorescent enzyme substrate. In some cases, the labeling group is biotin moiety, streptavidin moiety, bead, resin, a solid support, or a combination thereof. In some instances, the affinity handle moiety further comprises a chromophore. In some embodiments, the cysteine-reactive probe is a cysteine-reactive probe illustrated in FIG. 3.
[0231] Further described elsewhere herein, the cell or cell population is obtained from any mammal, such as human or non-human primates. In some embodiments, the cell or cell population is an epithelial cell, connective tissue cell, hormone secreting cell, a nerve cell, a skeletal muscle cell, a blood cell, or an immune system cell. In additional embodiments, the cell or cell population is cancerous or is obtained from a tumor site.
Polypeptides Comprising a Cysteine Interacting Site
[0232] Further disclosed herein are polypeptides that comprise one or more of the cysteine interacting sites identified by a method described herein. In some embodiments, described herein is an isolated and purified polypeptide that comprises at least 90% sequence identity to at least seven contiguous amino acids of an amino acid sequence selected from Tables 1-3 or 8-9. In some embodiments, the isolated and purified polypeptide comprises at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to at least seven contiguous amino acids of an amino acid sequence selected from Tables 1-3 or 8-9. In some embodiments, the isolated and purified polypeptide comprises 100% sequence identity to at least seven contiguous amino acids of an amino acid sequence selected from Tables 1-3 or 8-9. In some instances, the isolated and purified polypeptide consists 100% sequence identity to the full length of an amino acid sequence selected from Tables 1-3 or 8-9. In some instances, the isolated and purified polypeptide is at most 50 amino acids in length.
[0233] In some embodiments, additionally described herein include nucleic acid encoding a polypeptide that comprises at least 90% sequence identity at least seven contiguous amino acids of an amino acid sequence selected from Tables 1-3 or 8-9. In some embodiments, the nucleic acid encoding a polypeptide comprises at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity at least seven contiguous amino acids of an amino acid sequence selected from Tables 1-3 or 8-9. In some embodiments, the nucleic acid encoding a polypeptide comprises 100% sequence identity at least seven contiguous amino acids of an amino acid sequence selected from Tables 1-3 or 8-9. In some embodiments, the nucleic acid encoding a polypeptide consists 100% sequence identity to the full length of an amino acid sequence selected from Tables 1-3 or 8-9.
[0234] In some embodiments, further disclosed herein include a method of mapping a biologically active cysteine site on a protein, which comprises harvesting a set of cysteine-reactive probe-protein complexes from a sample wherein the cysteine-reactive probe comprises a reactive moiety capable of forming a covalent bond with a cysteine residue located on the cysteine containing protein; analyzing the set of cysteine-reactive probe-protein complexes by a proteomic analysis means; and based on the previous step, mapping the biologically active cysteine site on the protein.
[0235] In some embodiments, the analyzing further comprises treating the set of cysteine-reactive probe-protein complexes with a protease to generate a set of protein fragments. The protease is a serine protease, a threonine protease, a cysteine protease, an aspartate protease, a glutamic acid protease, or a metalloprotease. In some instances, the protease is a serine protease. In some instances, the protease is trypsin. In some instances, cysteine-reactive probe-protein complex is further attached to a labeling group such as a biotin moiety. In some instances, the labeling group such as a biotin moiety further comprises a linker. In some instances, the linker is a peptide. In some instances, the peptide linker is about 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid residues in length. In some instances, the peptide linker contains a cleavage site. A non-limiting list of cleavage sites includes Tobacco Etch Virus (TEV), thrombin (Thr), enterokinase (EKT), activated Factor X (Xa), or human Rhinovirus 3C protease (3C/PreScission). In some instances, the peptide linker contains a TEV protease cleavage site. In some instances, the TEV protease cleavage site comprises the following sequence Gly-Gln-Phe-Tyr-Leu-Asn-Glu (SEQ ID NO: 860). In some instances, the biotin moiety is further coupled to a bead (e.g. a streptavidin-coupled bead).
[0236] In some instances, the protein from the cysteine-reactive probe-protein complex attached to the bead (via a biotin moiety comprising a linker and attached to a streptavidin-coupled bead) is digested with trypsin, and the immobilized peptide or protein fragment is further separated and collected. In some instances, the collected peptide or protein fragment is then digested by a protease (e.g. TEV protease), and the treated protein fragment is then separated, and collected for analysis. In some instances, the analysis is a proteomic analysis as described above and elsewhere herein. In some instances, the sequence of the protein fragment is further determined. In some instances, the protein fragment correlates to a small molecule fragment binding site on the cysteine containing protein.
[0237] In some embodiments, the sequence of the protein fragment correlates to a sequence as illustrated in Tables 1-3 or 8-9. In some instances, the sequence as shown in Tables 1-3 or 8-9 correlate to a site on the full length protein as a drug binding site. In some instances, the sequence as shown in Tables 1-3 or 8-9 correlate to a drug binding site. In some instances, polypeptides comprising one or more of the sequences as shown in Tables 1-3 or 8-9 serve as probes for small molecule fragment screening.
[0238] In some instances after the generation of a polypeptide, the polypeptide is subjected to one or more rounds of purification steps to remove impurities. In some instances, the purification step is a chromatographic step utilizing separation methods such as affinity-based, size-exclusion based, ion-exchange based, or the like. In some cases, the polypeptide is at most 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.9%, or 100% pure or without the presence of impurities. In some cases, the polypeptide is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.9%, or 100% pure or without the presence of impurities.
[0239] As described above, nucleic acid encoding a polypeptide that is derived from a cysteine containing protein is subjected to one or more rounds of purification steps to remove impurities. In some instances, the purification step is a chromatographic step utilizing separation methods such as affinity-based, size-exclusion based, ion-exchange based, or the like. In some cases, the nucleic acid is at most 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.9%, or 100% pure or without the presence of impurities. In some cases, the nucleic acid is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.9%, or 100% pure or without the presence of impurities.
[0240] As used herein, a polypeptide includes natural amino acids, unnatural amino acids, or a combination thereof. In some instances, an amino acid residue refers to a molecule containing both an amino group and a carboxyl group. Suitable amino acids include, without limitation, both the D- and L-isomers of the naturally-occurring amino acids, as well as non-naturally occurring amino acids prepared by organic synthesis or other metabolic routes. The term amino acid, as used herein, includes, without limitation, .alpha.-amino acids, natural amino acids, non-natural amino acids, and amino acid analogs.
[0241] The term ".alpha.-amino acid" refers to a molecule containing both an amino group and a carboxyl group bound to a carbon which is designated the .alpha.-carbon.
[0242] The term ".beta.-amino acid" refers to a molecule containing both an amino group and a carboxyl group in a .beta. configuration.
[0243] "Naturally occurring amino acid" refers to any one of the twenty amino acids commonly found in peptides synthesized in nature, and known by the one letter abbreviations A, R, N, C, D, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y and V.
[0244] The following table shows a summary of the properties of natural amino acids:
TABLE-US-00001 3- 1- Side- Side-chain Letter Letter chain charge Hydropathy Amino Acid Code Code Polarity (pH 7.4) Index Alanine Ala A nonpolar neutral 1.8 Arginine Arg R polar positive -4.5 Asparagine Asn N polar neutral -3.5 Aspartic acid Asp D polar negative -3.5 Cysteine Cys C polar neutral 2.5 Glutamic acid Glu E polar negative -3.5 Glutamine Gln Q polar neutral -3.5 Glycine Gly G nonpolar neutral -0.4 Histidine His H polar positive(10%) -3.2 neutral(90%) Isoleucine Ile I nonpolar neutral 4.5 Leucine Leu L nonpolar neutral 3.8 Lysine Lys K polar positive -3.9 Methionine Met M nonpolar neutral 1.9 Phenylalanine Phe F nonpolar neutral 2.8 Proline Pro P nonpolar neutral -1.6 Serine Ser S polar neutral -0.8 Threonine Thr T polar neutral -0.7 Tryptophan Trp W nonpolar neutral -0.9 Tyrosine Tyr Y polar neutral -1.3 Valine Val V nonpolar neutral 4.2
[0245] "Hydrophobic amino acids" include small hydrophobic amino acids and large hydrophobic amino acids. "Small hydrophobic amino acid" are glycine, alanine, proline, and analogs thereof. "Large hydrophobic amino acids" are valine, leucine, isoleucine, phenylalanine, methionine, tryptophan, and analogs thereof. "Polar amino acids" are serine, threonine, asparagine, glutamine, cysteine, tyrosine, and analogs thereof. "Charged amino acids" are lysine, arginine, histidine, aspartate, glutamate, and analogs thereof. In some cases, aspartic acid and glutamic acid are referred to as acidic amino acids. In other cases, lysine, arginine and histidine are referred to as basic amino acids.
[0246] The term "amino acid analog" refers to a molecule which is structurally similar to an amino acid and which is substituted for an amino acid in the formation of a peptidomimetic macrocycle Amino acid analogs include, without limitation, .beta.-amino acids and amino acids where the amino or carboxy group is substituted by a similarly reactive group (e.g., substitution of the primary amine with a secondary or tertiary amine, or substitution of the carboxy group with an ester).
[0247] The term "non-natural amino acid" refers to an amino acid which is not one of the twenty amino acids commonly found in peptides synthesized in nature, and known by the one letter abbreviations A, R, N, C, D, Q, E, G, H, I, L, K, M, F, P, S, T, W Y and V.
[0248] In some instances, amino acid analogs include .beta.-amino acid analogs. Examples of .beta.-amino acid analogs include, but are not limited to, the following: cyclic .beta.-amino acid analogs; .beta.-alanine; (R)-.beta.-phenylalanine; (R)-1,2,3,4-tetrahydro-isoquinoline-3-acetic acid; (R)-3-amino-4-(1-naphthyl)-butyric acid; (R)-3-amino-4-(2,4-dichlorophenyl)butyric acid; (R)-3-amino-4-(2-chlorophenyl)-butyric acid; (R)-3-amino-4-(2-cyanophenyl)-butyric acid; (R)-3-amino-4-(2-fluorophenyl)-butyric acid; (R)-3-amino-4-(2-furyl)-butyric acid; (R)-3-amino-4-(2-methylphenyl)-butyric acid; (R)-3-amino-4-(2-naphthyl)-butyric acid; (R)-3-amino-4-(2-thienyl)-butyric acid; (R)-3-amino-4-(2-trifluoromethylphenyl)-butyric acid; (R)-3-amino-4-(3,4-dichlorophenyl)butyric acid; (R)-3-amino-4-(3,4-difluorophenyl)butyric acid; (R)-3-amino-4-(3-benzothienyl)-butyric acid; (R)-3-amino-4-(3-chlorophenyl)-butyric acid; (R)-3-amino-4-(3-cyanophenyl)-butyric acid; (R)-3-amino-4-(3-fluorophenyl)-butyric acid; (R)-3-amino-4-(3-methylphenyl)-butyric acid; (R)-3-amino-4-(3-pyridyl)-butyric acid; (R)-3-amino-4-(3-thienyl)-butyric acid; (R)-3-amino-4-(3-trifluoromethylphenyl)-butyric acid; (R)-3-amino-4-(4-bromophenyl)-butyric acid; (R)-3-amino-4-(4-chlorophenyl)-butyric acid; (R)-3-amino-4-(4-cyanophenyl)-butyric acid; (R)-3-amino-4-(4-fluorophenyl)-butyric acid; (R)-3-amino-4-(4-iodophenyl)-butyric acid; (R)-3-amino-4-(4-methylphenyl)-butyric acid; (R)-3-amino-4-(4-nitrophenyl)-butyric acid; (R)-3-amino-4-(4-pyridyl)-butyric acid; (R)-3-amino-4-(4-trifluoromethylphenyl)-butyric acid; (R)-3-amino-4-pentafluoro-phenylbutyric acid; (R)-3-amino-5-hexenoic acid; (R)-3-amino-5-hexynoic acid; (R)-3-amino-5-phenylpentanoic acid; (R)-3-amino-6-phenyl-5-hexenoic acid; (S)-1,2,3,4-tetrahydro-isoquinoline-3-acetic acid; (S)-3-amino-4-(1-naphthyl)-butyric acid; (S)-3-amino-4-(2,4-dichlorophenyl)butyric acid; (S)-3-amino-4-(2-chlorophenyl)-butyric acid; (S)-3-amino-4-(2-cyanophenyl)-butyric acid; (S)-3-amino-4-(2-fluorophenyl)-butyric acid; (S)-3-amino-4-(2-furyl)-butyric acid; (S)-3-amino-4-(2-methylphenyl)-butyric acid; (S)-3-amino-4-(2-naphthyl)-butyric acid; (S)-3-amino-4-(2-thienyl)-butyric acid; (S)-3-amino-4-(2-trifluoromethylphenyl)-butyric acid; (S)-3-amino-4-(3,4-dichlorophenyl)butyric acid; (S)-3-amino-4-(3,4-difluorophenyl)butyric acid; (S)-3-amino-4-(3-benzothienyl)-butyric acid; (S)-3-amino-4-(3-chlorophenyl)-butyric acid; (S)-3-amino-4-(3-cyanophenyl)-butyric acid; (S)-3-amino-4-(3-fluorophenyl)-butyric acid; (S)-3-amino-4-(3-methylphenyl)-butyric acid; (S)-3-amino-4-(3-pyridyl)-butyric acid; (S)-3-amino-4-(3-thienyl)-butyric acid; (S)-3-amino-4-(3-trifluoromethylphenyl)-butyric acid; (S)-3-amino-4-(4-bromophenyl)-butyric acid; (S)-3-amino-4-(4-chlorophenyl) butyric acid; (S)-3-amino-4-(4-cyanophenyl)-butyric acid; (S)-3-amino-4-(4-fluorophenyl) butyric acid; (S)-3-amino-4-(4-iodophenyl)-butyric acid; (S)-3-amino-4-(4-methylphenyl)-butyric acid; (S)-3-amino-4-(4-nitrophenyl)-butyric acid; (S)-3-amino-4-(4-pyridyl)-butyric acid; (S)-3-amino-4-(4-trifluoromethylphenyl)-butyric acid; (S)-3-amino-4-pentafluoro-phenylbutyric acid; (S)-3-amino-5-hexenoic acid; (S)-3-amino-5-hexynoic acid; (S)-3-amino-5-phenylpentanoic acid; (S)-3-amino-6-phenyl-5-hexenoic acid; 1,2,5,6-tetrahydropyridine-3-carboxylic acid; 1,2,5,6-tetrahydropyridine-4-carboxylic acid; 3-amino-3-(2-chlorophenyl)-propionic acid; 3-amino-3-(2-thienyl)-propionic acid; 3-amino-3-(3-bromophenyl)-propionic acid; 3-amino-3-(4-chlorophenyl)-propionic acid; 3-amino-3-(4-methoxyphenyl)-propionic acid; 3-amino-4,4,4-trifluoro-butyric acid; 3-aminoadipic acid; D-.beta.-phenylalanine; .beta.-leucine; L-.beta.-homoalanine; L-.beta.-homoaspartic acid .gamma.-benzyl ester; L-.beta.-homoglutamic acid .delta.-benzyl ester; L-.beta.-homoisoleucine; L-.beta.-homoleucine; L-.beta.-homomethionine; L-.beta.-homophenylalanine; L-.beta.-homoproline; L-.beta.-homotryptophan; L-.beta.-homovaline; L-N.omega.-benzyloxycarbony-.beta.-homolysine; N.omega.-L-.beta.-homoarginine; O-benzyl-L-.beta.-homohydroxyproline; O-benzyl-L-.beta.-homoserine; O-benzyl-L-.beta.-homothreonine; O-benzyl-L-.beta.-homotyrosine; .gamma.-trityl-L-.beta.-homoasparagine; (R)-.beta.-phenylalanine; L-.beta.-homoaspartic acid .gamma.-t-butyl ester; L-.beta.-homoglutamic acid .delta.-t-butyl ester; L-N.omega.-.beta.-homolysine; N.delta.-trityl-L-.beta.-homoglutamine; N.omega.-2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl-L-3-homoargin- ine; O-t-butyl-L-.beta.-homohydroxyproline; O-t-butyl-L-.beta.-homoserine; O-t-butyl-L-.beta.-homothreonine; O-t-butyl-L-.beta.-homotyrosine; 2-aminocyclopentane carboxylic acid; and 2-aminocyclohexane carboxylic acid.
[0249] In some instances, amino acid analogs include analogs of alanine, valine, glycine or leucine. Examples of amino acid analogs of alanine, valine, glycine, and leucine include, but are not limited to, the following: .alpha.-methoxyglycine; .alpha.-allyl-L-alanine; .alpha.-aminoisobutyric acid; .alpha.-methyl-leucine; .beta.-(1-naphthyl)-D-alanine; .beta.-(1-naphthyl)-L-alanine; .beta.-(2-naphthyl)-D-alanine; .beta.-(2-naphthyl)-L-alanine; .beta.-(2-pyridyl)-D-alanine .beta.-(2-pyridyl)-L-alanine; .beta.-(2-thienyl)-D-alanine; .beta.-(2-thienyl)-L-alanine; .beta.-(3-benzothienyl)-D-alanine; .beta.-(3-benzothienyl)-L-alanine; .beta.-(3-pyridyl)-D-alanine; .beta.-(3-pyridyl)-L-alanine; .beta.-(4-pyridyl)-D-alanine; .beta.-(4-pyridyl)-L-alanine; .beta.-chloro-L-alanine; .beta.-cyano-L-alanin; .beta.-cyclohexyl-D-alanine; .beta.-cyclohexyl-L-alanine; .beta.-cyclopenten-1-yl-alanine; .beta.-cyclopentyl-alanine; .beta.-cyclopropyl-L-Ala-OH.dicyclohexylammonium salt; .beta.-t-butyl-D-alanine; .beta.-t-butyl-L-alanine; .gamma.-aminobutyric acid; L-.alpha.,.beta.-diaminopropionic acid; 2,4-dinitro-phenylglycine; 2,5-dihydro-D-phenylglycine; 2-amino-4,4,4-trifluorobutyric acid; 2-fluoro-phenylglycine; 3-amino-4,4,4-trifluoro-butyric acid; 3-fluoro-valine; 4,4,4-trifluoro-valine; 4,5-dehydro-L-leu-OH.dicyclohexylammonium salt; 4-fluoro-D-phenylglycine; 4-fluoro-L-phenylglycine; 4-hydroxy-D-phenylglycine; 5,5,5-trifluoro-leucine; 6-aminohexanoic acid; cyclopentyl-D-Gly-OH.dicyclohexylammonium salt; cyclopentyl-Gly-OH.dicyclohexylammonium salt; D-.alpha.,.beta.-diaminopropionic acid; D-.alpha.-aminobutyric acid; D-.alpha.-t-butylglycine; D-(2-thienyl)glycine; D-(3-thienyl)glycine; D-2-aminocaproic acid; D-2-indanylglycine; D-allylglycine-dicyclohexylammonium salt; D-cyclohexylglycine; D-norvaline; D-phenylglycine; .beta.-aminobutyric acid; .beta.-aminoisobutyric acid; (2-bromophenyl)glycine; (2-methoxyphenyl)glycine; (2-methylphenyl)glycine; (2-thiazoyl)glycine; (2-thienyl)glycine; 2-amino-3-(dimethylamino)-propionic acid; L-.alpha.,.beta.-diaminopropionic acid; L-.alpha.-aminobutyric acid; L-.alpha.-t-butylglycine; L-(3-thienyl)glycine; L-2-amino-3-(dimethylamino)-propionic acid; L-2-aminocaproic acid dicyclohexyl-ammonium salt; L-2-indanylglycine; L-allylglycine.dicyclohexyl ammonium salt; L-cyclohexylglycine; L-phenylglycine; L-propargylglycine; L-norvaline; N-.alpha.-aminomethyl-L-alanine; D-.alpha.,.gamma.-diaminobutyric acid; L-.alpha.,.gamma.-diaminobutyric acid; .beta.-cyclopropyl-L-alanine; (N-.beta.-(2,4-dinitrophenyl))-L-.alpha.,.beta.-diaminopropionic acid; (N-.beta.-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl)-D-.alpha.,4-- diaminopropionic acid; (N-.beta.-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl)-L-.alpha.,.b- eta.-diaminopropionic acid; (N-.beta.-4-methyltrityl)-L-.alpha.,.beta.-diaminopropionic acid; (N-.beta.-allyloxycarbonyl)-L-.alpha.,.beta.-diaminopropionic acid; (N-.gamma.-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl)-D-.alpha.,.- gamma.-diaminobutyric acid; (N-.gamma.-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl)-L-.alpha.,.- gamma.-diaminobutyric acid; (N-.gamma.-4-methyltrityl)-D-.alpha.,.gamma.-diaminobutyric acid; (N-.gamma.-4-methyltrityl)-L-.alpha.,.gamma.-diaminobutyric acid; (N-.gamma.-allyloxycarbonyl)-L-.alpha.,.gamma.-diaminobutyric acid; D-.alpha.,.gamma.-diaminobutyric acid; 4,5-dehydro-L-leucine; cyclopentyl-D-Gly-OH; cyclopentyl-Gly-OH; D-allylglycine; D-homocyclohexylalanine; L-1-pyrenylalanine; L-2-aminocaproic acid; L-allylglycine; L-homocyclohexylalanine; and N-(2-hydroxy-4-methoxy-Bzl)-Gly-OH.
[0250] In some instances, amino acid analogs include analogs of arginine or lysine. Examples of amino acid analogs of arginine and lysine include, but are not limited to, the following: citrulline; L-2-amino-3-guanidinopropionic acid; L-2-amino-3-ureidopropionic acid; L-citrulline; Lys(Me).sub.2-OH; Lys(N.sub.3)--OH; N.delta.-benzyloxycarbonyl-L-omithine; N.omega.-nitro-D-arginine; N.omega.-nitro-L-arginine; .alpha.-methyl-omithine; 2,6-diaminoheptanedioic acid; L-omithine; (N.delta.-1-(4,4-dimethyl-2,6-dioxo-cyclohex-1-ylidene)ethyl)-D-omithine; (N.delta.-1-(4,4-dimethyl-2,6-dioxo-cyclohex-1-ylidene)ethyl)-L-omithine; (N.delta.-4-methyltrityl)-D-omithine; (N.delta.-4-methyltrityl)-L-omithine; D-omithine; L-omithine; Arg(Me)(Pbf)-OH; Arg(Me).sub.2-OH (asymmetrical); Arg(Me)2-OH (symmetrical); Lys(ivDde)-OH; Lys(Me)2-OH.HCl; Lys(Me3)-OH chloride; N.omega.-nitro-D-arginine; and N.omega.-nitro-L-arginine.
[0251] In some instances, amino acid analogs include analogs of aspartic or glutamic acids. Examples of amino acid analogs of aspartic and glutamic acids include, but are not limited to, the following: .alpha.-methyl-D-aspartic acid; .alpha.-methyl-glutamic acid; .alpha.-methyl-L-aspartic acid; .gamma.-methylene-glutamic acid; (N-.gamma.-ethyl)-L-glutamine; [N-.alpha.-(4-aminobenzoyl)]-L-glutamic acid; 2,6-diaminopimelic acid; L-.alpha.-aminosuberic acid; D-2-aminoadipic acid; D-.alpha.-aminosuberic acid; .alpha.-aminopimelic acid; iminodiacetic acid; L-2-aminoadipic acid; threo-.beta.-methyl-aspartic acid; .gamma.-carboxy-D-glutamic acid .gamma.,.gamma.-di-t-butyl ester; .gamma.-carboxy-L-glutamic acid .gamma.,.gamma.-di-t-butyl ester; Glu(OAll)-OH; L-Asu(OtBu)-OH; and pyroglutamic acid.
[0252] In some instances, amino acid analogs include analogs of cysteine and methionine. Examples of amino acid analogs of cysteine and methionine include, but are not limited to, Cys(farnesyl)-OH, Cys(farnesyl)-OMe, .alpha.-methyl-methionine, Cys(2-hydroxyethyl)-OH, Cys(3-aminopropyl)-OH, 2-amino-4-(ethylthio)butyric acid, buthionine, buthioninesulfoximine, ethionine, methionine methylsulfonium chloride, selenomethionine, cysteic acid, [2-(4-pyridyl)ethyl]-DL-penicillamine, [2-(4-pyridyl)ethyl]-L-cysteine, 4-methoxybenzyl-D-penicillamine, 4-methoxybenzyl-L-penicillamine, 4-methylbenzyl-D-penicillamine, 4-methylbenzyl-L-penicillamine, benzyl-D-cysteine, benzyl-L-cysteine, benzyl-DL-homocysteine, carbamoyl-L-cysteine, carboxyethyl-L-cysteine, carboxymethyl-L-cysteine, diphenylmethyl-L-cysteine, ethyl-L-cysteine, methyl-L-cysteine, t-butyl-D-cysteine, trityl-L-homocysteine, trityl-D-penicillamine, cystathionine, homocystine, L-homocystine, (2-aminoethyl)-L-cysteine, seleno-L-cystine, cystathionine, Cys(StBu)-OH, and acetamidomethyl-D-penicillamine.
[0253] In some instances, amino acid analogs include analogs of phenylalanine and tyrosine. Examples of amino acid analogs of phenylalanine and tyrosine include .beta.-methyl-phenylalanine, .beta.-hydroxyphenylalanine, .alpha.-methyl-3-methoxy-DL-phenylalanine, .alpha.-methyl-D-phenylalanine, .alpha.-methyl-L-phenylalanine, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, 2,4-dichloro-phenylalanine, 2-(trifluoromethyl)-D-phenylalanine, 2-(trifluoromethyl)-L-phenylalanine, 2-bromo-D-phenylalanine, 2-bromo-L-phenylalanine, 2-chloro-D-phenylalanine, 2-chloro-L-phenylalanine, 2-cyano-D-phenylalanine, 2-cyano-L-phenylalanine, 2-fluoro-D-phenylalanine, 2-fluoro-L-phenylalanine, 2-methyl-D-phenylalanine, 2-methyl-L-phenylalanine, 2-nitro-D-phenylalanine, 2-nitro-L-phenylalanine, 2;4;5-trihydroxy-phenylalanine, 3,4,5-trifluoro-D-phenylalanine, 3,4,5-trifluoro-L-phenylalanine, 3,4-dichloro-D-phenylalanine, 3,4-dichloro-L-phenylalanine, 3,4-difluoro-D-phenylalanine, 3,4-difluoro-L-phenylalanine, 3,4-dihydroxy-L-phenylalanine, 3,4-dimethoxy-L-phenylalanine, 3,5,3'-triiodo-L-thyronine, 3,5-diiodo-D-tyrosine, 3,5-diiodo-L-tryosine, 3,5-diiodo-L-thyronine, 3-(trifluoromethyl)-D-phenylalanine, 3-(trifluoromethyl)-L-phenylalanine, 3-amino-L-tyrosine, 3-bromo-D-phenylalanine, 3-bromo-L-phenylalanine, 3-chloro-D-phenylalanine, 3-chloro-L-phenylalanine, 3-chloro-L-tyrosine, 3-cyano-D-phenylalanine, 3-cyano-L-phenylalanine, 3-fluoro-D-phenylalanine, 3-fluoro-L-phenylalanine, 3-fluoro-tyrosine, 3-iodo-D-phenylalanine, 3-iodo-L-phenylalanine, 3-iodo-L-tyrosine, 3-methoxy-L-tyrosine, 3-methyl-D-phenylalanine, 3-methyl-L-phenylalanine, 3-nitro-D-phenylalanine, 3-nitro-L-phenylalanine, 3-nitro-L-tyrosine, 4-(trifluoromethyl)-D-phenylalanine, 4-(trifluoromethyl)-L-phenylalanine, 4-amino-D-phenylalanine, 4-amino-L-phenylalanine, 4-benzoyl-D-phenylalanine, 4-benzoyl-L-phenylalanine, 4-bis(2-chloroethyl)amino-L-phenylalanine, 4-bromo-D-phenylalanine, 4-bromo-L-phenylalanine, 4-chloro-D-phenylalanine, 4-chloro-L-phenylalanine, 4-cyano-D-phenylalanine, 4-cyano-L-phenylalanine, 4-fluoro-D-phenylalanine, 4-fluoro-L-phenylalanine, 4-iodo-D-phenylalanine, 4-iodo-L-phenylalanine, homophenylalanine, thyroxine, 3,3-diphenylalanine, thyronine, ethyl-tryosine, and methyl-tyrosine.
[0254] In some instances, amino acid analogs include analogs of proline. Examples of amino acid analogs of proline include, but are not limited to, 3,4-dehydro-proline, 4-fluoro-proline, cis-4-hydroxy-proline, thiazolidine-2-carboxylic acid, and trans-4-fluoro-proline.
[0255] In some instances, amino acid analogs include analogs of serine and threonine. Examples of amino acid analogs of serine and threonine include, but are not limited to, 3-amino-2-hydroxy-5-methylhexanoic acid, 2-amino-3-hydroxy-4-methylpentanoic acid, 2-amino-3-ethoxybutanoic acid, 2-amino-3-methoxybutanoic acid, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-amino-3-benzyloxypropionic acid, 2-amino-3-benzyloxypropionic acid, 2-amino-3-ethoxypropionic acid, 4-amino-3-hydroxybutanoic acid, and .alpha.-methylserine.
[0256] In some instances, amino acid analogs include analogs of tryptophan. Examples of amino acid analogs of tryptophan include, but are not limited to, the following: .alpha.-methyl-tryptophan; .beta.-(3-benzothienyl)-D-alanine; .beta.-(3-benzothienyl)-L-alanine; 1-methyl-tryptophan; 4-methyl-tryptophan; 5-benzyloxy-tryptophan; 5-bromo-tryptophan; 5-chloro-tryptophan; 5-fluoro-tryptophan; 5-hydroxy-tryptophan, 5-hydroxy-L-tryptophan; 5-methoxy-tryptophan; 5-methoxy-L-tryptophan; 5-methyl-tryptophan; 6-bromo-tryptophan; 6-chloro-D-tryptophan; 6-chloro-tryptophan; 6-fluoro-tryptophan; 6-methyl-tryptophan; 7-benzyloxy-tryptophan; 7-bromo-tryptophan; 7-methyl-tryptophan; D-1,2,3,4-tetrahydro-norharman-3-carboxylic acid; 6-methoxy-1,2,3,4-tetrahydronorharman-1-carboxylic acid; 7-azatryptophan; L-1,2,3,4-tetrahydro-norharman-3-carboxylic acid; 5-methoxy-2-methyl-tryptophan; and 6-chloro-L-tryptophan.
[0257] In some instances, amino acid analogs are racemic. In some instances, the D isomer of the amino acid analog is used. In some cases, the L isomer of the amino acid analog is used. In some instances, the amino acid analog comprises chiral centers that are in the R or S configuration. Sometimes, the amino group(s) of a .beta.-amino acid analog is substituted with a protecting group, e.g., tert-butyloxycarbonyl (BOC group), 9-fluorenylmethyloxycarbonyl (FMOC), tosyl, and the like. Sometimes, the carboxylic acid functional group of a .beta.-amino acid analog is protected, e.g., as its ester derivative. In some cases, the salt of the amino acid analog is used.
[0258] In some embodiments, nucleic acid molecules refer to at least two nucleotides covalently linked together. In some instances, a nucleic acid described herein contains phosphodiester bonds, although in some cases, as outlined below (for example in the construction of primers and probes such as label probes), nucleic acid analogs are included that have alternate backbones, comprising, for example, phosphoramide (Beaucage et al., Tetrahedron 49(10): 1925 (1993) and references therein; Letsinger, J. Org. Chem. 35:3800 (1970); Sprinzl et al., Eur. J. Biochem. 81:579 (1977); Letsinger et al., Nucl. Acids Res. 14:3487 (1986); Sawai et al, Chem. Lett. 805 (1984), Letsinger et al., J. Am. Chem. Soc. 110:4470 (1988); and Pauwels et al., Chemica Scripta 26:141 91986)), phosphorothioate (Mag et al., Nucleic Acids Res. 19:1437 (1991); and U.S. Pat. No. 5,644,048), phosphorodithioate (Bnu et al., J. Am. Chem. Soc. 111:2321 (1989), O-methylphosphoroamidite linkages (see Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxford University Press), and peptide nucleic acid (also referred to herein as "PNA") backbones and linkages (see Egholm, J. Am. Chem. Soc. 114:1895 (1992); Meier et al., Chem. Int. Ed. Engl, 31:1008 (1992); Nielsen, Nature, 365:566 (1993): Carlsson et al., Nature 380:207 (1996), all of which are incorporated by reference). Other analog nucleic acids include those with bicyclic structures including locked nucleic acids (also referred to herein as "LNA"), Koshkin et al., J. Am. Chem. Soc. 120.13252 3 (1998); positive backbones (Denpcy et al., Proc. Natl. Acad. Sci. USA 92:6097 (1995); non-ionic backbones (U.S. Pat. Nos. 5,386,023, 5,637,684, 5,602,240, 5,216,141 and 4,469,863; Kiedrowshi et al., Angew. Chem. Intl. Ed. English 30:423 (1991); Letsinger et al., J. Am. Chem. Soc. 110:4470 (1988): Letsinger et al., Nucleoside & Nucleotide 13:1597 (1994); Chapters 2 and 3, ASC Symposium Series 580, "Carbohydrate Modifications in Antisense Research", Ed. Y. S. Sanghui and P. Dan Cook; Mesmaeker et al., Bioorganic & Medicinal Chem. Lett. 4:395 (1994); Jeffs et al., J. Biomolecular NMR 34:17 (1994); Tetrahedron Lett. 37:743 (1996)) and non-ribose backbones, including those described in U.S. Pat. Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, "Carbohydrate Modifications in Antisense Research", Ed. Y. S. Sanghui and P. Dan Cook. Nucleic acids containing one or more carbocyclic sugars are also included within the definition of nucleic acids (see Jenkins et al., Chem. Soc. Rev. (1995) pp 169 176). Several nucleic acid analogs are described in Rawls, C & E News Jun. 2, 1997 page 35. "Locked nucleic acids" are also included within the definition of nucleic acid analogs. LNAs are a class of nucleic acid analogues in which the ribose ring is "locked" by a methylene bridge connecting the 2'-O atom with the 4'-C atom. All of these references are hereby expressly incorporated by reference. In some instances, these modifications of the ribose-phosphate backbone are done to increase the stability and half-life of such molecules in physiological environments. For example, PNA:DNA and LNA-DNA hybrids exhibit higher stability and thus are used in some embodiments. The target nucleic acids are single stranded or double stranded, as specified, or contain portions of both double stranded or single stranded sequence. Depending on the application, the nucleic acids are DNA (including, e.g., genomic DNA, mitochondrial DNA, and cDNA), RNA (including, e.g., mRNA and rRNA) or a hybrid, where the nucleic acid contains any combination of deoxyribo- and ribo-nucleotides, and any combination of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xathanine hypoxathanine, isocytosine, isoguanine, etc.
Samples, Analytical Techniques, and Instrumentation
[0259] In certain embodiments, one or more of the methods disclosed herein comprise a sample. In some embodiments, the sample is a cell sample or a tissue sample. In some instances, the sample is a cell sample. In some embodiments, the sample for use with the methods described herein is obtained from cells of an animal. In some instances, the animal cell includes a cell from a marine invertebrate, fish, insects, amphibian, reptile, or mammal. In some instances, the mammalian cell is a primate, ape, equine, bovine, porcine, canine, feline, or rodent. In some instances, the mammal is a primate, ape, dog, cat, rabbit, ferret, or the like. In some cases, the rodent is a mouse, rat, hamster, gerbil, hamster, chinchilla, or guinea pig. In some embodiments, the bird cell is from a canary, parakeet or parrots. In some embodiments, the reptile cell is from a turtles, lizard or snake. In some cases, the fish cell is from a tropical fish. In some cases, the fish cell is from a zebrafish (e.g. Danino rerio). In some cases, the worm cell is from a nematode (e.g. C. elegans). In some cases, the amphibian cell is from a frog. In some embodiments, the arthropod cell is from a tarantula or hermit crab.
[0260] In some embodiments, the sample for use with the methods described herein is obtained from a mammalian cell. In some instances, the mammalian cell is an epithelial cell, connective tissue cell, hormone secreting cell, a nerve cell, a skeletal muscle cell, a blood cell, or an immune system cell.
[0261] Exemplary mammalian cells include, but are not limited to, 293A cell line, 293FT cell line, 293F cells, 293 H cells, HEK 293 cells, CHO DG44 cells, CHO-S cells, CHO-K1 cells, Expi293F.TM. cells, Flp-In.TM. T-REx.TM. 293 cell line, Flp-In.TM.-293 cell line, Flp-In.TM.-3T3 cell line, Flp-In.TM.-BHK cell line, Flp-In.TM.-CHO cell line, Flp-In.TM.-CV-1 cell line, Flp-In.TM.-Jurkat cell line, FreeStyle.TM. 293-F cells, FreeStyle.TM. CHO-S cells, GripTite.TM. 293 MSR cell line, GS-CHO cell line, HepaRG.TM. cells, T-REx.TM. Jurkat cell line, Per.C6 cells, T-REx.TM.-293 cell line, T-REx.TM.-CHO cell line, T-REx.TM.-HeLa cell line, NC-HIMT cell line, and PC12 cell line.
[0262] In some instances, the sample for use with the methods described herein is obtained from cells of a tumor cell line. In some instances, the sample is obtained from cells of a solid tumor cell line. In some instances, the solid tumor cell line is a sarcoma cell line. In some instances, the solid tumor cell line is a carcinoma cell line. In some embodiments, the sarcoma cell line is obtained from a cell line of alveolar rhabdomyosarcoma, alveolar soft part sarcoma, ameloblastoma, angiosarcoma, chondrosarcoma, chordoma, clear cell sarcoma of soft tissue, dedifferentiated liposarcoma, desmoid, desmoplastic small round cell tumor, embryonal rhabdomyosarcoma, epithelioid fibrosarcoma, epithelioid hemangioendothelioma, epithelioid sarcoma, esthesioneuroblastoma, Ewing sarcoma, extrarenal rhabdoid tumor, extraskeletal myxoid chondrosarcoma, extraskeletal osteosarcoma, fibrosarcoma, giant cell tumor, hemangiopericytoma, infantile fibrosarcoma, inflammatory myofibroblastic tumor, Kaposi sarcoma, leiomyosarcoma of bone, liposarcoma, liposarcoma of bone, malignant fibrous histiocytoma (MFH), malignant fibrous histiocytoma (MFH) of bone, malignant mesenchymoma, malignant peripheral nerve sheath tumor, mesenchymal chondrosarcoma, myxofibrosarcoma, myxoid liposarcoma, myxoinflammatory fibroblastic sarcoma, neoplasms with perivascular epitheioid cell differentiation, osteosarcoma, parosteal osteosarcoma, neoplasm with perivascular epitheioid cell differentiation, periosteal osteosarcoma, pleomorphic liposarcoma, pleomorphic rhabdomyosarcoma, PNET/extraskeletal Ewing tumor, rhabdomyosarcoma, round cell liposarcoma, small cell osteosarcoma, solitary fibrous tumor, synovial sarcoma, telangiectatic osteosarcoma.
[0263] In some embodiments, the carcinoma cell line is obtained from a cell line of adenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma, anaplastic carcinoma, large cell carcinoma, small cell carcinoma, anal cancer, appendix cancer, bile duct cancer (i.e., cholangiocarcinoma), bladder cancer, brain tumor, breast cancer, cervical cancer, colon cancer, cancer of Unknown Primary (CUP), esophageal cancer, eye cancer, fallopian tube cancer, gastroenterological cancer, kidney cancer, liver cancer, lung cancer, medulloblastoma, melanoma, oral cancer, ovarian cancer, pancreatic cancer, parathyroid disease, penile cancer, pituitary tumor, prostate cancer, rectal cancer, skin cancer, stomach cancer, testicular cancer, throat cancer, thyroid cancer, uterine cancer, vaginal cancer, or vulvar cancer.
[0264] In some instances, the sample is obtained from cells of a hematologic malignant cell line. In some instances, the hematologic malignant cell line is a T-cell cell line. In some instances, B-cell cell line. In some instances, the hematologic malignant cell line is obtained from a T-cell cell line of: peripheral T-cell lymphoma not otherwise specified (PTCL-NOS), anaplastic large cell lymphoma, angioimmunoblastic lymphoma, cutaneous T-cell lymphoma, adult T-cell leukemia/lymphoma (ATLL), blastic NK-cell lymphoma, enteropathy-type T-cell lymphoma, hematosplenic gamma-delta T-cell lymphoma, lymphoblastic lymphoma, nasal NK/T-cell lymphomas, or treatment-related T-cell lymphomas.
[0265] In some instances, the hematologic malignant cell line is obtained from a B-cell cell line of: acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), acute monocytic leukemia (AMoL), chronic lymphocytic leukemia (CLL), high-risk chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), high-risk small lymphocytic lymphoma (SLL), follicular lymphoma (FL), mantle cell lymphoma (MCL), Waldenstrom's macroglobulinemia, multiple myeloma extranodal marginal zone B cell lymphoma, nodal marginal zone B cell lymphoma. Burkitt's lymphoma, non-Burkitt high grade B cell lymphoma, primary mediastinal B-cell lymphoma (PMBL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma. B cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, mediastinal (thymic) large B cell lymphoma, intravascular large B cell lymphoma, primary effusion lymphoma, or lymphomatoid granulomatosis.
[0266] In some embodiments, the sample for use with the methods described herein is obtained from a tumor cell line. Exemplary tumor cell line includes, but is not limited to, 600MPE, AU565, BT-20, BT-474, BT-483, BT-549, Evsa-T, Hs578T, MCF-7, MDA-MB-231, SkBr3, T-47D, HeLa, DU 145, PC3, LNCaP, A549, H1299, NCI-H460, A2780, SKOV-3/Luc, Neuro2a, RKO, RKO-AS45-1. HT-29, SW1417, SW948, DLD-1, SW480, Capan-1, MC/9, B72.3, B25.2, B6.2, B38.1, DMS153, SU.86.86, SNU-182, SNU-423, SNU-449, SNU-475, SNU-387, Hs 817.T, LMH, LMH/2A, SNU-398, PLHC-1, HepG2/SF, OCI-Ly1, OCI-Ly2, OCI-Ly3, OCI-Ly4, OCI-Ly6, OCI-Ly7, OCI-Ly10, OCI-Ly18, OCI-Ly19, U2932, DB, HBL-1, RIVA, SUDHL2, TMD8, MEC1, MEC2, 8E5, CCRF-CEM, MOLT-3, TALL-104, AML-193, THP-1, BDCM, HL-60, Jurkat, RPMI 8226, MOLT-4, RS4, K-562, KASUMI-1, Daudi, GA-10, Raji, JeKo-1, NK-92, and Mino.
[0267] In some embodiments, the sample for use in the methods is from any tissue or fluid from an individual. Samples include, but are not limited to, tissue (e.g. connective tissue, muscle tissue, nervous tissue, or epithelial tissue), whole blood, dissociated bone marrow, bone marrow aspirate, pleural fluid, peritoneal fluid, central spinal fluid, abdominal fluid, pancreatic fluid, cerebrospinal fluid, brain fluid, ascites, pericardial fluid, urine, saliva, bronchial lavage, sweat, tears, ear flow, sputum, hydrocele fluid, semen, vaginal flow, milk, amniotic fluid, and secretions of respiratory, intestinal or genitourinary tract. In some embodiments, the sample is a tissue sample, such as a sample obtained from a biopsy or a tumor tissue sample. In some embodiments, the sample is a blood serum sample. In some embodiments, the sample is a blood cell sample containing one or more peripheral blood mononuclear cells (PBMCs). In some embodiments, the sample contains one or more circulating tumor cells (CTCs). In some embodiments, the sample contains one or more disseminated tumor cells (DTC, e.g., in a bone marrow aspirate sample).
[0268] In some embodiments, the samples are obtained from the individual by any suitable means of obtaining the sample using well-known and routine clinical methods. Procedures for obtaining tissue samples from an individual are well known. For example, procedures for drawing and processing tissue sample such as from a needle aspiration biopsy is well-known and is employed to obtain a sample for use in the methods provided. Typically, for collection of such a tissue sample, a thin hollow needle is inserted into a mass such as a tumor mass for sampling of cells that, after being stained, will be examined under a microscope.
Sample Preparation and Analysis
[0269] In some embodiments, the sample is a sample solution. In some instances, the sample solution comprises a solution such as a buffer (e.g. phosphate buffered saline) or a media. In some embodiments, the media is an isotopically labeled media. In some instances, the sample solution is a cell solution.
[0270] In some embodiments, the sample (e.g., cells or a cell solution) is incubated with a cysteine-reactive probe for analysis of protein cysteine-reactive probe interactions. In some instances, the sample (e.g., cells or a cell solution) is further incubated in the presence of a small molecule fragment prior to addition of the cysteine-reactive probe. In some instances, the sample is compared with a control. In some instances, the control comprises the cysteine-reactive probe but not the small molecule fragment. In some instances, a difference is observed between a set of cysteine-reactive probe protein interactions between the sample and the control. In some instances, the difference correlates to the interaction between the small molecule fragment and the cysteine containing proteins.
[0271] In some embodiments, the sample (e.g. cells or a cell solution) is further labeled for analysis of cysteine-reactive probe protein interactions. In some instances, the sample (e.g. cells or a cell solution) is labeled with an enriched media. In some cases, the sample (e.g. cells or a cell solution) is labeled with isotope-labeled amino acids, such as .sup.13C or .sup.15N-labeled amino acids. In some cases, the labeled sample is further compared with a non-labeled sample to detect differences in cysteine-reactive probe protein interactions between the two samples. In some instances, this difference is a difference of a cysteine containing protein and its interaction with a small molecule fragment in the labeled sample versus the non-labeled sample. In some instances, the difference is an increase, decrease or a lack of protein cysteine-reactive probe interaction in the two samples. In some instances, the isotope-labeled method is termed SILAC, stable isotope labeling using amino acids in cell culture.
[0272] In some instances, the sample is divided into a first cell solution and a second cell solution. In some cases, the first cell solution is incubated with a small molecule fragment for an extended period of time prior to incubating the first cell solution with a first cysteine-reactive probe to generate a first group of cysteine-reactive probe-protein complexes. In some instances, the extended period of time is about 5, 10, 15, 20, 30, 60, 90, 120 minutes or longer. In some instances, the second cell solution comprises a second cysteine-reactive probe to generate a second group of cysteine-reactive probe-protein complexes. In some instances, the first cysteine-reactive probe and the second cysteine-reactive probe are the same. In some embodiments, cells from the second cell solution are further treated with a buffer, such as a control buffer, in which the buffer does not contain a small molecule fragment. In some embodiments, the control buffer comprises dimethyl sulfoxide (DMSO).
[0273] In some embodiments, the cysteine-reactive probe-protein complex is further conjugated to a chromophore, such as a fluorophore. In some instances, the cysteine-reactive probe-protein complex is separated and visualized utilizing an electrophoresis system, such as through a gel clectrophoresis, or a capillary electrophoresis. Exemplary gel electrophoresis includes agarose based gels, polyacrylamide based gels, or starch based gels. In some instances, the cysteine-reactive probe-protein is subjected to a native electrophoresis condition. In some instances, the cysteine-reactive probe-protein is subjected to a denaturing electrophoresis condition.
[0274] In some instances, the cysteine-reactive probe-protein after harvesting is further fragmentized to generate protein fragments. In some instances, fragmentation is generated through mechanical stress, pressure, or chemical means. In some instances, the protein from the cysteine-reactive probe-protein complexes is fragmented by a chemical means. In some embodiments, the chemical means is a protease. Exemplary proteases include, but are not limited to, serine proteases such as chymotrypsin A, penicillin G acylase precursor, dipeptidase E, DmpA aminopeptidase, subtilisin, prolyl oligopeptidase, D-Ala-D-Ala peptidase C, signal peptidase I, cytomegalovirus assemblin, Lon-A peptidase, peptidase Clp, Escherichia coli phage KIF endosialidase CIMCD self-cleaving protein, nucleoporin 145, lactoferrin, murein tetrapeptidase LD-carboxypeptidase, or rhomboid-1; threonine proteases such as omithine acetyltransferase; cysteine proteases such as TEV protease, amidophosphoribosyltransferase precursor, gamma-glutamyl hydrolase (Rattus norvegicus), hedgehog protein. DmpA aminopeptidase, papain, bromelain, cathepsin K, calpain, caspase-1, separase, adenain, pyroglutamyl-peptidase I, sortase A, hepatitis C virus peptidase 2, sindbis virus-type nsP2 peptidase, dipeptidyl-peptidase VI, or DeSI-1 peptidase; aspartate proteases such as beta-secretase 1 (BACE), beta-secretase 2 (BACE2), cathepsin D, cathepsin E, chymosin, napsin-A, nepenthesin, pepsin, plasmepsin, presenilin, or renin; glutamic acid proteases such as AfuGprA; and metalloproteases such as peptidase_M48.
[0275] In some instances, the fragmentation is a random fragmentation. In some instances, the fragmentation generates specific lengths of protein fragments, or the shearing occurs at particular sequence of amino acid regions.
[0276] In some instances, the protein fragments are further analyzed by a proteomic method such as by liquid chromatography (LC) (e.g. high performance liquid chromatography), liquid chromatography-mass spectrometry (LC-MS), matrix-assisted laser desorption/ionization (MALDI-TOF), gas chromatography-mass spectrometry (GC-MS), capillary electrophoresis-mass spectrometry (CE-MS), or nuclear magnetic resonance imaging (NMR).
[0277] In some embodiments, the LC method is any suitable LC methods well known in the art, for separation of a sample into its individual parts. This separation occurs based on the interaction of the sample with the mobile and stationary phases. Since there are many stationary/mobile phase combinations that are employed when separating a mixture, there are several different types of chromatography that are classified based on the physical states of those phases. In some embodiments, the LC is further classified as normal-phase chromatography, reverse-phase chromatography, size-exclusion chromatography, ion-exchange chromatography, affinity chromatography, displacement chromatography, partition chromatography, flash chromatography, chiral chromatography, and aqueous normal-phase chromatography.
[0278] In some embodiments, the LC method is a high performance liquid chromatography (HPLC) method. In some embodiments, the HPLC method is further categorized as normal-phase chromatography, reverse-phase chromatography, size-exclusion chromatography, ion-exchange chromatography, affinity chromatography, displacement chromatography, partition chromatography, chiral chromatography, and aqueous normal-phase chromatography.
[0279] In some embodiments, the HPLC method of the present disclosure is performed by any standard techniques well known in the art. Exemplary HPLC methods include hydrophilic interaction liquid chromatography (HILIC), electrostatic repulsion-hydrophilic interaction liquid chromatography (ERLIC) and reverse phase liquid chromatography (RPLC).
[0280] In some embodiments, the LC is coupled to a mass spectroscopy as a LC-MS method. In some embodiments, the LC-MS method includes ultra-performance liquid chromatography-electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC-ESI-QTOF-MS), ultra-performance liquid chromatography-electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS), reverse phase liquid chromatography-mass spectrometry (RPLC-MS), hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS), hydrophilic interaction liquid chromatography-triple quadrupole tandem mass spectrometry (HILIC-QQQ), electrostatic repulsion-hydrophilic interaction liquid chromatography-mass spectrometry (ERLIC-MS), liquid chromatography time-of-flight mass spectrometry (LC-QTOF-MS), liquid chromatography-tandem mass spectrometry (LC-MS/MS), multidimensional liquid chromatography coupled with tandem mass spectrometry (LC/LC-MS/MS). In some instances, the LC-MS method is LC/LC-MS/MS. In some embodiments, the LC-MS methods of the present disclosure are performed by standard techniques well known in the art.
[0281] In some embodiments, the GC is coupled to a mass spectroscopy as a GC-MS method. In some embodiments, the GC-MS method includes two-dimensional gas chromatography time-of-flight mass spectrometry (GC*GC-TOFMS), gas chromatography time-of-flight mass spectrometry (GC-QTOF-MS) and gas chromatography-tandem mass spectrometry (GC-MS/MS).
[0282] In some embodiments, CE is coupled to a mass spectroscopy as a CE-MS method. In some embodiments, the CE-MS method includes capillary electrophoresis-negative electrospray ionization-mass spectrometry (CE-ESI-MS), capillary electrophoresis-negative electrospray ionization-quadrupole time of flight-mass spectrometry (CE-ESI-QTOF-MS) and capillary electrophoresis-quadrupole time of flight-mass spectrometry (CE-QTOF-MS).
[0283] In some embodiments, the nuclear magnetic resonance (NMR) method is any suitable method well known in the art for the detection of one or more cysteine binding proteins or protein fragments disclosed herein. In some embodiments, the NMR method includes one dimensional (ID) NMR methods, two dimensional (2D) NMR methods, solid state NMR methods and NMR chromatography. Exemplary ID NMR methods include .sup.1Hydrogen, .sup.13Carbon, .sup.15Nitrogen, .sup.17Oxygen, .sup.19Fluorine, .sup.31Phosphorus, .sup.39Potassium, .sup.23Sodium, .sup.33Sulfur, .sup.87Strontium, .sup.27Aluminium, .sup.43Calcium, .sup.35Chlorine, .sup.37Chlorine, .sup.63Copper, .sup.65Copper, .sup.57Iron, .sup.25Magnesium, .sup.199Mercury or .sup.67Zinc NMR method, distortionless enhancement by polarization transfer (DEPT) method, attached proton test (APT) method and ID-incredible natural abundance double quantum transition experiment (INADEQUATE) method. Exemplary 2D NMR methods include correlation spectroscopy (COSY), total correlation spectroscopy (TOCSY), 2D-INADEQUATE, 2D-adequate double quantum transfer experiment (ADEQUATE), nuclear overhauser effect spectroscopy (NOSEY), rotating-frame NOE spectroscopy (ROESY), heteronuclear multiple-quantum correlation spectroscopy (HMQC), heteronuclear single quantum coherence spectroscopy (HSQC), short range coupling and long range coupling methods. Exemplary solid state NMR method include solid state .sup.13Carbon NMR, high resolution magic angle spinning (HR-MAS) and cross polarization magic angle spinning (CP-MAS) NMR methods. Exemplary NMR techniques include diffusion ordered spectroscopy (DOSY), DOSY-TOCSY and DOSY-HSQC.
[0284] In some embodiments, the protein fragments are analyzed by method as described in Weerapana et al., "Quantitative reactivity profiling predicts functional cysteines in proteomes," Nature, 468:790-795 (2010).
[0285] In some embodiments, the results from the mass spectroscopy method are analyzed by an algorithm for protein identification. In some embodiments, the algorithm combines the results from the mass spectroscopy method with a protein sequence database for protein identification. In some embodiments, the algorithm comprises ProLuCID algorithm, Probity, Scaffold, SEQUEST, or Mascot.
[0286] In some embodiments, a value is assigned to each of the protein from the cystein-reactive probe-protein complex. In some embodiments, the value assigned to each of the protein from the cysteine-reactive probe-protein complex is obtained from the mass spectroscopy analysis. In some instances, the value is the area-under-the curve from a plot of signal intensity as a function of mass-to-charge ratio. In some embodiments, a first value is assigned to the protein obtained from the first cell solution and a second value is assigned to the same protein obtained from the second cell solution. In some instances, a ratio is calculated between the two values. In some instances, a ratio of greater than 2 indicates that the protein is a candidate for interacting with a drug or that the protein is a cysteine binding protein. In some instances, the ratio is greater than 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some cases, the ratio is at most 20.
[0287] In some instances, the ratio is calculated based on averaged values. In some instances, the averaged value is an average of at least two, three, or four values of the protein from each cell solution, or that the protein is observed at least two, three, or four times in each cell solution and a value is assigned to each observed time. In some instances, the ratio further has a standard deviation of less than 12, 10, or 8.
[0288] In some instances, a value is not an averaged value. In some instances, the ratio is calculated based on value of a protein observed only once in a cell population. In some instances, the ratio is assigned with a value of 20.
[0289] In some embodiments, in the context of identifying a cysteine containing protein as a small fragment molecule binding target, a first ratio is obtained from two cell solutions in which both cell solutions have been incubated with a cysteine-reactive probe and the first cell solution is further incubated with a small molecule fragment. In some instances, the first ratio is further compared to a second ratio in which both cell solutions have been treated by cysteine-reactive probes in the absence of a small molecule fragment. In some instances, the first ratio is greater than 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some instances, the second ratio is greater than 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some instances, if the first ratio is greater than 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 and the second ratio is from about 0.5 to about 2, the two ratios indicate that a protein is a drug binding target.
[0290] In some embodiments, the value further enables calculating a percentage of inhibition of the cysteine-reactive probe to the cysteine containing protein. In some embodiments, the percentage of inhibition of greater than 50%, 60%, 70%, 80%, 90%, or at 100% indicates that the cysteine containing protein is a candidate for interacting with the small molecule fragment.
Kits/Article of Manufacture
[0291] Disclosed herein, in certain embodiments, are kits and articles of manufacture for use with one or more methods described herein. In some embodiments, described herein is a kit for identifying a cysteine containing protein as a small molecule fragment binding target. In some instances, also described herein is a kit for mapping binding sites on a cysteine containing protein. In some cases, described herein is a kit for identifying cysteine binding proteins. In some embodiments, also described herein is a kit for a high throughput screening of a small molecule fragment for interaction with a cysteine containing protein.
[0292] In some embodiments, such kit includes cysteine-reactive probes such as the cysteine-reactive probes described herein, test compounds such as small molecule fragments or libraries and/or controls, and reagents suitable for carrying out one or more of the methods described herein. In some instances, the kit further comprises samples, such as a cell sample, and suitable solutions such as buffers or media. In some embodiments, the kit further comprises recombinant proteins for use in one or more of the methods described herein. In some embodiments, additional components of the kit comprises a carrier, package, or container that is compartmentalized to receive one or more containers such as vials, tubes, and the like, each of the container(s) comprising one of the separate elements to be used in a method described herein. Suitable containers include, for example, bottles, vials, plates, syringes, and test tubes. In one embodiment, the containers are formed from a variety of materials such as glass or plastic.
[0293] The articles of manufacture provided herein contain packaging materials. Examples of pharmaceutical packaging materials include, but are not limited to, bottles, tubes, bags, containers, and any packaging material suitable for a selected formulation and intended mode of use.
[0294] For example, the container(s) include cysteine-reactive probes, test compounds, and one or more reagents for use in a method disclosed herein. Such kits optionally include an identifying description or label or instructions relating to its use in the methods described herein.
[0295] A kit typically includes labels listing contents and/or instructions for use, and package inserts with instructions for use. A set of instructions will also typically be included.
[0296] In one embodiment, a label is on or associated with the container. In one embodiment, a label is on a container when letters, numbers or other characters forming the label are attached, molded or etched into the container itself; a label is associated with a container when it is present within a receptacle or carrier that also holds the container, e.g., as a package insert. In one embodiment, a label is used to indicate that the contents are to be used for a specific therapeutic application. The label also indicates directions for use of the contents, such as in the methods described herein.
Services
[0297] In some embodiments, the methods provided herein also perform as a service. In some instances, a service provider obtain from the customer a plurality of small molecule fragment candidates for analysis with one or more of the cysteine-reactive probes for screening. In some embodiments, the service provider screens the small molecule fragment candidates using one or more of the methods described herein, and then provide the results to the customer. In some instances, the service provider provides the appropriate reagents to the customer for analysis utilizing one or more of the cysteine-reactive probes and one or more of the methods described herein. In some cases, the customer performs one or more of the methods described herein and then provide the results to the service provider for analysis. In some embodiments, the service provider then analyzes the results and provides the results to the costumer. In some cases, the customer further analyze the results by interacting with software installed locally (at the customer's location) or remotely (e.g., on a server reachable through a network). Exemplary customers include pharmaceutical companies, clinical laboratories, physicians, patients, and the like. In some instances, a customer is any suitable customer or party with a need or desire to use the methods, systems, compositions, and kits described herein.
Digital Processing Device
[0298] In some embodiments, the methods described herein include a digital processing device, or use of the same. In further embodiments, the digital processing device includes one or more hardware central processing units (CPU) that carry out the device's functions. In still further embodiments, the digital processing device further comprises an operating system configured to perform executable instructions. In some embodiments, the digital processing device is optionally connected to a computer network. In further embodiments, the digital processing device is optionally connected to the Internet such that it accesses the World Wide Web. In still further embodiments, the digital processing device is optionally connected to a cloud computing infrastructure. In other embodiments, the digital processing device is optionally connected to an intranet. In other embodiments, the digital processing device is optionally connected to a data storage device.
[0299] In accordance with the description herein, suitable digital processing devices include, by are not limited to, server computers, desktop computers, laptop computers, notebook computers, subnotebook computers, netbook computers, netpad computers, set-top computers, media streaming devices, handheld computers, Internet appliances, mobile smartphones, tablet computers, personal digital assistants, video game consoles, and vehicles. Suitable tablet computers include those with booklet, slate, or convertible configurations.
[0300] In some embodiments, the digital processing device includes an operating system configured to perform executable instructions. The operating system is, for example, software, including programs and data, which manages the device's hardware and provides services for execution of applications. Suitable server operating systems include, by way of non-limiting examples. FreeBSD, OpenBSD, NetBSD.RTM., Linux, Apple.RTM. Mac OS X Server.RTM., Oracle.RTM. Solaris.RTM., Windows Server.RTM., and Novell.RTM. NetWare.RTM.. Suitable personal computer operating systems include, by way of non-limiting examples, Microsoft.RTM. Windows.RTM., Apple.RTM. Mac OS X.RTM., UNIX.RTM., and UNIX-like operating systems such as GNU/Linux.RTM.. In some embodiments, the operating system is provided by cloud computing. Suitable mobile smart phone operating systems include, by way of non-limiting examples, Nokia.RTM. Symbian.RTM. OS, Apple.RTM. iOS.RTM., Research In Motion.RTM. BlackBerry OS.RTM., Google.RTM. Android.RTM., Microsoft.RTM. Windows Phone.RTM. OS, Microsoft.RTM. Windows Mobile.RTM. OS, Linux.RTM., and Palm.RTM. WebOS.RTM.. Suitable media streaming device operating systems include, by way of non-limiting examples, Apple TV.RTM., Roku.RTM., Boxee.RTM., Google TV.RTM., Google Chromecast.RTM., Amazon Fire.RTM., and Samsung.RTM. HomeSync.RTM.. Suitable video game console operating systems include, by way of non-limiting examples, Sony.RTM. PS3.RTM., Sony.RTM. PS4.RTM., Microsoft.RTM. Xbox 360.RTM., Microsoft Xbox One, Nintendo.RTM. Wii.RTM., Nintendo.RTM. Wii U.RTM., and Ouya.RTM..
[0301] In some embodiments, the device includes a storage and/or memory device. The storage and/or memory device is one or more physical apparatuses used to store data or programs on a temporary or permanent basis. In some embodiments, the device is volatile memory and requires power to maintain stored information. In some embodiments, the device is non-volatile memory and retains stored information when the digital processing device is not powered. In further embodiments, the non-volatile memory comprises flash memory. In some embodiments, the non-volatile memory comprises dynamic random-access memory (DRAM). In some embodiments, the non-volatile memory comprises ferroelectric random access memory (FRAM). In some embodiments, the non-volatile memory comprises phase-change random access memory (PRAM). In other embodiments, the device is a storage device including, by way of non-limiting examples, CD-ROMs, DVDs, flash memory devices, magnetic disk drives, magnetic tapes drives, optical disk drives, and cloud computing based storage. In further embodiments, the storage and/or memory device is a combination of devices such as those disclosed herein.
[0302] In some embodiments, the digital processing device includes a display to send visual information to a user. In some embodiments, the display includes a cathode ray tube (CRT), a liquid crystal display (LCD), a thin film transistor liquid crystal display (TFT-LCD), an organic light emitting diode (OLED) display, a plasma display, a video projector, or a combination thereof.
[0303] In some embodiments, the digital processing device includes an input device to receive information from a user. In some embodiments, the input device is a keyboard. In some embodiments, the input device is a pointing device including, by way of non-limiting examples, a mouse, trackball, track pad, joystick, game controller, or stylus. In some embodiments, the input device is a touch screen or a multi-touch screen. In other embodiments, the input device is a microphone to capture voice or other sound input. In other embodiments, the input device is a video camera or other sensor to capture motion or visual input. In further embodiments, the input device is a Kinect.TM., Leap Motion.TM., or the like. In still further embodiments, the input device is a combination of devices such as those disclosed herein.
[0304] In some embodiments, the systems and methods disclosed herein include one or more non-transitory computer readable storage media encoded with a program including instructions executable by the operating system of an optionally networked digital processing device. In further embodiments, a computer readable storage medium is a tangible component of a digital processing device. In still further embodiments, a computer readable storage medium is optionally removable from a digital processing device. In some embodiments, a computer readable storage medium includes, by way of non-limiting examples, CD-ROMs, DVDs, flash memory devices, solid state memory, magnetic disk drives, magnetic tape drives, optical disk drives, cloud computing systems and services, and the like. In some cases, the program and instructions are permanently, substantially permanently, semi-permanently, or non-transitorily encoded on the media.
[0305] In some embodiments, the systems and methods disclosed herein include at least one computer program, or use of the same. A computer program includes a sequence of instructions, executable in the digital processing device's CPU, written to perform a specified task. In some embodiments, computer readable instructions are implemented as program modules, such as functions, objects, Application Programming Interfaces (APIs), data structures, and the like, that perform particular tasks or implement particular abstract data types.
[0306] In some embodiments, the functionality of the computer readable instructions are combined or distributed as desired in various environments. In some embodiments, a computer program comprises one sequence of instructions. In some embodiments, a computer program comprises a plurality of sequences of instructions. In some embodiments, a computer program is provided from one location. In other embodiments, a computer program is provided from a plurality of locations. In various embodiments, a computer program includes one or more software modules. In various embodiments, a computer program includes, in part or in whole, one or more web applications, one or more mobile applications, one or more standalone applications, one or more web browser plug-ins, extensions, add-ins, or add-ons, or combinations thereof.
[0307] In some embodiments, a computer program includes a web application. A web application, in various embodiments, utilizes one or more software frameworks and one or more database systems. In some embodiments, a web application is created upon a software framework such as Microsoft.RTM. .NET or Ruby on Rails (RoR). In some embodiments, a web application utilizes one or more database systems including, by way of non-limiting examples, relational, non-relational, object oriented, associative, and XML database systems. In further embodiments, suitable relational database systems include, by way of non-limiting examples, Microsoft.RTM. SQL Server, mySQL.TM., and Oracle.RTM.. A web application, in various embodiments, is written in one or more versions of one or more languages. In some embodiments, a web application is written in one or more markup languages, presentation definition languages, client-side scripting languages, server-side coding languages, database query languages, or combinations thereof. In some embodiments, a web application is written to some extent in a markup language such as Hypertext Markup Language (HTML). Extensible Hypertext Markup Language (XHTML), or eXtensible Markup Language (XML). In some embodiments, a web application is written to some extent in a presentation definition language such as Cascading Style Sheets (CSS). In some embodiments, a web application is written to some extent in a client-side scripting language such as Asynchronous Javascript and XML (AJAX), Flash.RTM. Actionscript, Javascript, or Silverlight.RTM.. In some embodiments, a web application is written to some extent in a server-side coding language such as Active Server Pages (ASP), ColdFusion.RTM., Perl, Java.TM., JavaServer Pages (JSP), Hypertext Preprocessor (PHP), Python.TM., Ruby, Tel, Smalltalk, WebDNA.RTM., or Groovy. In some embodiments, a web application is written to some extent in a database query language such as Structured Query Language (SQL). In some embodiments, a web application integrates enterprise server products such as IBM.RTM. Lotus Domino.RTM.. In some embodiments, a web application includes a media player element. In various further embodiments, a media player element utilizes one or more of many suitable multimedia technologies including, by way of non-limiting examples, Adobe.RTM. Flash.RTM., HTML 5, Apple.RTM. QuickTime.RTM., Microsoft.RTM. Silverlight.RTM., Java.TM., and Unity.RTM..
[0308] In some embodiments, a computer program includes a mobile application provided to a mobile digital processing device. In some embodiments, the mobile application is provided to a mobile digital processing device at the time it is manufactured. In other embodiments, the mobile application is provided to a mobile digital processing device via the computer network described herein.
[0309] In view of the disclosure provided herein, a mobile application is created by techniques using hardware, languages, and development environments. Suitable programming languages include, by way of non-limiting examples, C, C++, C #, Objective-C, Java.TM., Javascript, Pascal, Object Pascal, Python.TM., Ruby, VB.NET, WML, and XHTML/HTML with or without CSS, or combinations thereof.
[0310] Suitable mobile application development environments are available from several sources. Commercially available development environments include, by way of non-limiting examples, AirplaySDK, alcheMo, Appcelerator.RTM., Celsius, Bedrock, Flash Lite, .NET Compact Framework, Rhomobile, and WorkLight Mobile Platform. Other development environments are available without cost including, by way of non-limiting examples, Lazarus, MobiFlex, MoSync, and Phonegap. Also, mobile device manufacturers distribute software developer kits including, by way of non-limiting examples, iPhone and iPad (iOS) SDK, Android.TM. SDK, BlackBerry.RTM. SDK, BREW SDK, Palm.RTM. OS SDK, Symbian SDK, webOS SDK, and Windows.RTM. Mobile SDK.
[0311] In some embodiments, commercial forums for distribution of mobile applications include, by way of non-limiting examples, Apple.RTM. App Store, Android.TM. Market, BlackBerry.RTM. App World, App Store for Palm devices, App Catalog for webOS, Windows.RTM. Marketplace for Mobile, Ovi Store for Nokia.RTM. devices, Samsung.RTM. Apps, and Nintendo.RTM. DSi Shop.
[0312] In some embodiments, a computer program includes a standalone application, which is a program that is run as an independent computer process, not an add-on to an existing process, e.g., not a plug-in. In some instances, standalone applications are compiled. A compiler is a computer program(s) that transforms source code written in a programming language into binary object code such as assembly language or machine code. Suitable compiled programming languages include, by way of non-limiting examples, C, C++, Objective-C, COBOL, Delphi, Eiffel, Java.TM., Lisp, Python.TM., Visual Basic, and VB .NET, or combinations thereof. Compilation is often performed, at least in part, to create an executable program. In some embodiments, a computer program includes one or more executable complied applications.
[0313] In some embodiments, the computer program includes a web browser plug-in. In computing, a plug-in is one or more software components that add specific functionality to a larger software application. Makers of software applications support plug-ins to enable third-party developers to create abilities which extend an application, to support easily adding new features, and to reduce the size of an application. When supported, plug-ins enable customizing the functionality of a software application. For example, plug-ins are commonly used in web browsers to play video, generate interactivity, scan for viruses, and display particular file types. In some instances, web browser plug-ins include Adobe.RTM. Flash.RTM. Player, Microsoft.RTM. Silverlight.RTM., and Apple.RTM. QuickTime.RTM.. In some embodiments, the toolbar comprises one or more web browser extensions, add-ins, or add-ons. In some embodiments, the toolbar comprises one or more explorer bars, tool bands, or desk bands.
[0314] In view of the disclosure provided herein, plug-in frameworks are available that enable development of plug-ins in various programming languages, including, by way of non-limiting examples, C++, Delphi, Java.TM., PHP, Python.TM., and VB .NET, or combinations thereof.
[0315] Web browsers (also called Internet browsers) are software applications, designed for use with network-connected digital processing devices, for retrieving, presenting, and traversing information resources on the World Wide Web. Suitable web browsers include, by way of non-limiting examples, Microsoft.RTM. Internet Explorer, Mozilla.RTM. Firefox.RTM., Google.RTM. Chrome, Apple.RTM. Safari.RTM., Opera Software.RTM. Opera.RTM., and KDE Konqueror. In some embodiments, the web browser is a mobile web browser. Mobile web browsers (also called mircrobrowsers, mini-browsers, and wireless browsers) are designed for use on mobile digital processing devices including, by way of non-limiting examples, handheld computers, tablet computers, netbook computers, subnotebook computers, smartphones, music players, personal digital assistants (PDAs), and handheld video game systems. Suitable mobile web browsers include, by way of non-limiting examples, Google.RTM. Android.RTM. browser, RIM Blackberry.RTM. Browser, Apple.RTM. Safari.RTM., Palm.RTM. Blazer, Palm.RTM. WebOS.RTM. Browser, Mozilla.RTM. Firefox.RTM. for mobile, Microsoft.RTM. Internet Explorer.RTM. Mobile, Amazon.RTM. Kindle.RTM. Basic Web, Nokia.RTM. Browser, Opera Software.RTM. Opera.RTM. Mobile, and Sony.RTM. PSP.TM. browser.
[0316] In some embodiments, the systems and methods disclosed herein include software, server, and/or database modules, or use of the same. In view of the disclosure provided herein, software modules are created and implemented in a multitude of ways. In various embodiments, a software module comprises a file, a section of code, a programming object, a programming structure, or combinations thereof. In further various embodiments, a software module comprises a plurality of files, a plurality of sections of code, a plurality of programming objects, a plurality of programming structures, or combinations thereof. In various embodiments, the one or more software modules comprise, by way of non-limiting examples, a web application, a mobile application, and a standalone application. In some embodiments, software modules are in one computer program or application. In other embodiments, software modules are in more than one computer program or application. In some embodiments, software modules are hosted on one machine. In other embodiments, software modules are hosted on more than one machine. In further embodiments, software modules are hosted on cloud computing platforms. In some embodiments, software modules are hosted on one or more machines in one location. In other embodiments, software modules are hosted on one or more machines in more than one location.
[0317] In some embodiments, the methods and systems disclosed herein include one or more databases, or use of the same. In view of the disclosure provided herein, databases are suitable for storage and retrieval of analytical information described elsewhere herein. In various embodiments, suitable databases include, by way of non-limiting examples, relational databases, non-relational databases, object oriented databases, object databases, entity-relationship model databases, associative databases, and XML databases. In some embodiments, a database is internet-based. In further embodiments, a database is web-based. In still further embodiments, a database is cloud computing-based. In other embodiments, a database is based on one or more local computer storage devices.
Server
[0318] In some embodiments, the methods provided herein are processed on a server or a computer server (FIG. 2). In some embodiments, the server 401 includes a central processing unit (CPU, also "processor") 405 which is a single core processor, a multi core processor, or plurality of processors for parallel processing. In some embodiments, a processor used as part of a control assembly is a microprocessor. In some embodiments, the server 401 also includes memory 410 (e.g. random access memory, read-only memory, flash memory); electronic storage unit 415 (e.g. hard disk); communications interface 420 (e.g. network adaptor) for communicating with one or more other systems; and peripheral devices 425 which includes cache, other memory, data storage, and/or electronic display adaptors. The memory 410, storage unit 415, interface 420, and peripheral devices 425 are in communication with the processor 405 through a communications bus (solid lines), such as a motherboard. In some embodiments, the storage unit 415 is a data storage unit for storing data. The server 401 is operatively coupled to a computer network ("network") 430 with the aid of the communications interface 420. In some embodiments, a processor with the aid of additional hardware is also operatively coupled to a network. In some embodiments, the network 430 is the Internet, an intranet and/or an extranet, an intranet and/or extranet that is in communication with the Internet, a telecommunication or data network. In some embodiments, the network 430 with the aid of the server 401, implements a peer-to-peer network, which enables devices coupled to the server 401 to behave as a client or a server. In some embodiments, the server is capable of transmitting and receiving computer-readable instructions (e.g., device/system operation protocols or parameters) or data (e.g., sensor measurements, raw data obtained from detecting metabolites, analysis of raw data obtained from detecting metabolites, interpretation of raw data obtained from detecting metabolites, etc.) via electronic signals transported through the network 430. Moreover, in some embodiments, a network is used, for example, to transmit or receive data across an international border.
[0319] In some embodiments, the server 401 is in communication with one or more output devices 435 such as a display or printer, and/or with one or more input devices 440 such as, for example, a keyboard, mouse, or joystick. In some embodiments, the display is a touch screen display, in which case it functions as both a display device and an input device. In some embodiments, different and/or additional input devices are present such an enunciator, a speaker, or a microphone. In some embodiments, the server uses any one of a variety of operating systems, such as for example, any one of several versions of Windows.RTM., or of MacOS.RTM., or of Unix.RTM., or of Linux.RTM..
[0320] In some embodiments, the storage unit 415 stores files or data associated with the operation of a device, systems or methods described herein.
[0321] In some embodiments, the server communicates with one or more remote computer systems through the network 430. In some embodiments, the one or more remote computer systems include, for example, personal computers, laptops, tablets, telephones. Smart phones, or personal digital assistants.
[0322] In some embodiments, a control assembly includes a single server 401. In other situations, the system includes multiple servers in communication with one another through an intranet, extranet and/or the Internet.
[0323] In some embodiments, the server 401 is adapted to store device operation parameters, protocols, methods described herein, and other information of potential relevance. In some embodiments, such information is stored on the storage unit 415 or the server 401 and such data is transmitted through a network.
Certain Terminology
[0324] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the claimed subject matter belongs. It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of any subject matter claimed. In this application, the use of the singular includes the plural unless specifically stated otherwise. It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. In this application, the use of "or" means "and/or" unless stated otherwise. Furthermore, use of the term "including" as well as other forms, such as "include". "includes," and "included," is not limiting.
[0325] As used herein, ranges and amounts can be expressed as "about" a particular value or range. About also includes the exact amount. Hence "about 5 .mu.L" means "about 5 .mu.L" and also "5 .mu.L." Generally, the term "about" includes an amount that would be expected to be within experimental error.
[0326] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.
[0327] The term "protein", as used herein, encompasses a full-length cysteine containing protein, a full-length functional cysteine containing protein, a cysteine containing protein fragment, or a functionally active cysteine containing protein fragment. In some instances, a protein described herein is also referred to as an "isolated protein", or a protein that by virtue of its origin or source of derivation is not associated with naturally associated components that accompany it in its native state; is substantially free of other proteins from the same species; is expressed by a cell from a different species, or does not occur in nature.
[0328] The term "polypeptide", as used herein, refers to any polymeric chain of amino acids. The term "polypeptide" encompasses native or modified cysteine containing protein, cysteine containing protein fragments, or polypeptide analogs comprising non-native amino acid residues. In some instances, a polypeptide is monomeric. In other instances, a polypeptide is polymeric. In some instances, a polypeptide described herein is also referred to as an "isolated polypeptide", or a polypeptide that by virtue of its origin or source of derivation is not associated with naturally associated components that accompany it in its native state; is substantially free of other proteins from the same species; is expressed by a cell from a different species; or does not occur in nature.
[0329] As used herein, the terms "individual(s)". "subject(s)" and "patient(s)" mean any mammal. In some embodiments, the mammal is a human. In some embodiments, the mammal is a non-human. None of the terms require or are limited to situations characterized by the supervision (e.g. constant or intermittent) of a health care worker (e.g. a doctor, a registered nurse, a nurse practitioner, a physician's assistant, an orderly or a hospice worker).
[0330] The term "alkyl" as used herein is a branched or unbranched saturated hydrocarbon group of 1 to 24 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, t-butyl, n-pentyl, isopentyl, s-pentyl, neopentyl, hexyl, heptyl, octyl, nonyl, decyl, dodecyl, tetradecyl, hexadecyl, eicosyl, tetracosyl, and the like. It is understood that the alkyl group is acyclic. In some instances, the alkyl group is branched or unbranched. In some instances, the alkyl group is also substituted or unsubstituted. For example, the alkyl group is substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, amino, ether, halide, hydroxy, nitro, silyl, sulfo-oxo, or thiol. A "lower alkyl" group is an alkyl group containing from one to six (e.g., from one to four) carbon atoms. In some instances, the term alkyl group is also a C1 alkyl, C1-C2 alkyl, C1-C3 alkyl, C1-C4 alkyl, C1-05 alkyl, C1-C6 alkyl, C1-C7 alkyl, C1-C8 alkyl, C1-C9 alkyl, C1-C10 alkyl, and the like up to and including a C1-C24 alkyl.
[0331] The term "aryl" as used herein is a group that contains any carbon-based aromatic group including, but not limited to, benzene, naphthalene, phenyl, biphenyl, anthracene, and the like. The aryl group can be substituted or unsubstituted. In some instances, the aryl group is substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, --NH.sub.2, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol. The term "biaryl" is a specific type of aryl group and is included in the definition of "aryl." In addition, the aryl group is optionally a single ring structure or comprises multiple ring structures that are either fused ring structures or attached via one or more bridging groups such as a carbon-carbon bond. For example, biaryl refers to two aryl groups that are bound together via a fused ring structure, as in naphthalene, or are attached via one or more carbon-carbon bonds, as in biphenyl.
EXAMPLES
[0332] These examples are provided for illustrative purposes only and not to limit the scope of the claims provided herein.
Example 1
Biological Methods
Preparation of Human Cancer Cell Line Proteomes
[0333] All cell lines were obtained from ATCC, were used with a low passage number and were grown at 37.degree. C. with 5% CO.sub.2. MDA-MB-231 cells and HEK-293T cells were grown in DMEM supplemented with 10% fetal bovine serum, penicillin, streptomycin and glutamine. Jurkat, Ramos and MUM2C cells were grown in RPMI-1640 medium supplemented with 10% fetal bovine serum, penicillin and streptomycin. For in vitro labeling, cells were grown to 100% confluence for MDA-MB-231 cells or until cell density reached 1.5 million cells/mL for Ramos and Jurkat cells. Cells were washed with cold PBS, scraped with cold PBS and cell pellets were isolated by centrifugation (1,400 g, 3 min, 4.degree. C.), and stored at -80.degree. C. until use. Cell pellets were lysed by sonication and fractionated (100,000 g, 45 min) to yield soluble and membrane fractions, which were then adjusted to a final protein concentration of 1.5 mg/mL for proteomics experiments and 1 mg/mL for gel-based ABPP experiments. The soluble lysate was prepared fresh from frozen pellets directly before each experiment. Protein concentration was determined using the Bio-Rad DC.TM. protein assay kit.
Screening of Fragment Electrophile Library by Gel-Based ABPP with IA-Rhodamine and Ac-Rho-DEVD-AMK ("DEVD" Disclosed as SEQ ID NO: 857)
[0334] 25 .mu.L of soluble proteome (1 mg/mL) was treated with fragment electrophiles (1 .mu.L of 25.times.stock solution in DMSO) at ambient temperature for 1 h. IA-rhodamine (1 .mu.L of 25 .mu.M, final concentration=1 .mu.M) was then added and allowed to react for an additional 1 h. The reactions were quenched with 8 .mu.L of 4.times.SDS-PAGE loading buffer and the quenched samples analyzed by SDS-PAGE (10% polyacrylamide; 15 .mu.L of sample/lane) and visualized by in-gel fluorescence using a flatbed fluorescent scanner (BioRad ChemiDoc.TM. MP or Hitachi FMBio IIe). To measure labeling of recombinant proteins expressed in E. coli, purified protein was added to soluble proteome to a final concentration of 1 .mu.M (CASP8, PRMT1, IMPDH2), 2 .mu.M (TIGAR, IDH1) or 4 .mu.M (IDH1 R132H) and the proteomes were treated as detailed above. IDH1 labeling by IA-rhodamine is relatively better in MDA-MB-231 soluble proteome when compared with Ramos and Jurkat soluble proteome. Recombinant, active CASP8 in soluble proteome was labeled with Rho-DEVD-AOMK ("DEVD" disclosed as SEQ ID NO: 857) (1 .mu.L of 50 .mu.M, final concentration=2 .mu.M), quenched and analyzed by SDS-PAGE on 14% polyacrylamide gels.
Gel-Based ABPP with Alkyne-Containing Click Probes
[0335] 25 .mu.L of soluble proteome (1 mg/mL) was labeled with the indicated concentration of 18 or 19 (1 .mu.L of 25.times. stock solution in DMSO) for 1 h at ambient temperature followed by copper-mediated azide-alkyne cycloaddition (CuAAC) conjugation to rhodamine-azide. CuAAC was performed with 20 .mu.M rhodamine-azide (50.times. stock in DMSO), 1 mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP; fresh 50.times. stock in water, final concentration=1 mM), ligand (17.times. stock in DMSO:t-butanol 1:4, final concentration=100 .mu.M) and 1 mM CuSO.sub.4 (50.times. stock in water, final concentration=1 mM). Samples were allowed to react for 1 h at ambient temperature before quenching with 8 .mu.L 4.times.SDS-PAGE loading buffer. Quenched reactions were analyzed by SDS-PAGE and visualized by in-gel fluorescence. For CASP8 and IMPDH2 25 .mu.L of soluble proteomes containing IMPDH2 or Pro-CASP8 (1 .mu.M each respectively) were treated with the indicated fragment for 1 h prior to incubation for 1 h with 18 (1 .mu.l, of 625 .mu.M, final concentration=25 .mu.M) for IMPDH2 or 61 (1 .mu.l, of 625 .mu.M, final concentration=25 .mu.M) for CASP8. For MLTK, HEK 293T cells stably overexpressing MTLK.sup.2 were treated with the indicated fragment electrophiles for 1 h, followed by labeling with 59 (1 .mu.l, of 125 .mu.M, final concentration=5 .mu.M) for 1 h. These were followed by CuAAC conjugation to rhodamine-azide and evaluation by SDS-PAGE as described above.
Determination of In Vitro IC.sub.50 Values
[0336] 25 .mu.L of proteomes containing the indicated protein were treated with fragment electrophiles for 1 h at ambient temperature, labeled with the probes detailed above for 1 h, quenched, and analyzed by SDS-PAGE and in-gel fluorescence visualization (n=3). IA-rhodamine was used as the probe for C161S-TIGAR, C409S-CASP8 and PRMT1, 59 was used as a probe for MLTK. The soluble proteome containing IMPDH2 was treated with ATP for 15 min prior to incubation with 18 (1 .mu.l, of 625 .mu.M, final concentration=25 .mu.M) for 1 h. MLTK and IMPDH2 were subjected to CuAAC conjugation to rhodamine-azide as detailed above. The percentage of labeling was determined by quantifying the integrated optical intensity of the bands, using ImageJ software. Nonlinear regression analysis was used to determine the IC50 values from a dose-response curve generated using GraphPad Prism 6.
IsoTOP-A BPP Sample Preparation
[0337] For in situ labeling, MDA-MB-231 cells were grown to 95% confluence and Ramos cells were grown to 1 million cells/mL. The media in all samples was replaced with fresh media, containing 200 .mu.M of the indicated fragments and the cells were incubated at 37.degree. C. for 2 h, washed with cold PBS, scraped into cold PBS and harvested by centrifugation (see prior section on "Preparation of human cancer cell line proteomes").
[0338] Fragments 2, 3, 8, 9, 10, 12, 13, 14, 21, 27, 28, 29, 31, 33, 38, 45, 51 and 56 were screened at 200 .mu.M in situ. Fragments 4 and 11 were screened at 100 .mu.M in situ. Fragments 2, 3, 8, and 20 were tested at 50 .mu.M in situ.
[0339] After in vitro or in situ fragment treatment, the samples were labeled for 1 h at ambient temperature with 100 .mu.M iodoacetamide alkyne (IA-alkyne, 5 .mu.L of 10 mM stock in DMSO). For direct labeling with 61, 61 (5 .mu.L of 1 or 10 mM stocks in DMSO, final concentration=10 or 100 .mu.M) was substituted for IA-alkyne. Samples were conjugated by CuAAC to either the light (fragment treated) or heavy (DMSO treated) TEV tags (10 .mu.L of 5 mM stocks in DMSO, final concentration=100 .mu.M), TCEP, TBTA ligand and CuSO.sub.4 as detailed above. The samples were allowed to react for 1 h at which point the samples were centrifuged (16,000 g, 5 min, 4.degree. C.). The resulting pellets were sonicated in ice-cold methanol (500 .mu.L) and the resuspended light- and heavy-labeled samples were then combined and centrifuged (16,000 g, 5 min, 4.degree. C.). The pellets were solubilized in PBS containing 1.2% SDS (1 mL) with sonication and heating (5 min, 95.degree. C.) and any insoluble material was removed by an additional centrifugation step at ambient temperature (14,000 g, 1 min).
[0340] For each sample, 100 .mu.L of streptavidin-agarose beads slurry (Pierce) was washed in 10 mL PBS and then resuspended in 5 mL PBS. The SDS-solubilized proteins were added to the suspension of streptavidin-agarose beads and the bead mixture was rotated for 3 h at ambient temperature. After incubation, the beads were pelleted by centrifugation (1.400 g, 3 min) and were washed (2.times.10 mL PBS and 2.times.10 mL water).
[0341] The beads were transferred to eppendorf tubes with 1 mL PBS, centrifuged (1,400 g, 3 min), and resuspended in PBS containing 6 M urea (500 .mu.L). To this was added 10 mM DTT (25 .mu.L of a 200 mM stock in water) and the beads were incubated at 65.degree. C. for 15 mins, 20 mM iodoacetamide (25 .mu.L of a 400 mM stock in water) was then added and allowed to react at 37.degree. C. for 30 mins with shaking. The bead mixture was diluted with 900 .mu.L PBS, pelleted by centrifugation (1,400 g, 3 min), and resuspended in 200 .mu.L PBS. To this was added 1 mM CaCl.sub.2 (2 .mu.L of a 200 mM stock in water) and trypsin (2 .mu.g, Promega, sequencing grade) and the digestion was allowed to proceed overnight at 37.degree. C. with shaking. The beads were separated from the digest with Micro Bio-Spin columns (Bio-Rad) by centrifugation (1,000 g, 1 min), washed (2.times.1 mL PBS and 2.times.1 mL water) and then transferred to fresh eppendorfs with 1 mL water. The washed beads were washed once further in 140 .mu.L TEV buffer (50 mM Tris, pH 8, 0.5 mM EDTA, 1 mM DTT) and then resuspended in 140 .mu.L TEV buffer, 5 .mu.L TEV protease (80 .mu.M) was added and the reactions were rotated overnight at 29.degree. C. The TEV digest was separated from the beads with Micro Bio-Spin columns by centrifugation (1,400 g, 3 min) and the beads were washed once with water (100 .mu.L). The samples were then acidified to a final concentration of 5% (v/v) formic acid and stored at -80.degree. C. prior to analysis.
Liquid-Chromatography-Mass-Spectrometry (LC-MS) Analysis of isoTOP-ABPP Samples
[0342] TEV digests were pressure loaded onto a 250 .mu.m (inner diameter) fused silica capillary column packed with C18 resin (Aqua 5 .mu.m, Phenomenex). The samples were analyzed by multidimensional liquid chromatography tandem mass spectrometry (MudPIT), using an LTQ-Velos Orbitrap mass spectrometer (Thermo Scientific) coupled to an Agilent 1200-series quaternary pump. The peptides were eluted onto a biphasic column with a 5 .mu.m tip (100 .mu.m fused silica, packed with C18 (10 cm) and bulk strong cation exchange resin (3 cm, SCX, Phenomenex,)) in a 5-step MudPIT experiment, using 0%, 30%, 60%, 90%, and 100% salt bumps of 500 mM aqueous ammonium acetate and using a gradient of 5-100% buffer B in buffer A (buffer A: 95% water, 5% acetonitrile, 0.1% formic acid; buffer B: 5% water, 95% acetonitrile, 0.1% formic acid) as has been described in Weerapana et al. Nat Protoc 2:1414-1425 (2007). Data was collected in data-dependent acquisition mode with dynamic exclusion enabled (20 s, repeat of 2). One full MS (MS1) scan (400-1800 m/z) was followed by 30 MS2 scans (ITMS) of the nth most abundant ions.
Peptide and Protein Identification
[0343] The MS2 spectra data were extracted from the raw file using RAW Xtractor (version 1.9.9.2; available at http://fields.scripps.edu/downloads.php). MS2 spectra data were searched using the ProLuCID algorithm (publicly available at http://fields.scripps.edu/downloads.php) using a reverse concatenated, nonredundant variant of the Human UniProt database (release-2012_11). Cysteine residues were searched with a static modification for carboxyamidomethylation (+57.02146) and up to one differential modification for either the light or heavy TEV tags (+464.28595 or +470.29976 respectively). Peptides were required to have at least one tryptic terminus and to contain the TEV modification. ProLuCID data was filtered through DTASelect (version 2.0) to achieve a peptide false-positive rate below 1%.
R Value Calculation and Processing
[0344] The ratios of heavy/light for each unique peptide (DMSO/compound treated; isoTOP-ABPP ratios, R values) were quantified with in-house CIMAGE software, using default parameters (3 ms 1 s per peak and signal to noise threshold 2.5). Site-specific engagement of electrophilic fragments was assessed by blockade of IA-alkyne probe labeling. For peptides that showed a .gtoreq.95% reduction in MS1 peak area from the fragment treated proteome (light TEV tag) when compared to the DMSO treated proteome (heavy TEV tag), a maximal ratio of 20 was assigned. Ratios for unique peptide entries were calculated for each experiment; overlapping peptides with the same modified cysteine (e.g. different charge states, MudPIT chromatographic steps or tryptic termini) were grouped together and the median ratio was reported as the final ratio (R). The peptide ratios reported by CIMAGE were further filtered to ensure the removal or correction of low quality ratios in each individual dataset. The quality filters applied were the following: removal of half tryptic peptides; for ratios with high standard deviations from the median (90% of the median or above) the lowest ratio was taken instead of the median; removal of peptides with R=20 and only a single ms2 event triggered during the elution of the parent ion; manual annotation of all the peptides with ratios of 20, removing any peptides with low-quality elution profiles that remained after the previous curation steps. Proteome reactivity values for individual fragments were computed as the percentage of the total quantified cysteine-containing peptides with R values.gtoreq.4 (defined as liganded cysteines) for each replicate experiment and the final proteome reactivity value was calculated as the mean for all replicate experiments for each fragment from both MDA-MB-231 and Ramos cellular proteomes.
Cross-Data Processing
[0345] Biological replicates of the same compound and cell-line were averaged if the standard deviation was below 60% of the mean; otherwise the lowest value of the ratio set was taken. For peptides with multiple modified cysteines, the cysteine with the highest number of quantification events was kept and the remaining, redundant peptides were discarded. Peptides included in the aggregate dataset (those used for further bioinformatics and statistical analyses) were required to have been quantified in 3 experiments. Cysteines were categorized as liganded if they had at least two ratios R.gtoreq.4 (hit fragments) and one ratio between 0.5 and 2 (control fragments). Although the majority (>75% of fragments) were profiled in at least two biological replicates, some data from single replicate MS experiments were included. Averaged filtered data for all fragments and representative individual filtered datasets are found in Tables 1-3.
In Situ Data Processing
[0346] R values were calculated and individual datasets were filtered as described above (R value calculation and processing). Two categories of hits in situ were defined: 1) cysteines liganded in situ that were also observed as hits in vitro and 2) cysteines that detected in vitro, but were only liganded in situ. For the first category. R values for the same cysteine containing peptide from in vitro and in situ experiments were compared and if both had ratios R.gtoreq.4, the cysteine was considered ligandable in situ. To qualify for the second category, two ratios R.gtoreq.4 for replicates of two different fragments were required to be detected in situ and at least one of these fragments must be quantified as a non-hit with R.ltoreq.2 in vitro. Additionally, another cysteine from the same protein was required to be unliganded in situ (R.ltoreq.2) by the same fragment to control for the possibility that changes in R values from changes in protein expression upon fragment treatment rather than from fragment competition.
Functional Annotation of Liganded Cysteines
[0347] Custom python scripts were used to compile functional annotations available in the UniProtKB/Swiss-Prot Protein Knowledge database (release-2012_11). Relevant Uniprot entries were mined for available functional annotations at the residue level, specifically for annotations regarding enzyme catalytic residues (active sites), disulfides (redox active and structural) and metal binding sites. Liganded proteins were queried against the Drugbank database (Version 4.2) and fractionated into DrugBank and non-Drugbank proteins. Functional keywords assigned at the protein level were collected from the Uniprot database and the Drugbank and non-drugbank categories were further classified into protein functional classes. Cysteine reactivity data was re-processed using ProLuCID as detailed above (Peptide and protein identification). Cysteines found in both the reactivity and ligandability datasets were sorted based on their reactivity values (lower ratio indicates higher reactivity). The moving average of the percentage of total liganded cysteines within each reactivity bin (step-size 50) was taken. Custom python scripts were developed to collect relevant NMR and X-ray structures from the RCSB Protein Data Bank (PDB). For proteins without available PDB structures, sequence alignments, performed with BLAST to proteins deposited in the PDB, were used to identify structural homologues. For annotation of active-site and non-active cysteines, enzymes with structures in the PDB were manually inspected to evaluate the location of the cysteine. Cysteines were considered to reside in enzyme active sites if they were within 10 .ANG. of active-site ligand or residue(s). Cysteines outside of the 10 .ANG. range were deemed non-active-site residues. Histograms of fragment hit-rates across high-coverage, ligandable cysteines, active-site and non-active site cysteines were calculated from the subset of ligandable cysteines quantified in 10 or more separate experiments. The fragment hit rate is reported as the percentage of the total quantification events with R.gtoreq.4. For analyses of trends within the whole data, including histograms and heatmaps, a cell-line merged dataset was used where data from the MDA-MB-231 experiments was taken first and the Ramos data was used if there was no data from MDA-MB-231 experiments for a particular fragment and cysteine. Heatmaps were generated in R (version 3.1.3) using the heatmap.2 algorithm. Protein structures were rendered using Pymol.
GSH Reactivity
[0348] Glutathione (GSH) was diluted to a final concentration of 125 .mu.M in assay buffer (100 mM Tris, pH 8.8, 10% ethanol as co solvent). In triplicate, to 100 .mu.L of the GSH mixture in a clear 96 well plate (Costar.RTM. Corning.RTM.), the indicated electrophile (2 .mu.L of a 50 mM stock solution in DMSO, final concentration=500 .mu.M) was added and the reaction mixture was incubated at room temperature for 1 h. 5 .mu.L of Ellman's reagent (100 mM stock in IM NaOH, final concentration=5 mM) was added and the absorbance was measure at 440 nm on a plate reader (Tecan Infinite F500). The concentration of GSH remaining was calculated from a standard curve.
Reactive Cysteine Docking
[0349] In silico fragment library containing all chloroacetamide and acrylamide fragments from FIG. 3 was prepared using Open Babel library with custom Python scripts. Fragments were modeled in their reactive form (i.e., with explicit chloroacetamide and acrylamide warheads). 3D coordinates were generated from SMILES strings, calculating their protonation state at pH 7.4, and then minimizing them using MMF94s forcefield (50K iterations steepest descent; 90K conjugate gradient); for chiral molecules with undefined configuration, all enantiomers were generated, resulting in 53 total fragments
[0350] For each protein, the UniProtKB ID was used to filter the PDB. Structures determined by X-ray crystallography were selected, privileging higher sequence coverage and structure resolution (See Table 5 for selected PDB IDs). When no human structures were available, the closest homologous organism available was selected (e.g. PRMT1: R. norvegicus). Protein structures were prepared following the standard AutoDock protocol. Waters, salts, and crystallographic additives were removed; AutoDockTools was used to add hydrogens, calculate Gasteiger-Marsili charges and generate PDBQT files.
[0351] MSMS reduced surface method was used to identify accessible cysteines. The protein volume was scanned using a probe radius of 1.5 .ANG.; residues were considered accessible if they had at least one atom in contact with either external surfaces or internal cavities.
[0352] The fragment library was docked independently on each accessible cysteine using AutoDock 4.2. A grid box of 24.4.times.24.4.times.24.4 .ANG. was centered on the geometric center of the residue; thiol hydrogen was removed from the side-chain, which was modeled as flexible during the docking; the rest of the structure was kept rigid. A custom 13-7 interaction potential was defined between the nucleophile sulfur and the reactive carbon in the ligands. The equilibrium distance (r.sub.eq) was set to the length of the C-S covalent bond (1.8 .ANG.); the potential well depth (.epsilon..sub.eq) varied between 1.0 and 0.175 to model to the reactivity of the different ligands. For each fragment, potential well depth was determined by dividing its proteomic reactivity percentage by 20, and the value for iodoacetamide was approximated as the maximum (2.5) for reference. The potential was implemented by modifying the force field table of AutoDock. Fragments were docked with no constraints, generating 100 poses using the default GA settings. For each fragment, the best docking score pose was analyzed: if the distance between the nucleophilic sulfur and the reactive carbon was .ltoreq.2.0 .ANG., the cysteine was considered covalently modified. If a residue was alkylated by at least one ligand, it was considered labeled. The docking score (i.e., negative binding energy) was calculated based on the estimated interaction energy of each fragment in its docked pose. The docking score of the best alkylating fragment defined the labeling score. The residue with the best labeling score was considered the most probable to be labeled.
Structural Modeling
[0353] IMPDH2 structure, including the Bateman domain, was modeled using I-TASSER.
Subcloning and Mutagenesis
[0354] Full length cDNAs encoding for IDH1 (Open Biosystems, Clone ID: 3880331) and IMPDH2 (Open Biosystems, Clone ID: 3447994) were subcloned into pET22b (+) (Novagen) with C-terminal His.sub.6-affinity tag (SEQ ID NO: 861). Full length cDNA encoding for TIGAR (Origene, Sc320794) was subcloned into pET28a (+) (Novagen) with N-terminal His-affinity tag (SEQ ID NO: 861). Full length PRMT1 subcloned into pET45b (+) (Novagen) was previously generated by the Cravatt lab. Full-length human CASP3 (residues 1-277) and a truncated CASP8 (residues 217-479) without the CARD domain was subcloned into pET23b (Novagen) with C-terminal His.sub.6-affinity tags (SEQ ID NO: 861). Cysteine mutants were generated using QuikChange site-directed mutagenesis, using primers containing the desired mutations and their respective compliments.
Recombinant Overexpression of TIGAR, IDH1, PRMT1 and IMPDH2
[0355] TIGAR, IDH1, PRMT1 and IMPDH2 were expressed in BL21(DE3) Chemically Competent Cells (NEB), grown on Terrific Broth supplemented with the desired antibiotic (50 .mu.g/mL Kanamycin or 50 .mu.g/mL Carbenicillin) to OD.sub.600 of 0.8 and induced with 0.5 mM IPTG for 16 h at 18.degree. C. Cells were immediately harvested and resuspended in 30 mL cold buffer A (25 mM Tris, pH 7.4, 200 mM NaCl, 10% glycerol, 1 mM BME), supplemented with lysozyme (Sigma), DNAase (NEB) and cOmplete protease inhibitor tablets (Roche), sonicated, and centrifuged (45,000 g, 30 min, 4.degree. C.). The soluble fractions were collected and rotated for 1 h with 1 mL Ni-NTA slurry (Qiagen) at 4.degree. C. The slurry was then transferred to a 50 mL volume, fritted column and collected by gravity flow. The resin was then washed with 100 mL buffer A containing 20 mM imidazole and eluted with 10 mL buffer A containing 200 mM imidazole. The eluant was concentrated to 2.5 mL (Amicon-Ultra-15, 10 kDa MW cutoff), buffer exchanged using PD10 columns (GE Amersham) into the storage buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 10% glycerol, 1 mM BME) and further concentrated (Amicon-Ultra-4, 10 kDa MW cutoff) to a final concentration of approximately 100 .mu.M protein. Protein concentration was determined using the Bio-Rad DC.TM. protein assay kit. Protein purity was assayed by SDS-PAGE under reducing conditions and were >95% pure.
Recombinant CASP3, CASP8 and TEV Protease Expression
[0356] CASP3, CASP8, pro-CASP8 (D374A, D384A) and an N-terminal MBP fusion-His.sub.6-TEV-Arg.sub.6 protease construct pRK793 ("His.sub.6" disclosed as SEQ ID NO: 861 and "Arg.sub.6" disclosed as SEQ ID NO: 862) were expressed in E. coli BL21(DE3)pLysS cells (Stratagene). Cells were grown in 2.times.YT medium supplemented with 200 .mu.g/ml ampicillin and 50 .mu.g/ml chloramphenicol at 37.degree. C. to an OD.sub.600 of 0.8-1.0. Overexpression of caspase was induced with 0.2 mM IPTG at 30.degree. C. for 4 h (CASP3) or at 12.degree. C. overnight (CASP8) or with 0.5 mM IPTG at 30.degree. C. for 4 h (TEV protease). Cells were immediately harvested and resuspended in ice cold buffer A (caspases: 100 mM Tris, pH 8.0, 100 mM NaCl; TEV protease: PBS) and subjected to 3 cycles of lysis by microfluidization (Microfluidics). The cell lysate was clarified by centrifugation (45,000 g, 30 min, 4.degree. C.) and soluble fractions were loaded onto a 1 mL HisTrap HP Ni-NTA affinity column (GE Amersham) pre-equilibrated with buffer A and eluted with buffer A containing 200 mM imidazole. The eluted protein was immediately diluted two-fold with buffer B (20 mM Tris, pH 8.0) and purified by anion-exchange chromatography (HiTrap Q HP, GE Amersham) with a 30-column volume gradient to 50% of buffer B containing 1 M NaCl. The caspases were injected over a Superdex 200 16/60 gel filtration column (GE Amersham) and TEV protease over a Superdex 75 gel filtration column (GE Amersham) in buffer C (caspases: 20 mM Tris, pH 8.0, 50 mM NaCl; TEV protease: PBS, 10 mM DTT) to buffer exchange and to remove any remaining contaminants. Fractions containing the desired protein were pooled and concentrated to approximately 1 mg/mL (Millipore Ultrafree-15, 10 kDa MW cutoff). The purified proteins were immediately frozen and stored at -80.degree. C. Protein concentrations were measured using both Bio-Rad colorimetric assay and A.sub.280 absorbance in denaturing conditions. Protein purity was assayed by SDS-PAGE under reducing conditions and were >98% pure.
Retroviral Overexpression of Flag-Tagged IDH1 Proteins
[0357] R132H-IDH1, including an additional K345K silent mutation to remove an unwanted restriction site and GFP were subcloned into a modified pCLNCX retroviral vector. Retrovirus was prepared by taking 1.5 .mu.g of each pCLNCX vector and 1.5 .mu.g pCMV-VSV-G and 20 .mu.L of Roche X-tremeGeneHP DNA transfection reagent to transfect HEK-293RTV cells. The medium was replaced after 1 day of transfection and the following day the culture supernatant was collected and filtered through 0.5 .mu.M filter, 10 mL of the filtrate, containing the desired virus, was used to infect MUM2C cells in the presence of polybrene (8 .mu.g/mL) for 48 h, at which point the infected cells were selected for in medium containing 100 .mu.g/mL hygromycin for 7-10 days. Surviving cells were expanded and cultured in complete RPMI-1640 medium containing hygromycin.
IDH1 NADP Assay
[0358] Recombinant IDH1 and C269S-IDH1 (100 .mu.M in storage buffer) were diluted 1:200 in MDA-MB-231 cellular proteome (1 mg/mL). To 25 .mu.L of this mixture was added 1 .mu.L of the indicated compound (25.times. stock solution in DMSO) and the lysates were incubated for 1 h at room temperature in clear 96 well plates (Corning.RTM., Costar.RTM.), 75 .mu.L per well of a stock solution of NADP (13.3 mM) and isocitrate (13.3 mM) in IDH1 buffer (40 mM Tris, pH 7.4, 2 mM MgCl.sub.2, 0.01% pluronic) was added immediately before measuring UV absorbance at 340 nm on a 96 well UV absorbance plate reader (TECAN). Absorbance was measured for 45 minutes and the relative activities were calculated from the change in absorbance for the linear portion of the curve.
IDH1 2-Hydroxyglutarate (2-HG) Formation Assay
[0359] MUM2C cells stably overexpressing IDH1 R132H were seeded 1.5.times.10.sup.6 cells/150 mm dish. The following day the indicated compounds (50 mM stock solutions in DMSO) or DMSO were added to the cells to the final concentrations indicated and were allowed to incubate for 2 h. Control cells overexpressing GFP were treated in parallel. The cells were washed in ice-cold PBS and collected by scraping in ice-cold PBS and centrifugation (1,400 g, 3 min, 4.degree. C.). The cell pellets were then resuspended in 100 .mu.L ice-cold PBS followed by sonication and centrifugation at 16,000 g for 10 min. Lysates were then buffer exchanged into IDH1 buffer (40 mM Tris, pH 7.4, 2 mM MgCl.sub.2) with 0.5 mL ZEBA spin desalting columns (Thermo Fisher, 89882). The protein concentrations were adjusted to 3.5 mg/mL and 25 .mu.L of the lysate was mixed with 25 .mu.L of the reaction mixture (2.5 mM NADPH and 2.5 mM .alpha.-ketoglutarate in IDH1 buffer) and the reaction was allowed to proceed for 4 h at which point the reaction mixtures were quenched with 50 .mu.L cold methanol, followed by a centrifugation (16,000 g, 10 min, 4.degree. C.). Formation of 2-HG was followed by targeted LC/MS analysis. The reaction mixture was separated with a Luna-NH.sub.2 column (5 .mu.m, 100 .ANG., 50.times.4.6 mm, Phenomenex) with a precolumn (NH.sub.2, 4.times.3.0 mm) using a gradient of mobile phases A and B (mobile phase A: 100% CH.sub.3CN, 0.1% formic acid; mobile phase B: 95:5 (v/v) H.sub.2O:CH.sub.3CN, 50 mM NH.sub.4OAc, 0.2% NH.sub.4OH). The flow rate started at 0.1 mL/min. and the gradient consisted of 5 min 0% B, a linear increase to 100% B over 20 min at a flow rate of 0.4 mL/min, followed by an isocratic gradient of 100% B for 2 min at 0.5 mL/min before equilibrating for 3 min at 0% B at 0.4 mL/min (30 min total). For each run, the injection volume was 25 .mu.L. MS analysis was performed on an Agilent G6410B tandem mass spectrometer with ESI source. The dwell time for 2-HG was set to 100 ms, and collision energy for 2-HG was set to 5. The capillary was set to 4 kV, and the fragmentor was set to 100 V. The drying gas temperature was 350.degree. C., the drying gas flow rate was 11 L/min and the nebulizer pressure was 35 psi. The mass spectrometer was run in MRM mode, monitoring the transition of m/z from 146.7 to 129 for 2-HG (negative ionization mode). Treatments were conducted in triplicate. Background 2-HG production, calculated from the `mock` GFP over expressing cells, was subtracted from the total 2-HG production. TIGAR activity assay
[0360] TIGAR activity assay was conducted as described in Gerin et al. The Biochemical Journal 458:439-448 (2014). Formation of 3PG (3-phosphoglycerate) production from 23BPG (2,3-bisphosphoglycerate) was measured spectrophotometrically on a TECAN plate reader, measuring decrease in absorbance at 340 nm in clear, flat-bottom 96 well microplate (Corning.RTM. Costar.RTM.). 2 .mu.L of recombinant TIGAR (10 mg/mL) was diluted into 1 mL dilution buffer (25 mM HEPES, pH 7.1, 25 mM KCl, 1 mM MgCl.sub.2). 25 .mu.L of diluted protein was incubated for 1 h with the indicated concentration of compound (1 .mu.L, 25.times. stock in DMSO). Then 75 .mu.L of assay mixture comprised of 25 mM HEPES (pH 7.1), 25 mM KCl, 1 mM MgCl.sub.2, 0.5 mM NADH, 1 mM DTT, 1 mM 23BPG, 1 mM ATP-Mg, the equivalent of 1 .mu.L each of rabbit muscle GAPDH (4000 units/mL, Sigma, G5537) and yeast PG kinase (6300 units/mL, Sigma, P7634) was added to the protein and decrease in absorbance was monitored at 340 nm. The background, calculated from samples lacking TIGAR, was subtracted from samples containing TIGAR. Experiments were performed in quadruplicate.
PRMT1 In Vitro Methylation Assays
[0361] PRMT1 assays were conducted as described in Weerapana et al. Nature 468:790-795 (2010). Recombinant human PRMT1 (0.85 .mu.M, wild type or C101S mutant) in 25 .mu.L methylation buffer (20 mM This, pH 8.0, 200 mM NaCl, 0.4 mM EDTA) was pre-incubated with indicated fragments for 1 h and methylation activity was monitored after addition of 1 mg of recombinant histone 4 (NEB, M2504S) and .sup.3H-SAM (2 .mu.Ci). Reactions were further incubated for 60 min at ambient temperature and stopped with 4.times.SDS sample buffer. SDS-PAGE gels were fixed with 10% acetic acid/10% methanol (v/v), washed, and incubated with Amplify reagent (Amersham) before exposing to film at -80.degree. C. for 3 days.
MLTK In Vitro Kinase Activity Assay
[0362] The kinase activity assay protocol was conducted as described in Wang et al. ACS Chemical biology 9:2194-2198 (2014). Kinase assay buffers, myelin basic protein (MBP) substrate and ATP stock solution were purchased from SignalChem. Radio-labeled [.gamma.-.sup.33P ATP was purchased from PerkinElmer. 250 .mu.L of HEK-293T soluble lysates (8 mg/mL), stably overexpressing WT, C22A or K45M MLTK were labeled for 1 h with 100 .mu.M fragment or DMSO. The samples were then individually immunoprecipitated with 20 .mu.L flag resin slurry per sample and then eluted with 15 .mu.L 3.times.Flag-peptide. To each sample was added 5 .mu.L of MBP and then 5 .mu.L of .gamma.-.sup.33P] ATP assay cocktail (250 .mu.M, 167 .mu.Ci/mL) was added to initiate the kinase reaction. Each reaction mixture was incubated at ambient temperature for 30 min, and the reactions were terminated by spotting 25 .mu.L of the reaction mixture onto individual precut phosphocellulose P81 paper. The spotted P81 strips were washed with 10 mL of 1% phosphoric acid (3.times.10 min). MLTK activity was measured by counting the radioactivity on the P81 paper in the presence of scintillation fluid in a scintillation counter. The background was determined from the K45M-inactive mutant MLTK activity level, which was subtracted from the WT and C22A samples. Relative activities for WT and C22A were normalized to their respective DMSO treated samples. Experiments were performed in triplicate.
CASP3 and CASP8 In Vitro Activity Assays
[0363] Caspase 3 and 8 assays were conducted with CASP8 activity assay kit (BioVision, K112-100) and Caspase 3 activity assay kit (Invitrogen, EnzChek.RTM. Caspase-3 Assay Kit), following the manufacturer's instructions. Briefly, recombinant Caspase 3 (10 .mu.M) was added to soluble Ramos lysates (1 mg/mL) to a 100 nM final concentration of protease. Caspase 8 (30 .mu.M) was added to soluble Ramos lysates to a 1 .mu.M final concentration of protease. In triplicate, 50 .mu.L lysate was treated with either DMSO, DEVD-CHO ("DEVD" disclosed as SEQ ID NO: 857) (20 .mu.M) or the indicated compounds (100 .mu.M) for 1 h. following which 50 .mu.L of 2.times. reaction buffer containing 10 mM DTT and 5 .mu.L substrate (4 mM stock in DMSO of IETD-AFC ("IETD" disclosed as SEQ ID NO: 858) for CASP8; 10 mM stock in DMSO of DEVD-AMC ("DEVD" disclosed as SEQ ID NO: 857) for CASP3) was added to each well and the samples were incubated at ambient temperature for 2 h. Caspase activity was measured from the increase in fluorescence (excitation 380 nm emission 460 nm). Experiments were performed in triplicate. Background was calculated from samples lacking the recombinant caspase.
Apoptosis Assays with Caspase 8 Inhibitors
[0364] 4 mL of Jurkat cells in RPMI (1.5 million cells/mL) were treated with the indicated compound at 30 .mu.M for 30 min (50 mM stock solution in DMSO). Z-VAD-FMK (EMD Millipore Biosciences, 627610) and was used at a final concentration of 100 .mu.M. After pre-incubation, FASL (4 .mu.L of 100 .mu.g/.mu.L stock solution of SuperFasLigand.TM. in water, final concentration=100 ng/mL, Enzo life Sciences) or staurosporine (8 .mu.L of 1 mM stock solution in DMSO, final concentration=2 .mu.M, Fisher Scientific, 50664333). After 6 hours, cells were harvested by centrifugation, washed and lysed in cell lysis buffer (BioVision, 1067-100) and 40 .mu.g of each sample were separated by SDS-Page on 14% polyacrylamide gels. The gels were transferred to nitrocellulose membranes and were immunoblotted overnight with the indicated antibodies. For measurements of cell viability, in quadruplicate for each condition, 150,000 cells (100 .mu.L of 1.5 million cells/mL) were plated in Nunc.TM. MicroWell.TM. 96-Well Optical-Bottom Plates with Polymer Base (Fisher Scientific). Compounds, FASL and STS were used at the same concentrations indicated above with a 30 minute pre-incubation with compound, followed by 6 hours with either STS or FASL or DMSO. Cell viability was measured with CellTiter-Glot.RTM. Luminescent Cell Viability Assay (Promega) and was read on a Biotech Synergy 4 plate reader.
Western Blotting
[0365] For CASP8, CASP3 and PARP, cell pellets were resuspended in cell lysis buffer from (BioVision, 1067-100) with 1.times. cOmplete protease inhibitor (Roche) and allowed to incubate on ice for 30 min prior to centrifugation (10 min, 16,000 g). For all other proteins, cell pellets were resuspended in PBS and lysed with sonication prior to centrifugation (10 min, 16,000 g). The proteins were then resolved by SDS-PAGE and transferred to nitrocellulose membranes, blocked with 5% BSA in TBST and probed with the indicated antibodies. The primary antibodies and the dilutions used are as follows: anti-parp (Cell Signaling, 9532, 1:1000), anti-casp3 (Cell Signaling, 9662, 1:500), anti-casp8 (Cell Signaling, 9746, 1:500), anti-IDH1 (Cell Signaling, 1:500, 3997s), anti-actin (Cell Signaling, 3700, 1:3000), anti-gapdh (Santa Cruz, sc-32233, 1:2000) anti-flag (Sigma Aldrich, F1804, 1:3000). Blots were incubated with primary antibodies overnight at 4.degree. C. with rocking and were then washed (3.times.5 min, TBST) and incubated with secondary antibodies (LICOR, IRDye.RTM. 800CW or IRDye.RTM. 800LT, 1:10,000) for 1 h at ambient temperature. Blots were further washed (3.times.5 min, TBST) and visualized on a LICOR Odyssey Scanner.
Statistical Analysis
[0366] Data are shown as mean.+-.SEM. P values were calculated using unpaired, two-tailed Student's t-test. P values of <0.05 were considered significant.
Prediction Failures in Reactive Docking
[0367] Prediction failures were due to the approximations of the rigid model used with highly flexible/solvent exposed loop regions (STAT1:C255, PDB ID: 1YVL; HAT1:C101. PDB ID:2P0W; ZAP70:C117, PDB ID:4K2R), or with partially buried residues (SARS:C438, PDB ID:4187; PAICS:C374, PDB ID:2H31). In some embodiments, the simulation of some degree of flexibility (such as flexible side chains) improves the success rate. In some embodiments, the method was limited by availability and quality of crystallographic structures, when sequences were not fully resolved in available models (XPO1:C34, C1070, PDB ID:3GB8, FNBP1:C511,C555,C609, PDB ID:2EFL; IMPDH2:C140, PDB ID: INF7), or when only orthologue sequences were available (PRMT1: R. norvegicus, PDB ID: 1ORI).
General Synthetic Methods
[0368] Chemicals and reagents were purchased from a variety of vendors, including Sigma Aldrich, Acros, Fisher, Fluka, Santa Cruz, CombiBlocks, BioBlocks, and Matrix Scientific, and were used without further purification, unless noted otherwise. Anhydrous solvents were obtained as commercially available pre-dried, oxygen-free formulations. Flash chromatography was carried out using 230-400 mesh silica gel. Preparative thin layer chromotography (PTLC) was carried out using glass backed PTLC plates 500-2000 .mu.m thickness (Analtech). All reactions were monitored by thin layer chromatography carried out on 0.25 mm E. Merck silica gel plates (60F-254) and visualized with UV light, or by ninhydrin, ethanolic phosphomolybdic acid, iodine, p-anisaldehyde or potassium permanganate stain. NMR spectra were recorded on Varian INOVA-400, Bruker DRX-600 or Bruker DRX-500 spectrometers in the indicated solvent. Multiplicities are reported with the following abbreviations: s singlet; d doublet; t triplet; q quartet; p pentet; m multiplet; br broad. Chemical shifts were reported in ppm relative to TMS and J values were reported in Hz. Mass spectrometry data were collected on a HP 1100 single-quadrupole instrument (ESI; low resolution) or an Agilent ESI-TOF instrument (HRMS).
[0369] In some embodiments, General Procedure A was used for the synthesis of one or more of the small molecule fragments and/or cysteine-reactive probes described herein. The amine was dissolved in anhydrous CH.sub.2Cl.sub.2 (0.2 M) and cooled to 0.degree. C. To this, anhydrous pyridine (1.5 equiv.) was added in one portion, then chloroacetyl chloride (1.5 equiv.) dropwise and the reaction was monitored by TLC until complete disappearance of starting material and conversion to product was detected (typically 1 h). If the reaction did not proceed to completion, additional aliquots of pyridine (0.5 equiv.) and chloroacetyl chloride (0.5 equiv.) were added. The reaction was quenched with H.sub.2O (1 mL), diluted with CH.sub.2Cl.sub.2 (20 mL), and washed twice with saturated NaHCO.sub.3 (100 mL). The organic layer was concentrated in vacuo and purified by preparatory thin layer or flash column chromatography to afford the desired product. In some embodiments, General Procedure A1 is similar to General Procedure A except triethylamine (3 equiv.) was used instead of pyridine. In some embodiments, General Procedure A2 is similar to General Procedure A except N-methylmorpholine (3 equiv.) was used instead of pyridine.
[0370] In some embodiments, General Procedure B was used for the synthesis of one or more of the small molecule fragments and/or cysteine-reactive probes described herein. The amine was dissolved in anhydrous CH.sub.2Cl.sub.2 (0.2 M) and cooled to 0.degree. C. To this, triethylamine (TEA, 1.5 equiv.), was added in one portion, then acryloyl chloride (1.5 equiv.) dropwise, and the reaction was monitored by TLC until complete disappearance of starting material and conversion to product was detected (typically 1 h). If the reaction did not proceed to completion, additional aliquots of TEA (0.5 equiv.) and acryloyl chloride (0.5 equiv.) were added. The reaction was quenched with H.sub.2O (1 mL), diluted with CH.sub.2Cl.sub.2 (20 mL), and washed twice with saturated NaHCO.sub.3 (100 mL). The organic layer was passed through a plug of silica, after which, the eluant was concentrated in vacuo and purified by preparatory thin layer or flash column chromatography to afford the desired product.
[0371] In some embodiments, General Procedure C was used for the synthesis of one or more of the small molecule fragments and/or cysteine-reactive probes described herein. Acryloyl chloride (80.4 .mu.L, 1.0 mmol, 2 equiv.) was dissolved in anhydrous CH.sub.2Cl.sub.2 (4 mL) and cooled to 0.degree. C. A solution of the amine (0.5 mmol, 1 equiv.) and N-methylmorpholine (0.16 mL, 1.5 mmol, 3 equiv.) in CH.sub.2Cl.sub.2 (2 mL) was then added dropwise. The reaction was stirred for 1 hr at 0.degree. C. then allowed to warm up to room temperature slowly. After TLC analysis showed disappearance of starting material, or 6 h, whichever was sooner, the reaction was quenched with saturated aqueous NaHCO.sub.3(5 mL) and extracted with CH.sub.2Cl.sub.2 (3.times.10 mL). The combined organic layers were dried over anhydrous Na.sub.2SO.sub.4, concentrated in vacuo, and the residue obtained was purified by preparatory thin layer chromatography to afford the desired product.
Synthesis of Probes and Fragments
Purchased Fragments
[0372] The following electrophilic fragments were purchased from the indicated vendors. 2 (Santa Cruz Biotechnology sc-345083), 3 (Key Organics JS-092C), 4 (Sigma Aldrich T142433-10 mg), 6 (Toronto Research Chemicals M320600), 8 (Alfa Aesar H33763), 10 (Santa Cruz Biotechnology sc-345060), 11 (Santa Cruz Biotechnology sc-354895), 12 (Santa Cruz Biotechnology sc-354966), 21 (Santa Cruz Biotechnology, sc-279681), 22 (Sigma Aldrich 699357-5G), 26 (Sigma Aldrich T109959), 27 (Santa Cruz Biotechnology sc-342184), 28 (Santa Cruz Biotechnology sc-335173), 29 (Santa Cruz Biotechnology sc-348978), 30 (Santa Cruz Biotechnology sc-355362), 32 (Santa Cruz Biotechnology sc-354613), 33 (Sigma Aldrich R996505). 34 (Santa Cruz Biotechnology sc-355477), 35 (Santa Cruz Biotechnology sc-328985), 41 (Sigma Aldrich L469769), 42 (Sigma Aldrich R901946), 43 (Santa Cruz Biotechnology sc-307626), 52 (Enamine, EN300-08075), 55 (Santa Cruz Biotechnology sc-354880), 57 (VWR 100268-442), 58 (Enzo Life Sciences ALX-430-142-M005), 62 (WuXi Apptec).
Synthesis of Isotopically-Labeled TEV-Tags:
##STR00065##
[0374] Isotopically-labeled heavy and light tags were synthesized with minor modifications to the procedure reported in Weerapana et al. Nat Protoc 2:1414-1425 (2007) and Weerapana et al. Nature 468:790-795 (2010). Fmoc-Rink-Amidc-MBHA resin (EMD Biosciences: 0.5 M, 830 mg, 0.6 mmol/g loading) was deprotected with 4-methylpiperidine in DMF (50% v/v, 2.times.5 mL, 1 min). Fmoc-Lys(N.sub.3)--OH (Anaspec) (500 mg, 1.26 mmol, 1.26 equiv.) was coupled to the resin overnight at room temperature with DIEA (113 .mu.l) and 2-(6-chloro-1H-benzotriazol-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate (HCTU; 1.3 mL of 0.5 M stock in DMF) followed by a second overnight coupling with Fmoc-Lys(N.sub.3)--OH (500 mg, 1.26 mmol, 1.26 equiv.), DIEA (113 .mu.l), O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU 1.3 mL of 0.5 M stock in DMF). Unmodified resin was then capped (2.times.30 min) with Ac.sub.2O (400 .mu.L) and DIEA (700 .mu.L) in DMF after which the resin was washed with DMF (2.times.1 min). Deprotection with 4-methylpiperidine in DMF (50% v/v, 2.times.5 mL, 1 min) and coupling cycles (4 equiv. Fmoc-protected amino acid (EMD biosciences) in DMF) with HCTU (2 mL, 0.5 M in DMF) and DIEA (347.7 .mu.L) were then repeated for the remaining amino acids. For the heavy TEV-tag. Fmoc-Valine-OH (.sup.13C.sub.5C.sub.15H.sub.21.sup.15NO.sub.4, .sup.13C.sub.5, 97-99, .sup.15N, 97-99%, Cambridge Isotope Laboratories, Inc.) was used. Reactions were monitored by ninhydrin stain and dual couplings were used for all steps that did not go to completion. Biotin (0.24 g, 2 equiv.) was coupled for two days at room temperature with NHS (0.1 g, 2 equiv.). DIC (0.16 g, 2 equiv.) and DIEA (0.175 g, 2 equiv.). The resin was then washed with DMF (5 mL, 2.times.1 min) followed by 1:1 CH.sub.2Cl.sub.2:MeOH (5 mL, 2.times.1 min), dried under a stream of nitrogen and transferred to a round-bottom flask. The peptides were cleaved for 90 minutes from the resin by treatment with 95:2.5:2.5 trifluoroacetic acid:water:triisopropylsilane. The resin was removed by filtration and the remaining solution was triturated with cold ether to provide either the light or heavy TEV-tag as a white solid. HPLC-MS revealed only minor impurities and the compounds were used without further purification. HRMS-ESI (m/z): calculated for C.sub.83H.sub.128N.sub.23O.sub.23S [M+H]: (Light-TEV-Tag) 1846.9268; found: 1846.9187; calculated for C.sub.78.sup.13C.sub.5H.sub.128N.sub.22.sup.15NO.sub.23S [M+H]: (Heavy-TEV-Tag): 1852.9237; found: 1852.9309.
Synthesis of Probes and Fragments
Synthesis of 1
##STR00066##
[0375] N-(hex-5-yn-1-yl)-2-chloroacetamide (SI-1)
##STR00067##
[0377] To a solution of 5-hexenylamine (63 mg, 0.65 mmol, 1.0 equiv.) in CH.sub.2Cl.sub.2 (3.2 mL, 0.2 M) at 0.degree. C. was added N-methylmorpholine (215 .mu.L, 3 equiv.) followed by chloroacetic anhydride portionwise (222 mg, 2 equiv.). The reaction was allowed to come to room temperature and then stirred overnight. The reaction was then diluted with ether (50 mL), washed with 1 M HCl, 1 M NaOH, then brine (20 mL each). The combined organic layers were dried over magnesium sulfate and concentrated to yield chloroacetamide SI-1 (74 mg, 66%). .sup.1H NMR (400 MHz. Chloroform-d) .delta. 6.79 (s, 1H), 4.09 (d, J=1.1 Hz, 2H), 3.34 (q, J=6.8 Hz, 2H), 2.23 (td, J=6.9, 2.7 Hz, 2H), 1.98 (t J=2.7 Hz, 1H), 1.75-1.62 (m, 4H), 1.62-1.51 (m, 2H).
N-(hex-5-yn-1-yl)-2-iodoacetamide (1)
##STR00068##
[0379] To a solution of chloroacetamide SI-1 (36.1 mg, 0.2 mmol) in acetone (1 mL, 0.2 M) was added sodium iodide (47 mg, 1.5 equiv.) and the reaction was stirred overnight. The next day the reaction was filtered through a plug of silica eluting with 20% ethyl acetate in hexanes, and the filtrate was concentrated to yield a 10:1 mixture of the desired iodoacetamide 1 and starting material. This mixture was re-subjected to the reaction conditions for one further day, at which point complete conversion was observed. The product was purified by silica gel chromatography, utilizing a gradient of 5 to 10 to 15 to 20% ethyl acetate in hexanes to yield the desired product (24 mg, 44%). In some embodiments, the reaction is performed with 2.5 equiv. of sodium iodide, in which case re-subjection is not necessary, and purification by PTLC is accomplished in 30% EtOAc/hexanes as eluent. .sup.1H NMR (500 MHz, Chloroform-d) .delta. 6.16 (s, 1H), 3.69 (s, 2H), 3.30 (q. J=6.8 Hz, 2H), 2.23 (td, J=6.8, 2.6 Hz, 2H), 1.97 (t, J=2.6 Hz, 1H), 1.75-1.61 (m, 2H), 1.61-1.52 (m, 2H).
N-(4-bromophenyl)-N-phe)-N-phenylacrylamide (5)
##STR00069##
[0381] The title compound was synthesized according to General Procedure C from 4-bromophenylaniline (18.9 mg, 0.0762 mmol, 1 equiv.). Purification of the crude product by prep. TLC (30% EtOAc/hexanes) provided the title compound as a white solid (12.5 mg, 54%). .sup.1H NMR (500 MHz, Chloroform-d) .delta. 7.47 (d. J=8.2 Hz, 2H), 7.39 (t, J=7.6 Hz, 2H), 7.32 (d. J=7.4 Hz, 1H), 7.21 (d, J=7.7 Hz, 2H), 7.12 (d, J=8.2 Hz, 2H), 6.48 (d, J=16.7 Hz, 1H), 6.17 (dd, J=16.8, 10.3 Hz, 1H), 5.65 (d, J=10.3 Hz, 1H); HRMS-ESI (m/z) calculated for C.sub.15H.sub.13BrNO [M+H]: 302.0175; found: 302.0176.
Synthesis of 7
##STR00070##
[0382] tert-butyl 4-(phenylaminopiperidine-1-carboxylate (SI-2)
##STR00071##
[0383] SI-2 was prepared according to Thoma et al, J. Med. Chem. 47:1939-1955 (2004). .sup.1H NMR (400 MHz, Chloroform-d) .delta. 7.24-7.12 (m, 2H), 6.75-6.68 (m, 1H), 6.66-6.58 (m, 2H), 3.88-3.81 (m, 1H), 3.44 (tt, J=10.4, 3.9 Hz, 2H), 3.00-2.88 (m, 2H), 2.10-1.99 (m, 2H), 1.48 (bs 9H), 1.41-1.27 (m, 2H).
tert-butyl 4-(2-chloro-N-phenylacetamido)piperidine-1-carboxylate (SI-3)
##STR00072##
[0385] To a solution of aniline SI-2 (65 mg, 0.24 mmol) at 0.degree. C. in CH.sub.2Cl.sub.2 (0.6 mL) was added pyridine (38 .mu.L, 2 equiv.) followed by chloroacetyl chloride (37.4 .mu.L, 2.0 equiv.) in CH.sub.2Cl.sub.2 (0.6 mL). The resulting solution was allowed to warm to room temperature and stirred overnight. The solution was then quenched with saturated aqueous sodium bicarbonate, extracted with Et.sub.2O (3.times.10 mL). The combined organic layers were dried over magnesium sulfate, filtered and concentrated to give an off-white solid, which was used without further purification (47 mg, 57%). .sup.1H NMR (400 MHz, Chloroform-d) .delta. 7.47-7.38 (m, 3H), 7.18-7.03 (m, 2H), 4.75-4.63 (m, 1H), 4.07 (s, 2H), 3.68 (s, 2H), 2.76 (s, 2H), 1.84-1.69 (m, 2H), 1.35 (s, 9H), 1.27-1.12 (m, 2H).
N-(1-benzoylpiperidin-4-yl)-2-chloro-N-phenylacetamide (7)
##STR00073##
[0387] To neat SI-3 (47 mg, 0.128 mmol) was added trifluoroacetic acid (0.7 mL, final 0.2 M). The resulting solution was concentrated under a stream of nitrogen until no further evaporation was observed, providing the deprotected amine as its trifluoroacetate salt. This viscous gum was then treated with triethylamine in ethyl acetate (10% v/v, 2 mL; solution smokes upon addition). The resulting solution was concentrated to afford the free base, which contained only triethylammonium trifluoroacetate and the free amine by proton NMR. A stock solution was prepared by dissolving the resulting gum in CH.sub.2Cl.sub.2 (1.2 mL, .about.0.1 M final).
[0388] The deprotected amine (0.3 mL of stock solution, 0.0319 mmol) was treated with Hunig's base (17.5 .mu.L, 3 equiv.) and benzoyl chloride (7.6 .mu.L, 2.0 equiv.). This solution was stirred overnight, quenched with saturated aqueous sodium bicarbonate, extracted with Et.sub.2O (3.times.10 mL). The resulting solution was dried over magnesium sulfate, filtered and concentrated. The resulting oil was purified by silica gel chromatography (20% EtOAc/hexanes) to afford chloroacetamide 7 as a white solid (8.6 mg, 75%). .sup.1H NMR (500 MHz, Chloroform-d) .delta. 7.55 (dd, J=5.5, 3.0 Hz, 3H), 7.50-7.32 (m, 5H), 7.21 (s, 2H), 4.92 (tt, J=12.3, 4.0 Hz, 1H), 4.87 (s, 1H), 3.87 (s, 1H), 3.78 (s, 2H), 3.21 (s, 1H), 2.97-2.90 (m, 1H), 2.01 (s, 1H), 1.90 (s, 1H), 1.45 (s, 1H), 1.36-1.26 (m, 1H), HRMS-ESI (m/z) calculated for C.sub.20H.sub.22ClN.sub.2O.sub.2 [M+H]: 357.1364; found: 357.1362.
1-(4-benzylpiperidin-1-yl)-2-chloroethan-1-one (9)
##STR00074##
[0390] Following General Procedure A, starting from 4-benzylpiperidine (840 mg, 5.2 mmol, 1 equiv.), the desired compound was obtained after column chromatography as a yellow oil (1 g, 81%). Spectroscopic data matches those reported previously reported in Papadopoulou et al. J. Med. Chem. 55:5554-5565 (2012). .sup.1H NMR (500 MHz, Chloroform-d) .delta. 7.42-7.14 (m, 5H), 4.61 (d, J=13.4 Hz, 1H), 4.14 (q. J=21.9, 11.5 Hz, 2H), 3.89 (d, J=13.5, 1H), 3.11 (td, J=13.1, 2.7 Hz, 1H), 2.69-2.57 (m, 3H), 1.92-1.75 (m, 3H), 1.40-1.21 (m, 2H); HRMS-ESI (m/z) calculated for C.sub.14H.sub.19ClNO [M+H]: 252.115; found: 252.115.
N-(2-(1H-indol-3-yl)ethyl)-2-chloroacetamide (13)
##STR00075##
[0392] Following General Procedure A, starting from tryptamine (400 mg, 2.5 mmol, 1 equiv.), the desired compound was obtained after column chromatography as a brownish solid (460 mg, 77%). .sup.1H NMR (500 MHz, Chloroform-d) .delta. 8.55 (s, 1H), 7.70 (d, J=7.9 Hz, 1H), 7.45 (d, J=8.1 Hz, 1H), 7.30 (t, J=7.5 Hz, 1H), 7.23 (t, J=7.4 Hz, 1H), 7.10 (s, 1H), 6.84 (s, 1H), 4.08 (s, 2H), 3.72 (q, J=6.4 Hz, 2H), 3.10 (t, J=6.8 Hz, 2H); HRMS-ESI (m/z) calculated for C.sub.12H.sub.14ClN.sub.2O.sub.2[M+H]: 237.0789; found: 237.0791.
N-(3,5-bis(trifluoromethyl)phenyl)acrylamide (14)
##STR00076##
[0394] Following General Procedure B, starting from 3,5-bis(trifluoromethyl)aniline (1.16 g, 5 mmol, 1 equiv.), the desired compound was obtained after column chromatography as a white solid (1.05 g, 74%). .sup.1H NMR (500 MHz, Chloroform-d) .delta. 8.33 (s, 1H), 8.18 (s, 2H), 7.68 (s, 1H), 6.57 (d, J=17.5 Hz, 1H), 6.38 (dd, J=16.9, 10.3 Hz, 1H), 5.93 (d, J=12.5 Hz, 1H); HRMS-ESI (m/z) calculated for C.sub.11H.sub.8F.sub.6NO.sub.2 [M+H]: 284.0505; found: 284.0504.
N-(4-phenoxy-3-(trifluoromethyl)phenyl)-N-(pyridin-3-ylmethyl)acrylamide (15)
##STR00077##
[0396] 4-phenoxy-3-(trifluoromethyl)aniline (260 mg, 1 mmol, 1 equiv.) (Combi-Blocks) was dissolved in TFA (5 mL). Following the reductive amination protocol reported by Boros et al. J Org. Chem 74:3587-3590 (2009), the reaction mixture was cooled to 0.degree. C. and to this sodium triacetoxyborohydride (STAB) (270 mg, 1.3 mmol, 1.3 equiv.) was added, 3-pyridinecarboxaldehyde (200 mg, 2 mmol, 2 equiv.) was dissolved in CH.sub.2Cl.sub.2 (5 mL) and slowly added to the reaction mixture. Upon complete conversion to product, the reaction was diluted with CH.sub.2Cl.sub.2 (20 mL) and washed with saturated sodium bicarbonate solution (3.times.20 mL) and the organic layer was dried then concentrated under reduced pressure. Without further purification the crude material was dissolved in anhydrous CH.sub.2Cl.sub.2 and subjected to General Procedure B. The resulting crude was purified by prep. TLC to give a white solid (31 mg, 10%). .sup.1H NMR (500 MHz, Chloroform-d) .delta. 8.52 (d, J=3.5 Hz, 1H), 8.39 (s, 1H), 7.68 (d, J=7.8 Hz, 1H), 7.40 (t, J=7.7 Hz, 2H), 7.34 (s, 1H), 7.28-7.18 (m, 2H), 7.07 (d, J=8.2 Hz, 2H), 6.98 (d, J=7.5 Hz, 1H), 6.82 (d, J=8.8 Hz, 1H), 6.46 (d. J=16.8 Hz, 1H), 6.01 (dd, J=16.2, 10.7 Hz, 1H), 5.64 (d, J=10.3 Hz, 1H), 4.96 (s, 2H). HRMS-ESI (m/z) calculated for C.sub.22H.sub.18F.sub.3N.sub.2O.sub.2 [M+H]: 399.1315; found: 399.1315.
Indoacetamide-rhodamine (16)
##STR00078##
[0398] 5-(and-6)-((N-(5-aminopentyl)amino)carbonyl)tetramethylrhodamine (tetramethylrhodamine cadaverine) mixed isomers (60 mg, 0.12 mmol, 1 equiv.) were dissolved in anhydrous DMF (500 .mu.L) with sonication. To this was added DIPEA (60 .mu.L, 0.34 mmol, 3 equiv.) and chloroacetyl chloride (10 .mu.L, 0.13 mmol, 1 equiv., diluted 1:10 in DMF) and the reaction was stirred at room temperature for 20 min until complete conversion to the product was detected by TLC. The DMF was removed under a stream of nitrogen and the reaction mixture was separated by PTLC in MeOH:CH.sub.2Cl.sub.2:TEA (15:85:0.001). The chloroacetamide rhodamine was then eluted in MeOH:CH.sub.2Cl.sub.2 (15:85), concentrated under reduced pressure and redissolved in acetone (500 .mu.L). NaI (150 mg, 1 mmol, 10 equiv.) was added to this and the reaction was stirred for 20 min at 50.degree. C. until complete conversion to product was detected and the crude reaction mixture was purified by reverse phase HPLC on a C18 column and concentrated to yield the title compound as a purple solid that is a mixture of 5 and 6 carboxamide tetramethylrhodamine isomers (ratio .about.6:1) (10 mg, 12%). .sup.1H NMR (600 MHz, Methanol-d.sub.4) .delta. 8.87 (t, J=4.8 Hz, 0.14H), 8.80-8.71 (m, 1H), 8.41 (dd, J=8.2, 1.1 Hz, 0.86H), 8.35 (br s, 1H), 8.27 (dt, J=7.9, 1.5 Hz, 0.164H), 8.20 (dt, J=8.2, 1.5 Hz, 0.86H), 7.81 (s, 0.86H), 7.53 (d, J=7.8 Hz, 0.14H), 7.18-7.11 (m, 2H), 7.07 (d, J=9.5 Hz, 2H), 7.00 (s, 2H), 3.68-3.62 (m, 2H), 3.46-3.37 (m, 2H), 3.31 (s, 12H, obscured by solvent) 3.21-3.12 (m, 2H), 1.81-1.21 (m, 6H); HRMS-ESI (m/z) calculated for C.sub.32H.sub.36IN.sub.4O.sub.5 [M+H]: 683.1725; found: 683.1716.
N-(3,5-bis(trifluoromethyl)phenyl)acetamide (17)
##STR00079##
[0400] Following General Procedure A, starting with 3,5-bis(trifluoromethyl)aniline (327 mg, 1.42 mmol, 1 equiv.) and acetic anhydride (200 .mu.L, 3 mmol, 2 equiv.), the title compound was obtained after PTLC as a white solid (302 mg, 78%). .sup.1H NMR (500 MHz, Chloroform-d) .delta. 8.10 (s, 2H), 7.72 (s, 1H), 7.68 (s, 1H), 2.32 (d, J=0.9 Hz, 3H). HRMS-ESI (m/z) calculated for C.sub.11H.sub.8F.sub.6NO.sub.2 [M+H]: 284.0505; found: 284.0504.
Synthesis of 18 and 19
##STR00080##
[0401] 3-amino-N-(hex-5-yn-1-yl)-5-(trifluoromethyl)benzamide (SI-5)
##STR00081##
[0403] To a solution of 3-amino-5-(trifluoromethyl)benzoic acid (74 mg, 0.36 mmol) in acetonitrile (3.6 mL, 0.1 M final) was added EDCI (83 mg, 1.2 equiv.) followed by hex-5-ynamine (35 mg, 1.0 equiv.) followed by 1-hydroxybenzotriazole hydrate (HOBt, 66.3 mg, 1.2 equiv.) and the resulting solution was stirred overnight. The reaction was diluted with ethyl acetate, washed with 1 M HCl twice and then brine. The organic layer was dried over magnesium sulfate and concentrated to yield aniline SI-5 (97.4 mg, 95%) as a white solid. .sup.1H NMR (400 MHz, Chloroform-d) .delta. 7.29-7.22 (m, 2H), 6.98 (t, J=1.8 Hz, 1H), 6.38 (t, J=5.5 Hz, 1H), 4.08 (s, 2H), 3.46 (td, J=7.1, 5.7 Hz, 2H), 2.25 (td, J=6.9, 2.6 Hz, 2H), 1.99 (t, J=2.7 Hz, 1H), 1.81-1.55 (m, 4H).
3-acrylamido-N-(hex-5-yn-1-yl)-5-(trifluoromethyl)benzamide (18)
##STR00082##
[0405] Following General Procedure B, starting with SI-5 (42 mg, 0.15 mmol, 1 equiv.), the title compound was obtained after column chromatography as a white solid (34 mg, 70%). .sup.1H NMR (500 MHz, Chloroform-d) .delta. 8.94 (s, 1H), 8.24 (d, J=11.9 Hz, 2H), 7.71 (s, 1H), 6.87 (t, J=5.7 Hz, 1H), 6.55 (dd, J=17.4, 0.7 Hz, 1H), 6.43 (dd, J=16.9, 10.1 Hz, 1H), 5.88 (dd, J=10.1, 1.3 Hz, 1H), 3.56 (q, J=6.7 Hz, 2H), 2.33 (td, J=6.9, 2.7 Hz, 2H), 2.06 (t, J=2.7 Hz, 1H), 1.87 (p, 0.1=7.3 Hz, 2H), 1.69 (p, J=7.8 Hz, 2H); HRMS-ESI (m/z) calculated for C.sub.17H.sub.18F.sub.3N.sub.2O.sub.2 [M+H]: 339.1314; found 339.1313.
3-acrylamido-N-(hex-5-yn-1-yl)-5-(trifluoromethyl)benzamide (19)
##STR00083##
[0407] Synthesized according to General Procedure A2, starting from SI-5. .sup.1H NMR (600 MHz, Chloroform-d) .delta. 8.57 (s, 1H), 8.16 (t, J=1.8 Hz, 1H), 8.05 (t, J=1.8 Hz, 1H), 7.79 (d, J=2.0 Hz, 1H), 6.38 (d, J=6.1 Hz, 1H), 4.23 (s, 2H), 3.51 (td, J=7.1, 5.7 Hz, 2H), 2.27 (td. J=6.9, 2.7 Hz, 2H), 2.00 (t, J=2.6 Hz, 1H), 1.82-1.74 (m, 2H), 1.71-1.59 (m, 2H); HRMS-ESI (m/z) calculated for C.sub.16H.sub.17ClF.sub.3N.sub.2O.sub.2 [M+H]: 361.0925; found: 361.0925.
2-chloro-1-(4-(hydroxydiphenylmethyl)piperidin-1-yl)ethan-1-one (20)
##STR00084##
[0409] Following General Procedure A, starting with .alpha.,.alpha.-diphenyl-4-piperidinomethanol (800 mg, 3 mmol, 1 equiv.), the title compound was obtained after column chromatography as a white solid (637 mg, 61%). .sup.1H NMR (500 MHz, Chloroform-d) .delta. 7.56 (d, J=7.6 Hz, 4H), 7.39 (q, J=7.1 Hz, 4H), 7.28 (q, J=6.8 Hz, 2H), 4.66 (d, J=13.3 Hz, 1H), 4.07 (dd, J=12.2, 4.2 Hz, 2H), 3.91 (d, J=13.4 Hz, 1H), 3.18 (t, J=12.9 Hz, 1H), 2.77-2.62 (m, 3H), 1.67 (t. J=12.5 Hz, 2H), 1.56 (q, J=11.8 Hz, 1H), 1.44 (q, J=12.4, 11.8 Hz, 1H); HRMS-ESI (m/z) calculated for C.sub.20H.sub.23ClNO.sub.2 [M+H]: 344.1412; found: 344.1412.
(E)-3-(3,5-bis(trifluoromethyl)phenyl)-2-cyanoacrylamide (23)
##STR00085##
[0411] 3,5-bis(trifluoromethyl)benzaldehyde (880 mg, 3.6 mmol, 1 equiv.) and 2-cyanoacetamide (460 mg, 5.5 mmol, 1.5 equiv.) were dissolved in MeOH (10 mL). To this was added piperidine (214 mg, 0.7 equiv.) and the reaction was stirred at room temperature for 30 minutes at which point starting material was consumed. After addition of an equivalent volume of water (10 mL), the precipitate was collected by filtration and washed with water/methanol (1:1) to yield the title compound as a white solid (534 mg, 47%); .sup.1H NMR (400 MHz, Acetone-d.sub.6) .delta. 8.78 (s, 2H), 8.61 (s, 1H), 8.41 (s, 1H), 7.57 (s, 1H), 7.42 (s, 1H); HRMS-ESI (m/z) calculated for C.sub.12H.sub.2F.sub.6N.sub.2O.sub.2 [M+H]: 309.0457; found: 309.0459.
N-(3,5-bis(trifluoromethyl)phenyl)-2-bromopropanamide (24)
##STR00086##
[0413] Following General Procedure A1, starting with 3,5-bis(trifluoromethyl)aniline (250 mg, 1.1 mmol, 1 equiv.) and 2-bromopropionyl chloride (200 .mu.L, 2 mmol, 1.8 equiv.) the title compound was obtained by PTLC as a white solid (130 mg, 35%). .sup.1H NMR (500 MHz, Chloroform-d) .delta. 8.34 (s, 1H), 8.06 (s, 2H), 7.66 (s, 1H), 4.58 (q, J=7.0 Hz, 1H), 1.98 (d, J=7.0 Hz, 3H); HRMS-ESI (m/z) calculated for C.sub.11H.sub.7BrF.sub.6NO [M-H]: 361.9621; found: 361.9623.
N-(3,5-bis(trifluoromethyl)phenyl)-2-chloropropanamide (25)
##STR00087##
[0415] Following General Procedure A1, starting with 3,5-bis(trifluoromethyl)aniline (327 mg, 1.42 mmol, 1 equiv.) and 2-chloropropionyl chloride (200 .mu.L, 2 mmol, 1.8 equiv.) the title compound was obtained by PTLC as a white solid (250 mg, 55%). .sup.1H NMR (500 MHz, Chloroform-d) .delta. 8.61 (s, 1H), 8.16 (s, 2H), 7.75 (s, 1H), 4.67 (q, J=7.1 Hz, 1H), 1.93 (d, J=7.1 Hz, 3H). HRMS-ESI (m/z) calculated for C.sub.11H.sub.7ClF.sub.6NO [M-H]: 318.0126; found: 318.0126.
N-(3,5-bis(trifluoromethyl)phenyl)-N-(pyridin-3-ylmethyl)acrylamide (31)
##STR00088##
[0417] 3,5-bis(trifluoromethyl)aniline (350 mg, 1.6 mmol, 1 equiv.) was dissolved in TFA (5 mL). The reaction mixture was cooled to 0.degree. C. and to this sodium triacetoxyborohydride (STAB) (400 mg, 2 mmol, 1.3 equiv.) was added, 3-pyridinecarboxaldehyde (244 mg, 1.5 mmol, 1 equiv.) was dissolved in CH.sub.2Cl.sub.2 (5 mL) and slowly added to the reaction mixture dropwise over 10 minutes. Upon complete conversion to product, the reaction mixture was diluted with CH.sub.2C2 (20 mL) and washed with saturated sodium bicarbonate solution (3.times.20 mL) and the organic layer was dried then concentrated under reduced pressure. Without further purification the crude material was dissolved in anhydrous CH.sub.2Cl.sub.2 and subjected to General Procedure B. The resulting crude was purified by PTLC to give a white solid (10 mg, 2%). .sup.1H NMR (500 MHz, Chloroform-d) .delta. 8.63 (d. J=3.8 Hz, 1H), 8.49 (s, 1H), 7.93 (s, 1H), 7.70 (d, J=7.7 Hz, 1H), 7.55 (s, 2H), 7.35 (dd, J=7.6, 5.3 Hz, 1H), 6.60 (dd, J=16.6, 1.6 Hz, 1H), 6.02 (dd, J=16.9, 10.2 Hz, 1H), 5.79 (dd, J=10.3, 1.6 Hz, 1H), 5.11 (s, 2H). HRMS-ESI (m/z) calculated for C.sub.17H.sub.13F.sub.6N.sub.2O [M+H]: 375.0927; found: 375.0928.
3-(2-chloroacetamido)-5-trifluoromethyl)benzoic acid (36)
##STR00089##
[0419] To a solution of 3-amino-5-(trifluoromethyl)benzoic acid (500 mg, 2.44 mmol) in 1.5 mL of dimethylacetamide (1.6 M) at 0.degree. C. was added chloroacetyl chloride (214 .mu.L, 2.69 mmol, 1.1 equiv.). The resulting solution was warmed to ambient temperature and stirred for 20 minutes, at which point ethyl acetate (40 mL) and water (30 mL) were added. The pH of the aqueous layer was adjusted to pH 10 via addition of 1 N NaOH, and the phases were separated. The aqueous layer was washed with 40 mL of ethyl acetate, then acidified by adding 1 N HCl. The product was extracted with ethyl acetate (40 mL), and the organic layer was washed with IM HCl (2.times.40 mL), brine (40 mL), dried over magnesium sulfate and concentrated to provide the desired product (456 mg, 66%). .sup.1H NMR (500 MHz, Chloroform-d) .delta. 8.31 (s, 1H), 8.27 (s, 1H), 8.14 (s, 1H), 4.13 (s, 2H); HRMS-ESI (m/z) calculated for C.sub.10H.sub.8ClF.sub.3NO.sub.3 [M+H]: 282.0139; found: 282.0141.
1-(4-(5-fluorobenzisoxazol-3-yl)piperidin-1-yl)prop-2-en-1-one (37)
##STR00090##
[0421] The title compound was obtained starting from 6-fluoro-3(4-piperidinyl)-1,2-benzisoxazole hydrochloride (53 mg, 0.2 mmol, 1 equiv.) according to General Procedure C as a colorless oil (49.1 mg, 87%). .sup.1H NMR (400 MHz, Chloroform-d) .delta. 7.64 (dd, J=8.7, 5.1 Hz, 1H), 7.27 (dd, J=8.4, 2.3 Hz, 1H), 7.08 (td, J=8.9, 2.1 Hz, 1H), 6.64 (dd, J=16.8, 10.6 Hz, 1H), 6.32 (dd, J=16.9, 1.9 Hz, 1H), 5.73 (dd, J=10.6, 1.9 Hz, 1H), 4.70 (d, J=13.4 Hz, 1H), 4.15 (d, J=12.4 Hz, 1H), 3.53-3.13 (m, 2H), 2.99 (t, J=13.1 Hz, 1H), 2.25-2.07 (m, 2H), 2.00 (ddd, J=23.1, 14.2, 7.8 Hz, 2H); HRMS-ESI (m/z) calculated for C.sub.15H.sub.16FN.sub.2O [M+H]: 275.119; found: 275.119.
tert-butyl 4-(4-acrylamido-2,6-difluorophenyl)piperazine-1-carboxylate (38)
##STR00091##
[0423] The title compound was obtained starting from tert-Butyl 4-(4-amino-2,6-difluorophenyl)piperazine-1-carboxylate according to General Procedure B. .sup.1H NMR (400 MHz, Chloroform-d) .delta. 8.12 (s, 11), 7.13 (d, J=10.4 Hz, 2H), 6.36 (d, J=16.9 Hz, 11), 6.19 (dd, J=16.8, 10.2 Hz, 1H), 5.70 (d, J=10.2 Hz, 1H), 3.45 (t, J=4.7 Hz, 4H), 3.00 (t, J=3.7 Hz, 4H), 1.41 (s, 9H); HRMS-ESI (m/z) calculated for C.sub.18H.sub.24F.sub.2N.sub.3O.sub.3[M+H]: 368.178; found: 368.178.
N-(4-bromo-2,5-dimethylphenyl)acrylamide (40)
##STR00092##
[0425] Following General Procedure B, starting from 4-bromo-2,5-dimethylaniline (900 mg, 4.5 mmol, 1 equiv.), the title compound was obtained after column chromatography and recrystallization from cold CH.sub.2Cl.sub.2 as a white solid (611 mg, 40%). .sup.1H NMR (500 MHz, Chloroform-d) .delta. 7.87 (s, 1H), 7.43 (s, 1H), 7.16 (s, 1H), 6.50 (d, J=16.7 Hz, 1H), 6.35 (dd, J=16.4, 10.3 Hz, 1H), 5.86 (d, J=10.3 Hz, 1H), 2.42 (s, 3H), 2.28 (s, 3H); HRMS-ESI (m/z) calculated for C.sub.11H.sub.13BrNO [M+H]: 254.0175; found: 254.0175.
2-Chloroacetamido-2-deoxy-.alpha./.beta.-D-glucopyranose (44)
##STR00093##
[0427] To a stirred solution of hexosamine hydrochloride (590 mg, 3.39 mmol, 1 equiv.) in anhydrous MeOH (200 mL) at room temperature was added sodium metal (60 mg, 2.6 mmol, 0.78 equiv.), TEA (400 .mu.L, 5.7 mmol, 1.8 equiv.). Chloroacetic anhydride (1 g, 5.9 mmol, 1 equiv.) was then added and the mixture stirred for 6 h, monitoring for completeness by TLC. After which, the reaction mixture was concentrated in vacuo. The crude product then was purified by two rounds of column chromatography to afford the pure title product as a white solid (610 mg, 72%). .sup.1H NMR (500 MHz, Methanol-d.sub.4) .delta. 5.20 (d, J=3.7 Hz, 1H.alpha.), 4.75 (d, J=8.3 Hz, 1H.beta.), 4.19 (dd, J=20.2, 13.9 Hz, 2H), 4.19 (d, J=12.6 Hz, 1H), 3.95 (dd. J=10.6, 3.5 Hz, 1H.alpha.), 3.83 (m, 3H.alpha., 3H.beta.), 3.74 (d, J=5.1 Hz, 1H.beta.), 3.70 (dd, J=11.4, 8.9 Hz, 1H.beta.), 3.60 (dd, J=10.7, 9.5 Hz, 1H.beta.), 3.46 (t, J=9.3 Hz, 11), 3.42 (t, J=10.0 Hz, 1H.beta.); HRMS-ESI (m/z) calculated for C.sub.8H.sub.15ClNO.sub.6 [M+H]: 256.0582; found: 256.0582.
2-chloro-1-(2-methyl-3,4-dihydroquinolin-1(2H)-yl)ethan-1-one (45)
##STR00094##
[0429] Chloroacetyl chloride (80.4 .mu.L, 0.9 mmol, 1.7 equiv.) was dissolved in anhydrous CH.sub.2Cl.sub.2 (3 mL) and cooled to 0.degree. C. A solution of 2-methyl-1,2,3,4-tetrahydroquinoline (80.1 mg, 0.544 mmol, 1 equiv.) and N-methylmorpholine (0.11 mL, 1.0 mmol, 1.8 equiv.) in CH.sub.2Cl.sub.2 (2 mL) was then added dropwise. After 6 h, the reaction was quenched with saturated aqueous NaHCO.sub.3 (5 mL) and extracted with CH.sub.2Cl.sub.2 (3.times.10 mL). The combined organic layers were dried over anhydrous Na.sub.2SO.sub.4 and concentrated under reduced pressure. The resultant residue was purified by prep. TLC (30% EtOAc/hexanes), providing the title compound as an off-white solid (108.8 mg, 89%). .sup.1H NMR (400 MHz, chloroform-d) .delta. 7.30-7.13 (m, 4H), 4.86-4.75 (m, 1H), 4.20 (d, J=12.5 Hz, 1H), 4.09 (d, J=12.5 Hz, 1H), 2.69-2.58 (m, 1H), 2.59-2.46 (m, 1H), 2.46-2.31 (m, 1H), 1.36-1.29 (m, 1H), 1.15 (d, J=6.5 Hz, 3H); HRMS-ESI (m/z) calculated for C.sub.12H.sub.15ClNO [M+H]: 224.0837; found: 224.0836.
N-cyclohexyl-N-phenylacrylamide (46)
##STR00095##
[0431] The title compound was synthesized according to General Procedure C from N-cyclohexylaniline (89.5 mg, 0.511 mmol, 1 equiv.). Purification of the crude product by flash column chromatography (10-20% EtOAc/hexanes) then prep. TLC (30% EtOAc/hexanes) provided the title compound as an off-white solid (53.1 mg, 45%). .sup.1H NMR (400 MHz, chloroform-d) .delta. 7.42-7.33 (m, 3H), 7.10-7.06 (m, 2H), 6.31 (dd, J=16.7, 2.1 Hz, 1H), 5.77 (dd, J=16.7, 10.3 Hz, 1H), 5.41 (dd, J=10.4, 2.1 Hz, 1H), 4.65 (tt, J=12.2, 3.7 Hz, 1H), 1.85 (dt, J=11.2, 1.8 Hz, 2H), 1.75-1.68 (m, 2H), 1.61-1.53 (m, 1H), 1.40 (qt, J=13.3, 3.6 Hz, 2H), 1.07 (qd, J=12.4, 3.6 Hz, 2H), 0.91 (qt, J=13.1, 3.8 Hz, 1H); HRMS-ESI (m/z) calculated for C.sub.15H.sub.20NO [M+H]: 230.1539; found: 230.1539.
1-(5-bromoindolin-1-yl)prop-2-en-1-one (47)
##STR00096##
[0433] The title compound was synthesized according to General Procedure C from 5-bromoindoline (41.7 mg, 0.211 mmol, 1 equiv.), acryloyl chloride (32 .mu.L, 0.40 mmol, 1.9 equiv.), and changing the base to pyridine (32 .mu.L, 0.40 mmol, 1.9 equiv.). Purification of the crude product by re-precipitation from EtOAc provided the title compound as a white solid (67.8 mg, 64%). .sup.1H NMR (400 MHz, chloroform-d) .delta. 8.16 (d, J=8.6 Hz, 1H), 7.33-7.25 (m, 2H), 6.60-6.42 (m, 2H), 5.84-5.76 (m, 1H), 4.15 (t, J=8.6 Hz, 2H), 3.17 (t, J=8.6 Hz, 2H); HRMS-ESI (m/z) calculated for C.sub.11H.sub.11BrNO [M+H]: 252.0018; found: 252.0017.
N-(1-benzylpiperidin-4-yl)-N-phenylacrylamide (48)
##STR00097##
[0435] The title compound was synthesized according to General Procedure C from 1-benzyl-N-phenylpiperidin-4-amine (30.0 mg, 0.113 mmol, 1 equiv.), acryloyl chloride (17 .mu.L, 0.21 mmol, 1.9 equiv.), and changing the base to pyridine (17 .mu.L, 0.21 mmol, 1.9 equiv.). Purification of the crude product by prep. TLC provided the title compound as a white solid (22.5 mg, 64%). .sup.1H NMR (400 MHz, chloroform-d) .delta. 7.62-7.56 (m, 2H), 7.43-7.36 (m, 6H), 7.05 (d, J=6.2 Hz, 2H), 6.29 (dd, J=16.8, 2.1 Hz, 1H), 5.79 (dd, J=16.8, 10.3 Hz, 1H), 5.46 (dd, J=10.3, 2.1 Hz, 1H), 4.81-4.70 (m, 1H), 4.09 (s, 2H), 3.41 (d, J=12.0 Hz, 2H), 2.82 (q, J=11.5 Hz, 2H), 2.21 (q. J=11.9 Hz, 2H), 1.94 (d, J=14.2 Hz, 2H); HRMS-ESI (m/z) calculated for C.sub.21H.sub.25N.sub.2O [M+H]: 321.1961; found: 321.1962.
2-chloro-N-(2-methyl-5-(trifluoromethyl)phenyl)acetamide (49)
##STR00098##
[0437] The title compound was synthesized according to General Procedure A1 from 2-methyl-5-(trifluoromethyl)aniline (35.0 mg, 0.2 mmol, 1 equiv.). Purification of the crude product by prep. TLC (20% EtOAc/hexanes) provided the title compound as a white solid (48.2 mg, 95%). .sup.1H NMR (600 MHz, chloroform-d) .delta. 8.31 (s, 1H), 8.25 (d, J=1.9 Hz, 1H), 7.37 (dd, J=7.9, 1.8 Hz, 1H), 7.32 (d, J=7.9 Hz, 1H), 4.25 (s, 2H), 2.36 (s, 3H); HRMS-ESI calculated for C.sub.10H.sub.10ClF.sub.3NO [M+H]: 252.0397; found: 252.0397.
1-(5-bromoindolin-1-yl)-2-chloroethan-1-one (50)
##STR00099##
[0439] The title compound was synthesized according to General Procedure A1 from 5-bromoindoline (39.6 mg, 0.2 mmol, 1 equiv.). Purification of the crude product by prep. TLC (25% EtOAc/hexanes) provided the title compound as an off-white solid (48.6 mg, 89%). .sup.1H NMR (600 MHz, CDCl.sub.3) .delta. 8.07 (d, J=8.4 Hz, 1H), 7.32 (d, J=8.8 Hz, 2H), 4.17 (t, J=8.6 Hz, 2H), 4.14 (s, 2H), 3.22 (t, J=8.4 Hz, 2H); HRMS-ESI (m/z) calculated for C.sub.10H.sub.10BrClNO [M+H]: 273.9629; found: 273.9629.
2-chloro-N-(quinolin-5-yl)acetamide (51)
##STR00100##
[0441] To a stirring suspension of 5-aminoquinoline (28.8 mg, 0.2 mmol, 1 equiv.) and potassium carbonate (82.9 mg, 0.6 mmol, 3 equiv.) in anhydrous CH.sub.2Cl.sub.2 (3 mL) at 0.degree. C. was added chloroacetyl chloride (24 .mu.L, 1.5 equiv.). The reaction was allowed to slowly warm up to room temperature. After 3 hours, the mixture was filtered, washed with EtOAc (10 mL) and CH.sub.2Cl.sub.2 (10 mL). The solid cake was then eluted with MeOH (20 mL) and the filtrate concentrated in vacuo. The residue was taken up in 10% MeOH/CH.sub.2Cl.sub.2 and passed through a pad of silica to provide the title compound as an off-white solid (42.6 mg, 82%). .sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 8.96 (d. J=2.5 Hz, 1H), 8.71 (s, 1H), 8.20 (d, J=8.6 Hz, 1H), 8.04 (d, J=8.5 Hz, 1H), 7.94 (d, J=7.5 Hz, 1H), 7.74 (t, =8.0 Hz, 1H), 7.48 (dd, J=8.5, 4.2 Hz, 1H), 4.35 (s, 2H); HRMS-ESI (m/z) calculated for C.sub.11H.sub.9ClN.sub.2O [M+H]: 221.0476; found: 221.0477.
1-(4-benzylpiperidin-1-yl)prop-2-en-1-one (53)
##STR00101##
[0443] Following General Procedure B, starting from 4-benzylpiperidine (1 g, 5.7 mmol, 1 equiv.), the title compound was obtained after column chromatography as a yellow oil (748 mg, 57%). .sup.1H NMR (500 MHz, Chloroform-d) .delta. 7.36 (t, J=7.4 Hz, 2H), 7.28 (t, J=7.4 Hz, 1H), 7.20 (d, J=7.1 Hz, 2H), 6.64 (dd, J=16.8, 10.6 Hz, 1H), 6.32 (dd, J=16.8, 1.9 Hz, 1H), 5.72 (dd, J=10.6, 1.9 Hz, 1H), 4.72 (d, J=12.7 Hz, 1H), 4.03 (d, J=13.0 Hz, 1H), 3.05 (t, J=12.7 Hz, 1H), 2.70-2.59 (m, 3H), 1.86 (ddp, J=14.6, 7.2, 3.5 Hz, 1H), 1.77 (m, 2H), 1.37-1.18 (m, 2H); HRMS-ESI (m/z) calculated for C.sub.15H.sub.20ClNO [M+H]: 230.1539; found: 230.1539.
2-chloro-N-((3-hydroxy-5-(hydroxymethyl)-2-methylpyridin-4-yl)methyl)aceta- mide (54)
##STR00102##
[0445] To a stirred solution of pyridoxamine hydrochloride (150 mg, 0.64 mmol, 1 equiv.) in anhydrous MeOH (20 mL) at room temperature was added sodium metal (30 mg, 1.5 mmol, 2.3 equiv.), TEA (100 .mu.L, 1 mmol, 1.6 equiv.). Chloroacetic anhydride (390 mg, 2.29 mmol, 3.5 equiv.) was added and the mixture stirred for 6 h, monitoring for completeness by TLC. After which, the reaction mixture was concentrated in vacuo. The crude product then was the purified by prep. TLC to afford the title compound as a white solid (46 mg, 30%). .sup.1H NMR (500 MHz, Methanol-d.sub.4) .delta. 7.97 (s, 1H), 4.81 (s, 2H), 4.61 (s, 2H), 4.17 (s, 3H), 4.06 (s, 1H), 3.35 (s, 1H), 2.52 (s, 3H); HRMS-ESI (m/z) calculated for C.sub.10H.sub.14ClN.sub.2O.sub.3[M+H]: 245.0687; found: 245.0688.
1-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)prop-2-en-1-one (56)
##STR00103##
[0447] To a stirring suspension of the 6,7-dimethoxy-3,4-dihydroisoquinoline (1 g, 5.2 mmol, 1 equiv.) and TEA (1800 .mu.L, 12.6 mmol, 2.5 equiv.) in anhydrous THF (10 mL) at 0.degree. C. was added acryloyl chloride (1320 .mu.L, 13.2 mmol, 2.6 equiv.) and the reaction was allowed to slowly warm up to room temperature. After 2 hours, the mixture was diluted with CH.sub.2Cl.sub.2 (2.times.50 mL) and washed with saturated brine (2.times.50 mL) and the combined organics were concentrated in vacuo. The residue was taken up in 10% MeOH/CH.sub.2Cl.sub.2 and purified by column chromatography to afford the title compound as a white solid (700 mg, 54%, mixture of E/Z isomers). .sup.1H NMR (500 MHz, Chloroform-d) .delta. 6.63 (m, 3H), 6.29 (d, J=16.8 Hz, 1H), 5.69 (dd, J=10.6, 1.8 Hz, 1H), 4.69 (s, 1H [major]), 4.63 (s, 0.8H [minor]), 3.82 (s, 7H), 3.73 (t, J=5.6 Hz, 1H), 2.84-2.77 (m, 2H); HRMS-ESI (m/z) calculated for C.sub.14H.sub.18NO.sub.3 [M+H]: 248.128; found: 248.1281.
2-chloro-N-(1-(3-ethynylbenzoyl)piperidin-4-yl)-N-phenylacetamide (61)
##STR00104##
[0449] To an excess of neat SI-3 was added 0.7 mL of trifluoroacetic acid (0.2 M). The resulting solution was concentrated under a stream of nitrogen until no further evaporation was observed, providing the deprotected amine as its trifluoroacetate salt. The trifluoroacetate amine salt (90.6 mg, 0.25 mmol) was taken up in DMF (0.5 mL, 0.5 M) and the resulting solution was cooled to 0.degree. C. 3-ethynyl benzoic acid (44 mg, 1.2 equiv.), HATU (113 mg, 1.2 equiv.), and Hunig's base (86 .mu.L, 2 equiv.) were sequentially added. The reaction was stirred for 2 hours at 0.degree. C., diluted with Et.sub.2O, and then washed with 1 M HCl. The organic layer was dried over magnesium sulfate, concentrated, and purified by flash chromatography (gradient from 40 to 70% ethyl acetate in hexanes) to provide the title compound (87 mg, 92%). .sup.1H NMR (400 MHz, Chloroform-d) .delta. 7.51 (dd, J=9.5, 5.4 Hz, 4H), 7.43 (d. J=1.9 Hz, 1H), 7.39-7.25 (m, 2H), 7.14 (d, J=10.4 Hz, 2H), 4.86 (tt, J=15.1, 5.3 Hz, 2H), 3.72 (s, 3H), 3.19 (d, J=14.0 Hz, 1H), 3.11 (s, 1H), 2.86 (s, 1H), 1.90 (d, J=36.6 Hz, 2H), 1.38 (s, 1H), 1.24 (d, J=19.9 Hz, 1H); HRMS-ESI (m/z) calculated for C.sub.22H.sub.22ClN.sub.2O.sub.2[M+H]: 381.1364; found: 381.1363.
Global Profiling of Cysteine-Reactive Fragments in Native Populations
[0450] Cysteine is unique among protein-coding amino acids owing to its high nucleophilicity and sensitivity to oxidative modification. Cysteine residues perform catalytic functions in diverse enzyme classes and represent sites for post-translational regulation of proteins through disulfide bonding, iron-sulfur cluster formation, conversion to sulfinic and sulfonic acid, nitrosylation, S-glutathionylation and lipid modification. Using a quantitative chemical proteomic method termed isoTOP-ABPP (isotopic Tandem Orthogonal Proteolysis-Activity-Based Protein Profiling), global measurements of the intrinsic reactivity of cysteine residues was carried out and their sensitivity to modification by lipid-derived electrophiles was assessed. In order to determine whether isoTOP-ABPP was adapted to perform covalent FBLD in native biological systems, a cell preparation (lysate or intact cells) was pre-treated with DMSO or one member of a library of electrophilic small-molecule fragments and then exposed to a broad-spectrum cysteine-reactive probe iodoacetamide (IA)-alkyne 1 (FIG. 1A). Proteins harboring IA-alkyne-labeled cysteine residues from DMSO- and fragment-treated samples were conjugated by copper-mediated azide-alkyne cycloaddition (CuAAC or click) chemistry to isotopically differentiated azide-biotin tags (heavy and light, respectively), combined, enriched by streptavidin, and proteolytically digested on-bead to yield isotopic peptide pairs that were analyzed by LC-MS. Quantification of MS1 chromatographic peak ratios for peptide pairs identified fragment-competed Cys residues as those displaying high competition ratios, or R values, in DMSO/fragment comparisons.
[0451] A 50+ member fragment library was constructed with most compounds containing either a chloroacetamide or acrylamide electrophile (FIG. 1B and FIG. 3), which are well-characterized cysteine-reactive groups found in many chemical probes and some clinically approved drugs. These electrophiles were appended to structurally diverse small-molecule fragments (<300 Da) intended to serve as recognition elements that promote interactions with different subsets of the human proteome. The library also contained some additional electrophiles, such as cyanoacrylamides and vinylsulfonamides, and known bioactive electrophilic compounds (e.g., the anti-cancer agent piperlongumine and anti-migratory agent locostatin) (FIG. 1B, and FIG. 3). The electrophile library was screened at a high concentration (500 .mu.M) comparable to the ligand concentrations used in typical FBLD experiments. A subset of the fragment library was initially assayed by competitive profiling in a human MDA-MB-231 breast cancer cell line proteome using an IA-rhodamine probe 16, which permitted facile SDS-PAGE detection of cysteine reactivity events. This experiment identified several proteins that showed reductions in IA-rhodamine labeling in the presence of one or more fragments (FIG. 1C, asterisks). Interestingly, the proteins exhibited distinct SARs across the test fragment set, indicating that the library recognition elements exert a strong influence over specific fragment-protein reactivity events.
[0452] Competitive isoTOP-ABPP was used to globally map human proteins and the cysteine residues within these proteins that were targeted by fragment electrophiles. Each fragment was tested, in general, against two distinct human cancer cell proteomes (MDA-MB-231 and Ramos cells) and most fragments were screened in duplicate against at least one of these proteomes. On average, 927 cysteines were quantified per data set, and it was required that individual cysteines were quantified in at least three data sets for interpretation. Based on these criteria, more than 6157 cysteines from 2885 proteins were quantified in aggregate across all data sets with an average quantification frequency of 22 data sets per cysteine (FIG. 4A). Fragment-competed cysteine residues, or "liganded" cysteines, were defined as those showing .gtoreq.75% reductions in IA-alkyne labeling (R values.gtoreq.4 for DMSO/fragment). To minimize the potential for false-positives, only cysteines that showed R values.gtoreq.4 in two or more data sets and met additional criteria for data quality control were considered as targets of the fragment electrophiles. The proteomic reactivity values, or liganded cysteine rates, of individual fragments were then calculated as the percentage of liganded/total quantified cysteines in isoTOP-ABPP experiments performed on that fragment.
[0453] Most fragment electrophiles showed a tempered reactivity across the human proteome, with a median liganded cysteine rate of 3.8% for the library (FIG. 4B). Substantial differences in reactivity were, however, observed, with individual electrophiles showing liganded cysteine rates of <0.1% and others displaying rates >15% (FIG. 4B). That piperlongumine and locostatin fell into the latter category indicated the intrinsic proteomic reactivity of the fragment electrophiles did not, in general, exceed that of previously described electrophilic probes. A subset of fragments was also screened at lower concentrations (25-50 .mu.M), which confirmed that their proteomic reactivities were concentration-dependent (FIG. 4C). The relative reactivity of fragment electrophiles was similar in MDA-MB-231 and Ramos cell proteomes (FIG. 4D), indicating that this parameter is an intrinsic property of the compounds. Fragments also showed consistent reactivity profiles when assayed in biological replicate experiments (FIG. 4E). Interestingly, it was found that the proteomic reactivity of fragment electrophiles was only marginally correlated with their glutathione adduction potential, which is a commonly used surrogate assay for measurements of proteinacious cysteine reactivity (FIG. 4F). These differences are attributed to the impact of the recognition element of fragment electrophiles on their interactions and, ultimately, reactivity with proteins.
[0454] A comparison of fragments 3, 14, 17, and 23-26 provided insights into the relative proteomic reactivity of different electrophilic groups coupled to a common recognition element (3,5-di(trifluoromethyl)phenyl group). Chloroacetamide 3 exhibited greater reactivity than acrylamide 14 (15% versus 3.4% liganded cysteines, respectively; FIG. 1D), with cyanoacrylamide 23 exhibiting similar reactivity to acrylamide 14 and other, more sterically congested electrophiles (24-26) showing reduced proteomic reactivity (FIG. 4G). Importantly, the non-electrophilic acetamide control fragment 17 showed negligible activity in competitive isoTOP-ABPP experiments (liganded cysteine rate <0.2%) (FIG. 1D), indicating that the vast majority of detected fragment-cysteine interactions reflected covalent reactions versus non-covalent binding events. Also in support of this conclusion, "clickable" alkyne analogues of 3 and 14 (compounds 19 and 18, respectively) exhibited different concentration-dependent proteome labeling profiles (19>18; FIG. 1E) that mirrored the respective liganded cysteine rates displayed by 3 and 14 in competitive isoTOP-ABPP experiments (3>14; FIG. 1D). Despite the greater overall proteomic reactivity of chloroacetamide 3 relative to acrylamide 14 and cyanoacrylamide 23, clear examples of cysteines were found that were preferentially liganded by the latter fragments (FIG. 1F).
[0455] In some instances, these findings demonstrate that the isoTOP-ABPP platform is one method for use to competitively profile fragment electrophiles against thousands of cysteine residues in native proteomes.
Cysteines Targeted by Fragment Electrophiles in Native Proteomes
[0456] Across all isoTOP-ABPP data sets combined, 758 liganded cysteines were identified on 637 distinct proteins, which corresponded to .about.12 and 22% of the total quantified cysteines and proteins, respectively (FIG. 5A and Tables 1-3). Only a modest fraction of the proteins harboring liganded cysteines were found in the DrugBank database (15%; FIG. 5B), indicating the fragment electrophiles targeted many proteins that lack small-molecule probes. Among protein targets with known covalent ligands, the fragment electrophiles frequently targeted the same cysteine residues as these known ligands (Table 4); examples include the protein kinase BTK, in which electrophilic fragments targeted an active-site cysteine that also reacts with the cancer drug ibrutinib, and XPO1 and ERCC3, in which electrophilic fragments targeted conserved cysteines that are modified by bioactive natural products and candidate anti-cancer agents. In the case of BTK, it was confirmed that the interaction of ibrutinib with this kinase was detected by isoTOP-ABPP, which also identified a known ibrutinib off-target--MAP2K7--in Ramos cell lysates (FIG. 7A).
[0457] DrugBank proteins with liganded cysteines mostly originated from classes that are regarded as "druggable", including enzymes, channels, and transporters (FIG. 5C). Non-DrugBank proteins with liganded cysteines, on the other hand, showed a broader class distribution that included proteins, such as transcription factors and adaptor/scaffolding proteins, that are considered challenging to target with small-molecule ligands (FIG. 5C). Even among the enzymes targeted by fragment electrophiles, many examples were noted where the liganded cysteine was a non-active site residue (FIG. 7B). These data indicated that the cysteines modified by fragment electrophiles were not restricted to classical ligand-binding pockets on proteins. Also consistent with this premise, only .about.6% of all of the liganded cysteines were functionally annotated as active-site residues (FIG. 5D). Active-site cysteines, as well as redox-active cysteines, were still, however, substantially enriched among the liganded cysteine group compared to unliganded cysteines quantified by isoTOP-ABPP (FIG. 5D). It had been previously found that active-site and redox-active cysteines also show, in general, greater intrinsic reactivity (as measured with the IA-alkyne probe) compared to other cysteines. While this heightened reactivity is a likely contributory factor to the ligandability of cysteines, as reflected in the high proportion of hyperreactive cysteines that were detected as targets of fragment electrophiles (FIG. 5E), liganded cysteines were also well-represented across a broad range of intrinsic reactivities (FIG. 5E). Finally, most proteins were found to harbor a single liganded cysteine among the several cysteines that were, on average, quantified per protein by isoTOP-ABPP (FIG. 5F). The nuclear export factor XPO1 and metabolic enzyme PHGDH provide compelling examples of the selectivity displayed by fragment electrophiles for individual cysteines within proteins (FIG. 5G and FIG. 7C). Among the six different XPO1 cysteine residues quantified by isoTOP-ABPP, a single cysteine, C528, was frequently targeted by fragment electrophiles (FIG. 5G), and this residue is also modified by electrophilic drugs in clinical development for cancer.sup.40. Similarly, among eight quantified cysteines in PHGDH, only C369, a non-active site residue, was targeted by electrophilic fragments (FIG. 7C).
[0458] Liganded cysteines displayed strikingly distinct SARs with the fragment electrophile library (FIG. 6A and Tables 1-3). While a handful of cysteines were targeted by a large number of fragments (>50%), most cysteines exhibited more restricted reactivity (FIG. 6A, B and Tables 1-3). The operational grouping of fragment electrophiles based on their relative proteomic reactivity values (group A, >10%; group B, <10%) revealed SAR features that emphasized both the recognition and reactivity components of cysteine-electrophile interactions. Certain cysteines, for instance, preferentially interacted with the less reactive (group B) fragments (e.g., GLRX5; MSTO1; SRP9; UCHL3; FIG. 6A), while others were mainly liganded by the most reactive (group A) fragments (e.g., ATXN7L3B; CRKL; C2ORF49; FIG. 6A), although, even in these cases, the interactions differed substantially across group A fragments. Liganded cysteines located in the active sites of proteins tended to show broader reactivity with the fragment electrophiles compared to other cysteines (FIG. 6C), possibly reflecting their greater ligandability, but clear SARs were observed for many non-active site cysteines and these residues were not disproportionately targeted by group A fragments (FIG. 6D). These principles applied across different protein classes and were well-exemplified in kinases, for which >20 liganded cysteines were identified that distributed near-evenly between active- and non-active-site residues (FIG. 7D-F). Even cysteines found in proteins considered challenging to drug, such as transcription factors/regulators, showed distinct SARs indicative of specific interactions involving both binding and reactivity (FIG. 6D and FIG. 9G). In addition, about greater than 60% of liganded cysteines, electrophile (IA-alkyne or fragment) reactivity was blocked by heat denaturation of the proteome, while about a fraction of unliganded cysteines (about 20%) showed decreased IA-alkyne labeling following heat denaturation (FIGS. 15 and 16). In some instances, these results showed that the ligand-cysteine interactions are specific in that they depend on both the binding groups of ligands and structured sites in protein.
[0459] The availability of three-dimensional structures for a subset of proteins with liganded cysteines provided an opportunity to test whether docking predicts sites of fragment electrophile reactivity. Covalent docking programs have recently been introduced to discover ligands that target pre-specified cysteines in proteins; here, however, the aim was to computationally assess the relative ligandability of all cysteines within a protein and match these outputs to the data acquired in isoTOP-ABPP experiments. First, 29 representative protein targets were scanned and 99 solvent-accessible cysteines were identified. Then, the fragment electrophile library was docked on each residue independently using a modified potential to simulate non-covalent interactions preceding the alkylation event. In cases where the fragment electrophile bound favorably near a cysteine and the reactive group was within covalent bond distance of the cysteine, the cysteine was considered to be modified by the fragment. Docking scores were then calculated based on the estimated interaction energy of each fragment in its docked pose, and the ranking of these predictions matched the experimental data in 19 out of the 29 systems (i.e., cases where the top predicted ligandable cysteine matched the liganded cysteine determined by isoTOP-ABPP) (FIG. 6E, F and Table 5). In six out of the remaining 10 systems, the liganded cysteines were ranked second by reactive docking. In the remaining four systems, reactive docking failed to predict the liganded cysteine due to limitations in the docking scoring function or structural issues in the models used. Notably, across the entire 29 proteins evaluated by reactive docking, it was found that cysteines predicted to be ligandable were much more likely to have been detected by isoTOP-ABPP compared to cysteines not predicted to be ligandable (FIG. 6E and FIG. 7H). It was also found that cysteines predicted to be ligandable were more likely to have been detected by isoTOP-ABPP and exhibited heat-sensitive IA-alkyne reactivity (FIG. 17A and FIG. 17B). These results indicate that reactive docking provides a good overall prediction of the ligandability of proteinaceous cysteines and suggest that IA-alkyne reactivity itself provides an independent experimental parameter useful for designating potentially ligandable cysteines in proteins.
Functional Analysis of Ligand-Cysteines Interactions
[0460] The next step was to confirm and determine the functional impact of ligand-cysteine interactions mapped by isoTOP-ABPP using recombinant proteins. Two proteins were selected for which the functional significance of the liganded cysteines had been previously demonstrated. The protein methyltransferase PRMT1 possesses a non-catalytic active-site cysteine (C109) that, when modified by electrophilic small molecules like 4-hydroxynonenal (HNE), results in the inhibition of PRMT1 activity.sup.27. Competitive isoTOP-ABPP revealed a very selective SAR for ligand engagement of C109 of PRMT1, with only three fragments (2, 11, and 51) blocking IA-alkyne labeling of this residue (FIG. 6A and FIG. 8A and Tables 1-3). Even though several additional cysteines in PRMT1 were quantified in isoTOP-ABPP experiments (none of which showed sensitivity to the tested fragment electrophiles; FIG. 8A and Tables 1-3), it was found that IA-rhodamine labeling of recombinant PRMT1 was blocked by mutating C109 to serine (FIG. 8B). These data are consistent with past studies indicating that C109 is the most reactive cysteine in PRMT1 and is selectively labeled by low concentrations of electrophilic probes. Using a convenient SDS-PAGE readout, it was confirmed that fragment 11 blocked IA-rhodamine labeling of PRMT1 with an IC.sub.50 value of 36 .mu.M, whereas control fragment 3 was inactive (FIG. 8B, C), despite displaying similar overall proteome reactivity to 11 (FIG. 4B). Pre-treatment with 11, but not 3, also inhibited PRMT1-catalyzed methylation of histone 4 in a C109-dependent manner (FIG. 8D). These data indicate that electrophilic ligands targeting C109 act as PRMT1 inhibitors.
[0461] MLTK, or ZAK, which is a MAP3 kinase that possesses an active site-proximal cysteine residue C22 that is modified by HNE to feedback-inhibit JNK pathways under conditions of oxidative stress, was then examined. MLTK has also recently been implicated as an oncogenic driver in gastric cancer and is an off-target for ibrutinib, which reacts with C22 of MLTK. Competitive isoTOP-ABPP experiments identified a subset of fragment electrophiles that blocked IA-alkyne labeling of C22 in MLTK (FIG. 9A and Tables 1-3). The SAR provided by isoTOP-ABPP was verified and extended by testing fragments for blockade of labeling of recombinant MLTK using an ibrutinib-derived activity probe (FIG. 8E and FIG. 9B), which identified the benzofuran fragment 60 as having good potency for inhibiting MLTK (IC.sub.50 value of 2.6 .mu.M) and 3 as an inactive control probe (FIG. 8E, F and FIG. 9A, B). Fragment 60, but not 3, also blocked the catalytic activity of MLTK using a substrate phosphorylation assay, and this inhibitory effect was not observed with a C22A-MLTK mutant (FIG. 8G and FIG. 18).
[0462] Next, proteins were evaluated that possessed previously uncharacterized liganded cysteines. IMPDH2, which is the rate-limiting enzyme in de novo synthesis of guanine nucleotides and regulates immune cell proliferation and cancer, contained two liganded cysteines--C140 and C331--that showed overlapping, but distinct SARs in competitive isoTOP-ABPP experiments (FIG. 9C, D; FIG. 19 and Tables 1-3). C331 serves as a catalytic nucleophile and active site-directed inhibitors of IMPDH2 have been described. C140, on the other hand, is found in a separate Bateman domain of IMPDH2, which serves as a module for allosteric regulation by sensing nucleotides (FIG. 9D) and has not been shown to react with electrophilic small molecules. Therefore focused was placed on the characterization of C140. It was first confirmed that fragment 14 directly labeled C140 of recombinant IMPDH2 by MS methods (Table 6). An alkyne analogue of 14 (18: FIG. 8H) was then synthesized, which provided a means to directly monitor ligand interactions at C140 by click chemistry conjugation to a rhodamine-azide tag and SDS-PAGE analysis. Click probe 18 labeled WT-IMPDH2 and a C331S-IMPDH2 mutant, but not the C140S or C140S/C331S mutants of this enzyme (FIG. 8H). Using this assay, it was confirmed that 14, but not control fragment 8, inhibited the labeling of IMPDH2 by 18 (FIG. 9E). IMPDH2 labeling by 18 was also inhibited by nucleotides ATP, AMP, and GTP, but not UTP or IMP (FIG. 8I and FIG. 9F). ATP blocked 18 labeling of IMPDH2 with an IC.sub.50 value of 45 .mu.M (FIG. 8J). Thus, covalent ligands targeting the Bateman domain of IMPDH2 serves not only as inhibitors, but also probes of nucleotide binding to this enzyme.
[0463] Two liganded cysteines--C114 and C161--were also identified in the p53-induced phosphatase TIGAR (FIG. 9G, H). In some instances, TIGAR acts as both a fructose-2,6-bisphosphatase and 2,3-bisphosphoglycerate phosphatase to shape the metabolic state of cancer cells and protect them from ROS-induced apoptosis. Inhibitors of TIGAR have not been described. C114 is found on the lid of the TIGAR active site, .about.15 .ANG. from the phosphate substrate binding site (FIG. 9H). C161 resides on the opposite side of the protein. Focus was placed on the characterization of fragment labeling of C114 given its proximity to the TIGAR active site. It was first confirmed that both C114 and C161 of recombinant TIGAR were labeled by the IA-rhodamine probe and this labeling was partly diminished in C114S and C161S single mutants and fully blocked in a C114S/C116S double mutant of TIGAR (FIG. 9I). It was also verified interactions of hit fragment 5 with C114 of TIGAR by LC-MS analysis (Table 6) and by showing that the fragment blocked IA-rhodamine labeling of a C161 S-TIGAR mutant with an IC.sub.50 value of 16 .mu.M (FIG. 8K, L); in contrast, the control fragment 3 showed much lower potency (FIG. 8K, L), 5 also blocked the catalytic activity of WT- and C161S-, but not C114S- or C114S/C161 S-TIGAR using a substrate assay (FIG. 8M). Control fragment 3 did not affect TIGAR catalytic activity (FIG. 8L). Inhibition of TIGAR substrate turnover by 5 plateaued at 70% (FIG. 9J), which indicates that the covalent ligand acts by an allosteric mechanism or does not extend fully into the active site of TIGAR to produce complete inhibition.
Electrophilic Ligands that Inhibit IDH1 Activity in Cancer Cells
[0464] Isocitrate dehydrogenase 1 (IDH1) and 2 (IDH2) are mutated in a number of human cancers to produce enzyme variants with a neomorphic catalytic activity that converts isocitrate to 2-hydroxyglutarate (2-HG). Increases in 2-HG inhibit .alpha.-ketoglutarate-dependent dioxygenases that function as tumor suppressors, in particular, by methylating DNA and proteins. Competitive isoTOP-ABPP experiments identified distinct subsets of ligands that targeted a conserved cysteine in IDH1 and IDH2 (C269 and C308, respectively; Tables 1-3). This cysteine is an active site-proximal residue that is 13 .ANG. from the NADP.sup.+ molecule in a crystal structure of IDH1 (FIG. 10A); glutathionylation of C308 has previously been shown to block IDH2 activity, but, to our knowledge, irreversible inhibitors of IDH enzymes have not been characterized.
[0465] The functional significance of ligand interactions with IDH enzymes by recombinantly expressing wild type (WT) and a C269S mutant of IDH1 was explored. WT-, but not C269S-IDH1 reacted with the IA-rhodamine probe as detected by SDS-PAGE, and fragment electrophiles blocked this reaction with an SAR that mirrored that observed for endogenous IDH1 in competitive isoTOP-ABPP experiments (FIG. 11A and Tables 1-3). Fragment 20 inhibited IA-rhodamine labeling of WT-IDH1 with an IC.sub.50 value of 2.9 .mu.M (FIG. 11B and FIG. 10B) and showed similar activity with the R132H oncogenic mutant of IDH1 (FIG. 10C and FIG. 20). It was also confirmed by isoTOP-ABPP that 20 (25 .mu.M) completely blocked IA-alkyne labeling of endogenous IDH1 in MDA-MD-231 proteomes (R value=20; FIG. 10D) and, by MS analysis, that 20 directly modifies C269 of IDH1 (Table 6). Fragment 2 showed much less activity against C269 of IDH1 (IC.sub.50>50 .mu.M; FIG. 11B and FIG. 10B) and was therefore selected as a control probe. It was found that 20 blocked in a concentration-dependent manner the catalytic activity of WT-IDH1 (as measured by the reduction of NADP.sup.+ to NADPH in the presence of isocitrate), but did not inhibit the activity of the C269S-IDH1 mutant (FIG. 11C). The in situ activity of 20 was also tested by generating a human cancer cell line that stably overexpressed R132H-IDH1 (FIG. 10E). The R132H-IDH1 cells were treated with fragments 20 and 2 for 2 h, lysed, and assayed ex situ for 2-HG production, 20 (50 .mu.M) near-completely blocked 2-HG production by R132H cell lysates, while 2 (50 .mu.M) only caused a slight decrease in this activity (FIG. 11D). Parallel competitive isoTOP-ABPP experiments confirmed that fragment 20, but not fragment 2 inhibited IA-alkyne labeling of C269 of IDH1 in situ (FIG. 10F).
Global Profiling of Cysteine-Reactive Fragments in Cells
[0466] Encouraged by the cellular activity of the IDH1 ligand 20, the capacity of fragment electrophiles to modify proteinaceous cysteines in situ was more broadly assessed. MDA-MB-231 and Ramos cells were treated with representative members of the fragment library (23 compounds tested in total; each compound tested at 200 .mu.M, 2 h in situ treatment), and the cells were then harvested, lysed, and analyzed by isoTOP-ABPP. A handful of fragments were cytotoxic to cells and re-tested at lower (50 or 100 .mu.M) concentrations. The tested fragments showed a broad range of in situ reactivities that generally matched their respective reactivities in vitro (FIG. 11E and Tables 1-3). Some fragments, however, showed somewhat greater reactivity in cells, while fragment 11 was notably devoid of activity in situ (FIG. 11E). These differences reflect the impact of transport and/or metabolic pathways on the cellular concentrations of fragment electrophiles. A substantial fraction (64%) of the liganded cysteines identified in cell lysates were also sensitive to the same electrophilic fragments in cells (FIG. 11F). A handful of fragment-cysteine interactions were also observed selectively in situ, but not in lysates, including C182 of p53 (TP53), a redox-regulated residue at the dimerization interface of the DNA binding domain.sup.50 (FIG. 11G). In some instances, these liganded cysteines require an intact cellular environment to preserve their interactions with fragment electrophiles. Taken together, these findings indicate that the ligandability of cysteine residues is generally similar in lysates and cells, although exceptional cases underscore the importance of having the capability to perform ligand discovery experiments in situ.
Electrophilic Ligands that Target Pro-Caspase-8 and Block Extrinsic Apoptosis
[0467] Several fragments targeted the catalytic cysteine nucleophile C360 of the protease caspase-8 (CASP8) in isoTOP-ABPP experiments performed in vitro and in situ (FIG. 12A and Tables 1-3). CASP8 plays important roles apoptosis, immune cell proliferation, and embryonic development, but selective, non-peptidic, and cell-active inhibitors for this protease are lacking. Representative fragment hits against recombinant, active CASP8 were screened using substrate and activity-based probe (Rho-DEVD-AOMK probe ("DEVD" disclosed as SEQ ID NO: 857)) assays and observed marginal to no inhibition with most fragments (FIG. 12B). Initially puzzled by this outcome, it was hypothesized that fragment labeling of CASP8 in isoTOP-ABPP experiments might reflect reaction with the inactive zymogen (pro-) rather than active form of this protease. Western blots confirmed that most, if not all of the CASP8 in MDA-MB-231 cell lysates existed in the pro-form (FIG. 12C). Next a recombinant form of pro-CASP8 was expressed with mutated cleavage sites (D374A and D384 .ANG.) to prevent processing and activation. A non-catalytic cysteine C409S of pro-CASP8 was also mutated, which enabled detection of C360 labeling with IA-rhodamine by SDS-PAGE analysis (FIG. 13A). Several hit fragments detected in isoTOP-ABPP experiments completely blocked IA-rhodamine labeling of pro-CASP8 (FIG. 12D). Fragment 7 displayed the highest potency, with an IC.sub.5s value of .about.5 M (FIG. 13A, B), which, when combined with the low overall proteome reactivity of this fragment (3%), designated it as suitable tool compound for further studies.
[0468] Fragment 7 (50 .mu.M) fully blocked IA-alkyne labeling of C360 of CASP8 in isoTOP-ABPP experiments performed in both Ramos and Jurkat cell lysates (FIG. 13C). Next, a clickable analogue of 7 (61) was synthesized and it was found that this probe (25 .mu.M) strongly labeled pro-CASP8, but not a C360S-pro-CASP8 mutant (FIG. 13D and FIG. 12E). 7 (50 .mu.M) blocked labeling of pro-CASP8 by 61, but did not inhibit labeling of active CASP8 by the Rho-DEVD-AOMK probe ("DEVD" disclosed as SEQ ID NO: 857) developed to target active caspases (FIG. 13D and FIG. 12F). Conversely, the general caspase inhibitor Ac-DEVD-CHO ("DEVD" disclosed as SEQ ID NO: 857) (20 .mu.M) blocked Rho-DEVD-AOMK ("DEVD" disclosed as SEQ ID NO: 857) labeling of active CASP8, but not 61 labeling of pro-CASP8 (FIG. 13D, FIG. 12F, and FIG. 21A). Similar results were obtained in substrate assays, where DEVD-CHO ("DEVD" disclosed as SEQ ID NO: 857), but not 7, blocked CASP8 activity (FIG. 13E). Cross-reactivity of 7 with other caspases was not observed, including recombinant, active CASP3 assayed with a substrate (FIG. 13E) or the Rho-DEVD-AOMK probe ("DEVD" disclosed as SEQ ID NO: 857) (FIG. 12F) or CASP2 and CASP7 in cell lysates measured by isoTOP-ABPP (FIG. 12G). Finally, to further verify that 7 preferentially reacts with pro-CASP8 over active CASP8 in complex biological systems, recombinant forms of these proteins were doped into MDA-MB-231 cell lysates followed by treatment with 7 (30 .mu.M, 1 h) or DMSO and analysis by isoTOP-ABPP, 7 produced a near-complete blockade of IA-alkyne labeling of C360 for pro-CASP8 (R=10), but had little effect on IA-alkyne reaction with C360 of active CASP8 (R=1.9) (FIG. 13F).
[0469] Treatment of Jurkat cell lysates with 10 or 100 .mu.M of 61, followed by analysis of the combined samples by isoTOP-ABPP, confirmed direct labeling of C360 of CASP8 by 61 (FIG. 12H). The low R value observed for C360 in this analysis (R=2) indicated near complete labeling of this cysteine by 61 at 10 .mu.M in cell lysates, consistent with the low .mu.M IC.sub.50 value displayed by the parent fragment 7 for inhibiting IA-rhodamine labeling of C360 of CASP8 (FIG. 13B). The effect of pro-CASP8 inhibition in cellular apoptosis assays was next to be evaluated. Because C360 is the catalytic nucleophile of CASP8, mutation of this residue was not possible to create a control protein for evaluating the pharmacological effects of 7 in cells. Instead, a structurally related inactive probe was developed for this purpose. It was found that bulky substituents placed on the aniline ring of 7 furnished compounds such as 62 that did not inhibit pro-CASP8 labeling by IA-rhodamine (FIG. 13B, G). It was confirmed that 62 also did not inhibit active CASP3 or CASP8 using substrate (FIG. 13E) and activity-probe (FIG. 12F) assays and was inactive against endogenous CASP8, CASP2, or CASP7 in Jurkat lysates as determined by isoTOP-ABPP (FIG. 12G). Based on these data, 62 was designed as a suitable inactive control probe for studying the inhibition of pro-CASP8 by 7. Jurkat cells were treated with 7 or 62 (30 .mu.M, 30 min) prior to addition of FASL or staurosporine (STS) to induce extrinsic and intrinsic apoptosis, respectively, 7, but not 62, completely blocked FASL-induced apoptosis (FIG. 13H and FIG. 21B-C), as well as the proteolytic processing of CASP3, CASP8, and the apoptosis marker PARP (FIG. 13I). In contrast, 7 did not block STS-induced intrinsic apoptosis (FIG. 13H) or the cleavage of PARP and CASP3, although the compound did substantially inhibit cleavage of CASP8 in these cells (FIG. 13I). The non-selective caspase inhibitor VAD-FMK prevented both FASL- and STS-induced apoptosis and associated proteolytic processing events (FIG. 13H, I). Chemical proteomic experiments revealed that 7 fully inhibited CASP8, as well as the related initiator caspase CASP10 (but not other caspases, including CASP2, 3, 6, and 9) in Jurkat cells (FIG. 14A and FIG. 22A). It was confirmed that 7 blocked labeling of pro-CASP10 by 61 with an apparent IC50 value of 4.5 .mu.M (FIG. 22B-D), but did not inhibit active CASP10 as measured by labeling with the Rho-DEVD-AOMK probe ("DEVD" disclosed as SEQ ID NO: 857) (FIG. 21A) or a substrate assay (FIG. 22E). As such, in some instances, 7 blocking CASP8 processing in both FASL- and STS-treated cells supports a model where CASP8 activation mainly occurs through auto-processing in either extrinsic or intrinsic apoptosis, but is only required for the former type of programmed cell death.
[0470] In some instances, the respective functions of CASP8 and CASP10 in extrinsic apoptosis and other cellular processes remain poorly understood in large part due to a lack of selective, non-peptidic, and cell-active inhibitors for these enzymes and the absence of animal models for CASP10 (which is not expressed in rodents). In some cases, the potency and selectivity of 7 was improved to address this issue. Conversion of the 4-piperidino moiety to a 3-piperidino group and addition of a p-morpholino substituent to the benzoyl ring of 7 furnished compound 63 that was separated by chiral chromatography into its two purified enantiomers, 63-R (FIG. 4c) and 63-S, the former of which showed substantially improved activity against CASP8 (apparent IC.sub.50 value of 0.7 .mu.M (95% CI, 0.5-0.8); FIG. 22F-H) and negligible cross-reactivity with CASP10 (IC.sub.50 value>100 .mu.M; FIG. 22C, D, F), 63-S was much less active against CASP8 (apparent IC.sub.50 value of 15 .mu.M, FIG. 22G, H) and also inactive against CASP10 (FIG. 14A). With dual CASP8/10 (7) and CASP8-selective (63-R) ligands in hand, we next set out to investigate the biological functions of these proteases.
[0471] The effects of caspase ligands in human T cells were evaluated, where both CASP8 and CASP10 are highly expressed (FIG. 22I) in Jurkat cells, which are a commonly studied immortalized human T cell line. It was found that 63-R fully blocked FasL-induced apoptosis in Jurkat cells and did so with greater potency than 7 (FIG. 14B and FIG. 22J) or 63-S (FIG. 22K). Similar results were obtained in HeLa cells, which express CASP8, but not CASP1026 (FIG. 22L). In contrast to these cell line results, FasL-induced apoptosis in primary human T cells showed substantial resistance to 63-R at all tested concentrations and instead was completely inhibited by the dual CASP8/10 ligand 7 (FIG. 14B). It was confirmed by chemical proteomics with probe 61 that 7 blocked both CASP8 and CASP10, while 63-R inhibited CASP8, but not CASP10, in primary human T cells and Jurkat cells (FIG. 14A). Consistent with these cell death results, 7, but not 63-R, prevented proteolytic processing of CASP3 and CASP10 in primary human T cells (FIG. 22M). In some instances, the processing of both CASP8 and the initiator caspase substrate RIP kinase were also preferentially inhibited by 7 versus 63-R (FIG. 22M, indicating that CASP10 also contribute to these proteolytic events in T cells, as has been suggested by biochemical studies.
Example 2
[0472] Dimethyl fumarate (DMF) is a drug used to treat autoimmune conditions, including multiple sclerosis and psoriasis. In some instances, the mechanism of action of DMF is unclear, but is proposed to involve covalent modification of proteins and/or serving as a pro-drug that is converted to monomethyl fumarate (MMF). Using an isoTOP-ABPP approach, the mechanism of action of DMF is examined.
Chemical Reagents
[0473] Assays were performed with the following reagents: dimethyl fumarate (DMF; 242926; Sigma Aldrich), monomethyl fumarate (MMF; 651419; Sigma Aldrich), dimethyl succinate (DMS: W239607; Sigma Aldrich), and buthionine sulfoximine (BSO; 14484; Cayman Chemical).
Isolation of Primary Human T Cells
[0474] All studies using samples from human volunteers follow protocols approved by the TSRI institutional review board. Blood from healthy donors (females aged 30-49) were obtained after informed consent. Peripheral blood mononuclear cells (PBMCs) were purified over Histopaque-1077 gradients (10771; Sigma) following the manufacturer's instructions. Briefly, blood (20.times.25 mL blood aliquots) were layered over Histopaque-1077 (12.5 mL) and the samples were then fractionated by centrifugation (2000 rpm, 20 min, 20.degree. C., no brake). PBMC's were harvested from the Histopaque-plasma interface and washed twice with PBS. After that time, the T cells were isolated using an EasySep.TM. Human T Cell Isolation Kit (17951; STEMCELL) per the manufacturer's instructions.
Mice
[0475] C57BL/6J and Nrf2' mice (Stock No:017009; Nfe212.sup.tm1Ywk; Jackson Labs) were bred and maintained in a closed breeding facility at The Scripps Research Institute and were 6-8 weeks old when used in experiments. All mice were used in accordance with guidelines from the Institutional Animal Care and Use Committee of The Scripps Research Institute.
[0476] For the PKC.theta. studies, C57BL/6 mice and Prkcq-/- mice were housed under specific pathogen-free conditions and used in accordance with a protocol approved by the La Jolla Institute for Allergy and Immunology Animal Care Committee.
Isolation of Primary Mouse T Cells
[0477] Spleens were harvested from female mice, perfused with collagenase, and incubated at 37.degree. C. with 5% CO.sub.2 for 30 min. After this time, the spleens were homogenized. Cells that passed through a 100 mun cell strainer were collected and washed with RPMI. T cells were isolated from the splenocytes using the EasySep.TM. Mouse T cell Isolation Kit (19851; STEMCELL) according to manufacturer's instructions.
[0478] For the PKC.theta. studies, CD4' T cells were isolated by anti-mouse CD4 magnetic particles (L3T4; BD IMag) and were cultured in RPMI-1640 medium (Gibco) supplemented with 10% (vol/vol) heat-inactivated FBS, 2 mM glutamine, 1 mM sodium pyruvate, 1 mM MEM nonessential amino acids, 100 U/mL each of penicillin G and streptomycin (Life Technologies) and recombinant IL-2 (100 U/mL, Biolegend).
T Cell Stimulation
[0479] 96-well plates were coated with anti-CD3 (1:200; BioXcell) and anti-CD28 (1:500; 302933; BioLegend) in PBS (100 .mu.L/well) overnight at 4.degree. C. The plates were then washed twice with PBS and to each well was added 500,000 primary T cells in 100 .mu.L of RPMI supplemented with 10% FBS, glutamine, and Pen-Strep. Cells were then treated with 100 .mu.L of media containing compound at the indicated concentrations (final well volume of 200 .mu.L). Cells were left at 37.degree. C. in a 5% CO.sub.2 incubator for the indicated periods of time and harvested by centrifugation (500 g, 8 min, 4.degree. C.), followed by washing with PBS.
Cellular Analysis and Sorting by Flow Cytometry
[0480] Cells were transferred to a round bottom 96-well plate (0720095; Fisher Scientific), harvested by centrifugation (500 g, 3 min, 4.degree. C.), washed with PBS, and stained with LIVE/DEAD fixable cell stain (L23105; ThermoFisher) according to the manufacturer's instructions. Briefly, one vial of LIVE/DEAD stain was resuspended in 50 uL of DMSO and added to 20 mL of PBS. To each well of the 96-well plate was added 200 .mu.L of the stain, and the cells were incubated on ice for 30 min in the dark. After this time, cells were pelleted and washed once with PBS, then stained for cell surface antigens.
[0481] Flow cytometry analysis of cell surface antigens was performed with the following antibodies: Pacific Blue-conjugated anti-CD8 (1:25 dilution; clone RPA-T8; BD Biosciences), APC-conjugated anti-CD4 (1:25 dilution; clone RPA-T4; eBioscience), phycoerythrin-conjugated anti-CD25 (1:25 dilution; clone BC96; eBioscience or PC61; BioLegend (PKC.theta. studies)), FITC-conjugated anti-CD69 (1:25 dilution; clone FN50; eBioscience). All antibodies were diluted in 1% FBS in PBS, and 50 .mu.L of the stain solution was added to each well. Cells were stained for 15 min on ice in the dark, after which cells were harvested by centrifugation (500 g, 3 min, 4.degree. C.), washed with 1% FBS in PBS, and resuspended in 200 .mu.L/well of 4% PFA in PBS. Flow cytometrv acquisition was performed with BD FACSDiva.TM.-driven BD.TM. LSR II flow cytometer (Becton, Dickinson and Company). Data was then analyzed with FlowJo software (Treestar Inc.). Data represent mean.+-.SE for four-five experiments per group.
Quantification of Secreted Cytokines by Enzyme-Linked Immunosorbent Assay (ELISA)
[0482] T cells were harvested and stimulated as described above. At the indicated time points, cell culture supernatants were collected and IL-2 levels were measured in clear microplates (991427; R&D Systems) according to the manufacturer's instructions (Human IL-2 DuoSet ELISA; DY202; R&D Systems). Plates were read in a Gemini SpectraMax 250 microplate reader set to 450 nm. Data represent mean.+-.SE for four experiments per group.
[0483] For the PKC.theta. studies, aliquots of transduced Prkcq.sup.-/-CD4+ T cells (1.times.10.sup.6) were stimulated for 48 h with anti-CD3 alone or anti-CD3 plus anti-CD28, and the concentration of IL-2 in culture supernatants was determined by enzyme-linked immunosorbent assay according to the manufacturer's instructions (BioLegend). Briefly, a 96-well plate (Corning Costar) was coated overnight at 4.degree. C. with mAb to IL-2. Triplicates of IL-2 standards and supernatants from cultured cells were then added to the plate, followed by 2h incubation at room temperature. A biotinylated polyclonal antibody to IL-2 was added to the plate, followed by incubation for 1 h at room temperature, and then Avidin-HRP was added, followed by incubation for 30 min at room temperature. The amount of bound avidin was then assessed with TMB peroxidase that was acidified by 2 N H.sub.2SO.sub.4. The absorbance of each well at 450 nm was then measured with a spectrophotometric plate reader (BioTek).
Quantification of Cellular Glutathione (GSH) Levels
[0484] Primary human T cells (2.5 million cells/mL, 20 mL per condition) were treated as indicated, harvested by centrifugation (500 g, 8 min, 4.degree. C.), and washed twice with PBS. To the cell pellet was added 75 .mu.L of lysis buffer. After vortexing, the samples were incubated on ice for 15 min, then harvested by centrifugation (16,000 g, 10 min, 4.degree. C.). Protein concentrations were adjusted to at least 5 mg/mL and the assay performed according to manufacturer's instructions (Sigma-Aldrich, CS1020). Data represent mean.+-.SE for two biological replicates.
Protein Labeling and Click Chemistry
[0485] Cells were lysed by sonication and diluted to a concentration of 2 mg protein/mL. Protein concentrations were measured with the Bio-Rad DC.TM. protein assay reagents A and B (5000113, 5000114; Bio-Rad). 500 .mu.L of proteome sample was treated with 100 .mu.M of IA-alkyne probe using 10 .mu.L of a 10 mM DMSO stock. The labeling reactions were incubated at room temperature for 1 h upon which time the samples were conjugated to isotopically-labeled TEV-cleavable tags (TEV tags) by copper-catalyzed azide-alkyne cycloaddition (CuACC or `click chemistry`). 60 .mu.L of heavy click chemistry reaction mixture was added to the DMSO-treated control sample and 60 .mu.L of the light reaction mixture was added to the compound-treated sample. The click reaction mixture comprised TEV tags (10 .mu.L of a 5 mM stock, light (fragment treated) or heavy (DMSO treated)), CuSO.sub.4 (10 .mu.L of a 50 mM stock in water), and TBTA (30 .mu.L of a 1.7 mM stock in 4:1 tBuOH:DMSO). To this was added TCEP (10 .mu.L of a 50 mM stock). The reaction was performed for 1 h at room temperature.
[0486] The light- and heavy-labeled samples were then centrifuged (16.000 g, 5 min, 4.degree. C.) to harvest the precipitated proteins. The resulting pellets were resuspended in 500 .mu.L of cold methanol by sonication and the heavy and light samples combined pairwise. Combined pellets were then washed with cold MeOH, after which the pellet was solubilized in PBS containing 1.2% SDS by sonication. The samples were heated at 90.degree. C. for 5 min and subjected to streptavidin enrichment of probe-labeled proteins, sequential on-bead trypsin and TEV digestion, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) according to the published isoTOP-ABPP protocols.
Peptide and Protein Identification
[0487] RAW Xtractor (version 1.9.9.2) was used to extract the MS2 spectra data from the raw files. MS2 data were searched against a reverse concatenated, nonredundant variant of the Human UniProt database (release-2012_11) using the ProLuCID algorithm. Cysteine residues were searched with a static modification for carboxyamidomethylation (+57.02146) and up to one differential modification for either the light or heavy TEV tags (+464.28595 or +470.29976, respectively). Peptides were required to have at least one tryptic terminus and to contain the TEV modification. ProLuCID data was filtered through DTASelect (version 2.0) to achieve a peptide false-positive rate below 1%.
R Value Calculation and Processing
[0488] The quantification of heavy/light ratios (isoTOP-ABPP ratios, R values) was performed by in-house CIMAGE software using default parameters (3 MS1's per peak and signal to noise threshold of 2.5). Site-specific engagement of electrophilic compounds was assessed by blockade of IA-alkyne probe labeling. For peptides that showed a=95% reduction in MS1 peak area from the compound-treated proteome (light TEV tag) when compared to the DMSO treated proteome (heavy TEV tag), a maximal ratio of 20 was assigned. Overlapping peptides with the same labeled cysteine (for example, same local sequence around the labeled cysteines but different charge states, MudPIT segment numbers, or tryptic termini) were grouped together, and the median ratio from each group was recorded as the R value of the peptide for that run.
Analysis of Cysteine Conservation
[0489] For each human protein containing a DMF-sensitive cysteine, the mouse homolog was identified and the human and mouse sequences aligned using the Align tool on UniProt.
Immunofluorescent Analysis of NF-kB Translocation
[0490] Primary human T cells were harvested and stimulated as described above (500,000 cells/well), with concomitant treatment with DMSO or DMF for 60 min. Cells were pelleted (500 g, 3 min, 4.degree. C.), then each well was resuspended in 50 .mu.L PBS and added to Poly-D-lysine coated covcrslips (12 mm; 354087; Corning.RTM. BioCoat.TM.). Cells were allowed to adhere to the coverslips for 30-60 min at 4.degree. C. Coverslips were transferred to a 6 well plate and fixed with 4% PFA (157-4-100; Electron Microscopy Sciences) at room temperature for 10 min. After washing three times with PBS, cells were permeabilized with 0.1% Triton X-100 in PBS at room temperature for 10 min. Cells were washed three times with PBS, then placed cell-side-up on Parafilm. To each cover slip was added 150 .mu.L of blocking buffer (2% BSA in PBS), and the slides were blocked for 30 min at room temperature.
[0491] The blocking buffer was aspirated, coverslips placed face down in 40 .mu.L of antibody buffer (anti-human p65; p65Ab; FivePhoton Biochemicals; 1:500 dilution in blocking buffer), and allowed to stain overnight at 4.degree. C. in a wet chamber. Cover slips were washed three times with PBS, then incubated with 150 .mu.L of secondary antibody (anti-rabbit Alexa Fluor 488; A21441; Life Technologies; 1:200 dilution in PBS) for 2 h at room temperature. After washing three times with PBS, 150 .mu.L of Hoechst counter stain was added (5 .mu.g/mL in PBS) and coverslips were left at room temperature for 30-60 min. Cells were again washed with PBS three times, then stained with Alexa Fluor 555 Phalloidin red (8953S; Cell Signaling; 1:20 dilution in PBS). The coverslips were washed with PBS a final three times, then transferred to SuperFrost Plus slides (12-550-15, Fisherbrand) spotted with 10 .mu.L of Prolong, Gold Antifade Mountant (P36934. ThermoFisher). The circumference of each coverslip was sealed with clear nail polish (72180; Electron Microscopy Sciences).
[0492] Images were acquired using a Zeiss 780 laser scanning confocal microscope with a 63.times. Objective (0.3 um image step size) and the automated stitching module to merged (10% overlap) and create a three dimensional multi-paneled mega image composite. The composite image was gathered as a z-series of at least 9 individual image panels that were auto-merged using zen software. The mega-image composite was projected into a maximum image projection in the zen software then analyzed using the colocalization modual in Zen (Zeiss Inc) and Image Pro Premier (Media Cybernetics). The Mander's Correlation Coefficients (MCC), specifically M1 and M2 between the various combination of fluorescent label (Rhodamine Phalloidin vs NFkB-P65 and Hoechst vs NFkB-p65) are calculated in ZEN (Zeiss inc) per cell and displayed as a percent. Each cell was outlined using the region of interest module and the software then calculated the M1 and M2 correlation coefficients between the two fluorophores and tabulated the results. The fluorescent signal dynamic range and threshold cutoffof real signal was defined by multiple background and secondary controls. Correlation coefficient values were compared using Image Pro Premier (IPP) (Media Cybernetics), where images were imported as raw calibrated czi files and analyzed using a similar module in IPP. Similar results were obtained with both platforms (not shown). Data represent mean.+-.SE for two-three biological replicates.
Subcloning and Mutagenesis
[0493] QuikChange site-directed mutagenesis was performed on a pEF4 His A plasmid containing the full length human PKC.theta. (residues 1-707). The PKC.theta. insert was excised using BamHI and Xhol, then ligated into a pMIG vector.
PKC.theta. Retroviral Transduction and Stimulation
[0494] Platinum-E packaging cells were plated in a six-well plate in 2 mL RPMI-1640 medium plus 10% FBS. After 24 h, cells were transfected with empty pMIG vector or the appropriate PKC.theta.-expressing vector DNA (3 .mu.g) with TranslT-LTI transfection reagent (Mirns Bio). After overnight incubation, the medium was replaced and cultures were maintained for another 24 h. Retroviral supernatants were then collected and filtered, supplemented with 8 .mu.g/mL of polybrene and used to infect CD4.sup.+ T cells that had been pre-activated for 24 h with plate-bound monoclonal antibody to CD3 (8 .mu.g/mL) and CD28 (8 .mu.g/mL). After centrifuging plates for 1.5-2 h at 2,000 r.p.m., cell supernatants were replaced by fresh RPMI-1640 supplemented with 10% FBS and recombinant IL-2 (100 U/mL). Cells were incubated for another 24h at 37.degree. C. On day 3, cells were washed, moved to new plates and cultured in RPMI-1640 medium containing 10% FBS and recombinant IL-2 (100 U/mL) without stimulation for 2 additional days before restimulation with mAb to CD3 alone or plus mAb to CD28.
PKC.theta. Immunoprecipitation and Immunoblot Analysis
[0495] Cells were lysed in 1% (wt/vol) digitonin (D141, Sigma) lysis buffer (20 mM Tris-HCl. pH7.5, 150 mM NaCl, 5 mM EDTA) supplemented with protease inhibitors (10 .mu.g/mL aprotinin, 10 .mu.g/mL leupeptin and 1 mM PMSF) and phosphatase inhibitors (5 mM sodium pyrophosphate and 1 mM Na.sub.3VO.sub.4). Supernatants were incubated 2h with 1 .mu.g anti-CD28 mAb, and proteins were immunoprecipitated overnight at 4.degree. C. with protein G-Sepharose beads (GE Healthcare). The immunoprecipitated proteins were resolved by SDS-PAGE, transferred onto a PVDF membrane and probed overnight at 4.degree. C. with primary antibodies, followed by incubation for 1 h at room temperature with horseradish peroxidase (HRP)-conjugated secondary antibodies. Signals were visualized by enhanced chemiluminescence (ECL; GE Healthcare) and were exposed to X-ray film. Densitometry analysis was performed with ImageJ software. Immunoblotting antibodies to CD28 (C-20) and PKC.theta. (C-19) were obtained from Santa Cruz Biotechnology.
DMF, but not MMF, Inhibits T Cell Activation
[0496] Multiple sclerosis is an autoimmune disease with a prominent T cell component; as such, it was reasoned that DMF in some cases impact primary T cell activation. Consistent with this, previous reports have shown that DMF inhibits cytokine release from mouse splenocytes and promotes a Th2 phenotype via induction of IL-10-producing type II dendritic cells. The effects of DMF and MMF (FIG. 23A) were tested on cytokine release from primary human T cells activated with anti-CD3, anti-CD28 antibodies. Secretion of IL-2 was strongly inhibited by DMF, but not MMF (FIG. 23B). DMF, but not MMF or the non-electrophilic analogue dimethyl succinate (DMS, FIG. 23A) also blocked the expression of the early activation markers CD25 (FIG. 23C, D) and CD69 (FIG. 23E) in anti-CD3, anti-CD28-stimulated T cells. The blockade of T cell activation by DMF was concentration-dependent, with 10, 25 and 50 .mu.M of the drug producing marginal/negligible, partial, and near-complete inhibition, respectively (FIG. 23B, D, E). In some instances, the effects of DMF on cytokine release and activation markers occurred at concentrations of the drug that did not impair T cell viability (FIG. 24). Similar results were obtained with primary splenic T cells from C57BL/6 mice, the activation of which was also suppressed by DMF, but not MMF or DMS (FIG. 25). Of note, the inhibitory effects of DMF were reduced if the drug was added two hours after anti-CD3, anti-CD28 stimulation and completely ablated if the drug was added six hours after stimulation (FIG. 23F), suggesting that DMF inhibits an early event(s) in the T cell activation pathway
DMF Effects on T Cell Activation are Independent of Nrf2 and GSH
[0497] DMF is thought to produce neuroprotective effects through activating the Nrf2-Keap1 pathway, but whether this pathway contributes to the immunomodulatory effects of DMF is unclear. A recent study showed that DMF inhibits pro-inflammatory cytokine release from primary mouse splenocytes and this effect was comparable in wild type and Nrf2(-/-) splenocytes (Gillard, et al., "DMF, but not other fumarates, inhibits NF-kappaB activity in vitro in an Nrf2-independent manner," J. Neuroimmunol. 283, 74-85 (2015)). Consistent with this, it was found that the activation of Nrf2(+/+) and (-/-) T cells was similarly sensitive to inhibition by DMF (FIG. 26A). In some instances, DMF also impair T cell activation through depleting glutathione (GSH), and, indeed, DMF-treated primary human T cells showed a significant decrease in cellular GSH content (FIG. 26B). Significant reductions in GSH were, however, also observed with the GSH synthesis inhibitor buthionine sulfoximine (BSO), which had no effect on T cell activation (FIG. 26C, D). In some cases, these data indicate that the blockade of T cell activation by DMF involves processes other than Nrf2 activation or GSH depletion.
Chemical Proteomic Discovery of DMF-Sensitive Cys Residues in T Cells
[0498] The inhibition of T cell activation by DMF, but not the non-electrophilic analogues MMF and DMS, pointed to a mechanism that involves covalent reactivity with one or more proteins important for T cell function. As such, a globally inventory of DMF-sensitive Cys residues in primary human and mouse T cells were examined using the quantitative chemical proteomic platform isoTOP-ABPP. In this method, DMF is evaluated for its ability to block the reactivity of proteinaceous Cys residues with the general electrophilic probe iodoacetamide-alkyne (IA-alkyne). Using isotopically differentiated azide-biotin tags (containing a TEV protease-cleavable linker), Cys residues are identified and comparatively quantified for their IA-reactivity in cells treated with DMF versus DMSO control. Primary advantages of the isoTOP-ABPP platform include: 1) the competing electrophile does not itself need to be chemically altered for target identification, which is particularly beneficial when studying very small compounds like DMF; and 2) isotopic labeling occurs late in the sample processing, which facilitates the quantitative analysis of primary cells and tissues that are not readily amenable to metabolic labeling.
[0499] The isoTOP-ABPP method was performed on primary human T cells treated with DMSO or DMF (50 .mu.M, 4 h). Five independent replicates were performed, and the total aggregate number of unique quantified peptides and proteins began to plateau by the fourth and fifth replicate (FIG. 28), indicating that we approached maximal proteomic coverage of IA-reactive Cys residues in human T cells under the conditions employed. Of the more than 2400 quantified Cys residues, a small fraction (.about.40) showed substantial reductions (>four-fold; isoTOP-ABPP ratio (R value)>4) in IA-alkyne labeling in DMF-treated T cells (FIG. 27A, and Tables 7-9). Similar isoTOP-ABPP analyses revealed that none of the .about.40 DMF-sensitive Cys residues were altered by MMF (50 .mu.M, 4 h) or BSO (2.5 mM, 4 h) treatment, which, in general, affected the reactivity of very few Cys residues across the T cell proteome (FIG. 27A, B and FIG. 29, respectively). The Cys residues targeted by DMF exhibited concentration- (FIG. 27C and Tables 8-9) and time (FIG. 27D) dependent increases in DMF sensitivity, as revealed by isoTOP-ABPP experiments performed with human T cells treated with lower concentrations of DMF (10 and 25 .mu.M, 4 h) or for shorter periods of time (50 .mu.M DMF, 1 or 2 h). Of note, very few DMF-sensitive Cys residues were detected in T cells treated with 10 .mu.M DMF, a concentration of the drug that also had limited impact T cell activation (FIG. 23B, D, E). These concentration- and time-dependent studies uncovered another .about.10 DMF sensitive Cys residues that were not detected in the original 50 .mu.M/4 h isoTOP-ABPP experiments, likely reflecting the stochastic nature of peptide discovery in data dependent MS experiments.
[0500] The possibility that some of the alterations in Cys reactivity following DMF treatment could reflect changes in protein expression was considered; however, multiple Cys residues were quantified by isoTOP-ABPP for the majority of proteins harboring DMF-sensitive Cys residues, and, in most of these cases, the additional quantified Cys residues were clearly unaffected by DMF treatment (FIG. 27E). The DNA activated protein kinase PRKDC was shown as one representative example, for which IA-alkyne reactivity was quantified for several Cys residues, only one of which (C4045) was blocked by DMF (FIG. 27F). These results indicate that DMF directly impaired the IA alkyne reactivity of specific Cys residues rather than indirectly affecting protein expression in human T cells.
Conservation of DMF-Sensitive Cys Residues in Human and Mouse T Cells
[0501] Considering that DMF impaired the activation of both human and mouse T cells, it was surmised that at least a subset of Cys residues potentially important for mediating DMF action were conserved in humans and mice. Consistent with this, approximately two-thirds of the DMF-sensitive Cys residues discovered in human T cells are conserved in mice (FIG. 30A and Table 7). The isoTOP-ABPP experiments were performed on mouse T cells treated with DMF (50 .mu.M, 4 h) and found that the vast majority (>80%) of the conserved, quantified Cys residues sensitive to DMF in human T cells were also blocked (R values>4) by this drug in mouse T cells (FIG. 30B and Tables 8-9). These results indicate that DMF targets a similar array of Cys residues in human and mouse T cells, pointing to a specific set of proteins as candidate sites of action for this electrophilic drug.
[0502] The proteins containing DMF-sensitive Cys residues, as a whole, originated from several functional classes, including enzymes, channels, transporters, scaffolding proteins, and transcriptional regulators (FIG. 30C). Among these proteins were several with important immune functions (Table 7). DMF-sensitive Cys residues were found, for instance, in multiple proteins that are either components or regulators of the NF-.kappa.B signaling pathway, including I.kappa.B kinase .beta. (IKK.beta. or IKBKB), protein kinase C-.theta. (PKC.theta. or PRKCQ), and TNFAIP3 (Table 7). Consistent with these sites of DMF action and potentially others within the NF-.kappa.B pathway, it was found that DMF treatment blocked p65 nuclear translocation (FIG. 31), as has been shown in other cell types. DMF-sensitive Cys residues were also found in: 1) the adenosine deaminase enzyme ADA, deleterious mutations in which cause severe combined immunodeficiency in humans, 2) the transcription factors interferon regulatory factors-4 (IRF4) and -8 (IRF8), and 3) the immunomodulatory cytokine IL-16 (Table 7).
DMF Perturbs a CXXC Motif Critical for PKC.theta.-CD28 Interactions and T Cell Activation
[0503] PKC.theta. is a key kinase involved in T cell signaling at the immunological synapse where engagement of the T cell receptor and CD28 co-receptor initiates activation of multiple downstream pathways, including NF-.kappa.B. T cells from PKC.theta.(-/-) mice are defective in early activation. The isoTOP-ABPP analysis identified two DMF sensitive Cys residues--C14 and C17--in human (FIG. 32A) and mouse (FIG. 33A) T cells, and these Cys residues showed time- and concentration-dependent increases in DMF sensitivity (FIG. 33B, C), but were not affected by MMF treatment (FIG. 33D). Because C14 and C17 are found on the same tryptic peptide, it was difficult to distinguish whether one or both residues was sensitive to DMF treatment, but, in certain isoTOP-ABPP experiments, this tryptic peptide appeared to migrate as two adjacent peaks, both of which showed DMF sensitivity (FIG. 32A), suggesting that the IA-alkyne reactivity of both C14 and C17 is blocked by DMF treatment. The isoTOP-ABPP experiments also identified a third Cys in PKC.theta. (C322) that was unaffected by DMF treatment (FIG. 32A), indicating that DMF caused reductions in C14/17 reactivity rather than changes in PKC.theta. expression. C14 and C17 form a CXXC motif found in the C2 domain of PKC.theta., but not other PKC isoforms (FIG. 32B, C). The C2 domain of PKC.theta. was recently shown to bind phosphotyrosine-containing peptides and has been postulated to stabilize plasma membrane association of PKC.theta. at the immunological synapse. Upon TCR/CD28 stimulation, PKC.theta. is recruited to the immunological synapse where it interacts with the CD28 co-receptor by associating with the CD28 cytoplasmic tail. It was found that DMF, but not MMF, blocked the interaction between PKC.theta. and CD28 in mouse T cells (FIG. 32D). A retroviral transduction was used to reconstitute PKC.theta.(-/-) T cells with either WT- or a C14S/C17S-PKC.theta. mutant and found that the mutant protein failed to associate with CD28 (FIG. 32E). PKC.theta.(-/-) T cells reconstituted with the C14S/C17SPKC.theta. mutant also showed impaired expression of CD25 (FIG. 32F) and IL-2 release (FIG. 32G) compared to cells reconstituted with WT PKC.theta. following anti-CD3, anti-CD28 treatment. Taken together, these data indicate that the C14/C17 motif within the C2 domain of PKC.theta. regulates localization of this kinase to the immunological synapse, and disruption of this motif by DMF or genetic mutation impairs T cell activation.
Sensitive Cysteine Residue Sites in DMF Toward Probe ADA
[0504] The DMFsensitive Cys residue C75 is located between two amino acids--G74 and R76--that, when mutated in humans, contribute to an immunosuppressive phenotype. The amino acid 74-76 region of ADA is over 25 angstroms from the active site of the enzyme (FIG. 34), suggesting that it performs a non-catalytic function possibly perturbed by DMF reactivity. The DMF-sensitive Cys in IKBKB is located in the leucine-zipper domain and is distinct from another electrophile-sensitive Cys residue C179 found in the active site of this kinase.
[0505] Table 1 illustrates a list of liganded cysteines and their reactivity profiles with the fragment electrophile library from isoTOP-ABPP experiments performed in cell lysates (in vitro). Table 1 further shows the accession number (or the protein identifier) of the protein.
TABLE-US-00002 TABLE 1A SEQ ID 2_500.mu.M.sub.-- 2_500.mu.M.sub.-- Identifier Protein NO: invitro_231 invitro_ramos Q99873_C109 PRMT1 Protein arginine 17 3.1 12.6 N-methyltransferase 1 P24752_C119 ACAT1 Acetyl-CoA 22 3.9 3.2 acetyltransferase, mitochondrial P09211_C48 GSTP1 Glutathione S- 25 3.1 1.6 transferase P O14980_C34 XPO1 Exportin-1 28 2.8 4.6 P24752_C196 ACAT1 Acetyl-CoA 33 12.4 9.3 acetyltransferase, mitochondrial Q15084_C55 PDIA6 Protein disulfide-isomerase A6 51 6.3 7.1 P24752_C413 ACAT1 Acetyl-CoA 56 18.1 -- acetyltransferase, mitochondrial P63244_C182 GNB2L1 Guanine nucleotide-binding 85 1.2 1.2 protein subunit beta-2- P24752_C126 ACAT1 Acetyl-CoA 89 20.0 -- acetyltransferase, mitochondrial Q15084_C190 PDIA6 Protein disulfide-isomerase 96 -- 15.4 A6 Q8TAQ2_C145 SMARCC2 SWI/SNF complex 119 8.5 14.0 subunit SMARCC2 P68036_C86 UBE2L3 Ubiquitin-conjugating 120 2.8 2.5 enzyme E2 L3 P15374_C95 UCHL3 Ubiquitin carboxyl-terminal 146 -- 1.8 hydrolase isozyme L3 Q16763_C118 UBE2S Ubiquitin-conjugating 187 4.2 6.9 enzyme E2 S Q16822_C306 PCK2 Phosphoenolpyruvate 192 -- -- carboxykinase O14980_C528 XPO1 Exportin-1 DLLGLCEQK 218 4.9 3.1 K.DLLGLC*EQKR.G O00170_C122 AIP AH receptor-interacting 240 12.5 6.7 protein O75874_C269 IDH1 Isocitrate dehydrogenase 260 20.0 -- O75362_C286 ZNF217 Zinc finger protein 217 268 20.0 20.0 P40763_C259 STAT3 Signal transducer and 283 -- -- activator of transcription 3 Q9Y3Z3_C522 SAMHD1 SAM domain and HD 288 -- 4.0 domain-containing protein 1 P16455_C150 MGMT Methylated-DNA--protein-cysteine 291 -- 20.0 methyltransferase Q96GG9_C115 DCUN1D1 DCN1-like protein 1 293 -- 20.0 P00813_C75 ADA Adenosine deaminase 296 -- 7.3 Q14790_C360 CASP8 Caspase-8 335 9.8 -- Q15306_C194 IRF4 Interferon regulatory 338 -- -- factor 4 Q6L8Q7_C108 PDE12 2,5-phosphodiesterase 12 339 -- 5.1 P48735_C308 IDH2 Isocitrate dehydrogenase 360 1.4 -- Q86UV5_C39 USP48 Ubiquitin carboxyl-terminal 381 -- 7.1 hydrolase 48 P50851_C1704 LRBA. Lipopolysaccharide-responsive 388 -- 3.4 and beige-like ancho O94953_C694 KDM4B Lysine-specific 395 20.0 5.1 demethylase 4B P19447_C342 ERCC3 TFIIH basal 402 20.0 -- transcription factor complex helicase Q00535_C157 CDK5 Cyclin-dependent kinase 5 407 -- 3.2 Q9UPT9_C171 USP22 Ubiquitin carboxyl- 413 -- 7.1 terminal hydrolase 22 Q9HB90_C377 RRAGC Ras-related GTP-binding 417 20.0 -- protein C P50851_C2675 LRBA Lipopolysaccharide-responsive 426 -- 3.0 and beige-like ancho Q9NYL2_C22 MLTK Mitogen-activated protein 430 20.0 -- kinase kinase kinase MLT Q5T1V6_C414 DDX59 Probable ATP-dependent 439 20.0 20.0 RNA helicase DDX59 Q9HB90_C358 RRAGC Ras-related GTP-binding 452 20.0 20.0 protein C P16455_C145 MGMT Methylated-DNA--protein-cysteine 470 -- 20.0 methyltransferase Q9Y5T5_C205 USP16 Ubiquitin carboxyl-terminal 474 20.0 -- hydrolase 16 Q02556_C306 IRF8 Interferon regulatory factor 8 513 -- -- Q15910_C503 EZH2 Histone-lysine 557 -- 2.8 N-methyltransferase EZH2 Q96RU2_C171 USP28 Ubiquitin carboxyl- 569 -- 1.1 terminal hydrolase 28 Q16877_C159 PFKFB4 6-phosphofructo-2- 582 -- -- kinase/fructose-2,6-bisphosphata P04150_C302 NR3C1 Glucocorticoid receptor 600 2.3 -- Q96JH7_C219 VCPIP1 Deubiquitinating protein 601 -- 1.1 VCIP135 P48200_C137 IREB2 Iron-responsive 603 -- 10.5 element-binding protein 2 O00622_C39 CYR61 Protein CYR61 612 -- -- P24752_C196 ACAT1 Acetyl-CoA 33 12.4 9.3 acetyltransferase, mitochondrial Q15084_C55 PDIA6 Protein disulfide-isomerase 51 6.3 7.1 A6 P24752_C413 ACAT1 Acetyl-CoA 56 18.1 -- acetyltransferase, mitochondrial P63244_C182 GNB2L1 Guanine nucleotide-binding 85 1.2 1.2 protein subunit beta-2- P24752_C126 ACAT1 Acetyl-CoA 89 20.0 -- acetyltransferase, mitochondrial Q15084_C190 PDIA6 Protein disulfide-isomerase 96 -- 15.4 A6 Q8TAQ2_C145 SMARCC2 SWI/SNF complex 119 8.5 14.0 subunit SMARCC2 P68036_C86 UBE2L3 Ubiquitin-conjugating 120 2.8 2.5 enzyme E2 L3 P15374_C95 UCHL3 Ubiquitin carboxyl-terminal 146 -- 1.8 hydrolase isozyme L3 Q16763_C118 UBE2S Ubiquitin-conjugating 187 4.2 6.9 enzyme E2 S Q16822_C306 PCK2 Phosphoenolpyruvate 192 -- -- carboxy kinase O14980_C528 XPO1 Exportin-1 DLLGLCEQK 218 4.9 3.1 K.DLLGLC*EQKR.G O00170_C122 AIP AH receptor-interacting 240 12.5 6.7 protein O75874_C269 IDH1 Isocitrate dehydrogenase 260 20.0 -- O75362_C286 ZNF217 Zinc finger protein 217 268 20.0 20.0 P40763_C259 STAT3 Signal transducer and 283 -- -- activator of transcription 3 Q9Y3Z3_C522 SAMHD1 SAM domain and HD 288 -- 4.0 domain-containing protein 1 P16455_C150 MGMT Methylated-DNA-protein-cysteine 291 -- 20.0 methyltransferase Q96GG9_C115 DCUN1D1 DCN1-like protein 1 293 -- 20.0 P00813_C75 ADA Adenosine deaminase 296 -- 7.3 Q14790_C360 CASP8 Caspase-8 335 9.8 -- Q15306_C194 IRF4 Interferon regulatory factor 4 338 -- -- Q6L8Q7_C108 PDE12 2,5-phosphodiesterase 12 339 -- 5.1 P48735_C308 IDH2 Isocitrate dehydrogenase 360 1.4 -- Q86UV5_C39 USP48 Ubiquitin carboxyl-terminal 381 -- 7.1 hydrolase 48 P50851_C1704 LRBA Lipopolysaccharide- 388 -- 3.4 responsive and beige-like ancho O94953_C694 KDM4B Lysine-specific 395 20.0 5.1 demethylase 4B P19447_C342 ERCC3 TFIIH basal transcription 402 20.0 -- factor complex helicase Q00535_C157 CDK5 Cyclin-dependent kinase 5 407 -- 3.2 Q9UPT9_C171 USP22 Ubiquitin carboxyl-terminal 413 -- 7.1 hydrolase 22 Q9HB90_C377 RRAGC Ras-related GTP-binding 417 20.0 -- protein C P50851_C2675 LRBA Lipopolysaccharide-responsive 426 -- 3.0 and beige-like ancho Q9NYL2_C22 MLTK Mitogen-activated protein 430 20.0 -- kinase kinase kinase MLT Q5T1V6_C414 DDX59 Probable ATP-dependent 439 20.0 20.0 RNA helicase DDX59 Q9HB90_C358 RRAGC Ras-related GTP-binding 452 20.0 20.0 protein C P16455_C145 MGMT Methylated-DNA-protein-cysteine 470 -- 20.0 methyltransferase Q9Y5T5_C205 USP16 Ubiquitin carboxyl-terminal 474 20.0 -- hydrolase 16 Q02556_C306 IRF8 Interferon regulatory factor 513 -- -- 8 Q15910_C503 EZH2 Histone-lysine 557 -- 2.8 N methyltransferase EZH2 Q96RU2_C171 USP28 Ubiquitin carboxyl-terminal 569 -- 1.1 hydrolase 28 Q16877_C159 PFKFB4 6-phosphofructo-2- 582 -- -- kinase/fructose-2,6-bisphosphata P04150_C302 NR3C1 Glucocorticoid receptor 600 2.3 -- Q96JH7_C219 VCPIP1 Deubiquitinating protein 601 -- 1.1 VCIP135 P48200_C137 IREB2 Iron-responsive 603 -- 10.5 element-binding protein 2 O00622_C39 CYR61 Protein CYR61 612 -- -- Q5T1V6_C453 DDX59 Probable ATP-dependent 620 20.0 20.0 RNA helicase DDX59 P51617_C608 IRAK1 Interleukin-1 656 -- 2.5 receptor-associated kinase 1 P42575_C370 CASP2 Caspase-2 661 -- -- P09086_C346 POU2F2 POU domain, class 2, 663 -- -- transcription factor 2 Q09472_C1738 EP300 Histone acetyltransferase 676 -- 12.7 p300 Q01201_C109 RELB Transcription factor RelB 681 20.0 20.0 Q70CQ2_C741 USP34 Ubiquitin carboxyl-terminal 688 -- -- hydrolase 34 P41226_C599 UBA7 Ubiquitin-like 702 -- -- modifier-activating enzyme 7 P14598_C378 NCF1 Neutrophil cytosol factor 1 705 -- 4.4 Q9C0C9_C375 UBE2O Ubiquitin-conjugating 707 -- -- enzyme E2O O00622_C134 CYR61 Protein CYR61 713 -- -- O00541_C361 PES1 Pescadillo homolog 718 -- -- P43403_C117 ZAP70 Tyrosine-protein kinase 726 -- -- ZAP-70 Q96FA3_C282 PELI1 E3 ubiquitin-protein ligase 729 -- -- pellino homolog 1 Q9UPT9_C44 USP22 Ubiquitin carboxyl-terminal 737 -- -- hydrolase 22 Q9Y4C1_C251 KDM3A Lysine-specific 753 -- -- demethylase 3A Q70CQ2_C1090 USP34 Ubiquitin carboxyl-terminal 761 -- -- hydrolase 34 O00622_C70 CYR61 Protein CYR61 762 -- -- P04150_C622 NR3C1 Glucocorticoid receptor 765 -- -- 3_500.mu.M.sub.-- 3_500.mu.M.sub.-- 4_250.mu.M.sub.-- 4_250.mu.M.sub.-- Identifier invitro_231 invitro_ramos invitro_231 invitro_ramos Q99873_C109 1.3 1.8 1.1 0.2 P24752_C119 2.1 5.3 1.8 0.2 P09211_C48 2.9 1.7 3.2 0.5 O14980_C34 1.4 1.6 1.0 0.2 P24752_C196 1.7 3.1 1.9 0.5 Q15084_C55 17.9 14.9 1.3 1.2 P24752_C413 15.3 20.0 3.3 -- P63244_C182 20.0 4.7 0.9 -- P24752_C126 2.6 2.4 2.1 1.2 Q15084_C190 19.9 13.1 1.8 1.2 Q8TAQ2_C145 9.7 6.6 1.3 -- P68036_C86 1.2 3.0 1.2 -- P15374_C95 1.7 1.4 -- 0.9 Q16763_C118 1.3 1.5 1.2 -- Q16822_C306 10.6 0.9 2.2 -- O14980_C528 20.0 20.0 0.9 1.1 O00170_C122 7.0 3.1 2.5 0.3 O75874_C269 20.0 1.0 1.6 0.6 O75362_C286 1.4 -- 1.7 -- P40763_C259 2.5 2.9 2.0 1.9 Q9Y3Z3_C522 2.6 -- 1.5 -- P16455_C150 -- 17.1 -- 6.9 Q96GG9_C115 -- 5.5 -- -- P00813_C75 -- 1.5 -- 0.1 Q14790_C360 3.3 2.3 12.3 -- Q15306_C194 -- 20.0 -- -- Q6L8Q7_C108 3.6 1.8 -- -- P48735_C308 2.2 -- 0.7 -- Q86UV5_C39 1.8 1.5 1.4 1.4 P50851_C1704 -- 3.6 -- 1.5 O94953_C694 20.0 7.4 20.0 -- P19447_C342 12.1 -- 1.6 1.1 Q00535_C157 -- 20.0 -- 1.3 Q9UPT9_C171 -- -- -- 4.0 Q9HB90_C377 3.7 -- 3.5 -- P50851_C2675 -- 5.1 1.1 -- Q9NYL2_C22 1.8 -- 20.0 -- Q5T1V6_C414 -- 6.0 -- -- Q9HB90_C358 1.2 -- 1.5 -- P16455_C145 -- 20.0 -- 20.0 Q9Y5T5_C205 -- 8.8 -- 20.0 Q02556_C306 -- 5.6 -- -- Q15910_C503 -- 2.0 -- 1.5 Q96RU2_C171 -- 1.7 -- --
Q16877_C159 -- 12.9 1.4 -- P04150_C302 7.6 -- -- -- Q96JH7_C219 20.0 15.1 -- -- P48200_C137 -- -- -- -- O00622_C39 4.0 -- 2.8 -- P24752_C196 1.7 3.1 1.9 0.5 Q15084_C55 17.9 14.9 1.3 1.2 P24752_C413 15.3 20.0 3.3 -- P63244_C182 20.0 4.7 0.9 -- P24752_C126 2.6 2.4 2.1 1.2 Q15084_C190 19.9 13.1 1.8 1.2 Q8TAQ2_C145 9.7 6.6 1.3 -- P68036_C86 1.2 3.0 1.2 -- P15374_C95 1.7 1.4 -- 0.9 Q16763_C118 1.3 1.5 1.2 -- Q16822_C306 10.6 0.9 2.2 -- O14980_C528 20.0 20.0 0.9 1.1 O00170_C122 7.0 3.1 2.5 0.3 O75874_C269 20.0 1.0 1.6 0.6 O75362_C286 1.4 -- 1.7 -- P40763_C259 2.5 2.9 2.0 1.9 Q9Y3Z3_C522 2.6 -- 1.5 -- P16455_C150 -- 17.1 -- 6.9 Q96GG9_C115 -- 5.5 -- -- P00813_C75 -- 1.5 -- 0.1 Q14790_C360 3.3 2.3 12.3 -- Q15306_C194 -- 20.0 -- -- Q6L8Q7_C108 3.6 1.8 -- -- P48735_C308 2.2 -- 0.7 -- Q86UV5_C39 1.8 1.5 1.4 1.4 P50851_C1704 -- 3.6 -- 1.5 O94953_C694 20.0 7.4 20.0 -- P19447_C342 12.1 -- 1.6 1.1 Q00535_C157 -- 20.0 -- 1.3 Q9UPT9_C171 -- -- -- 4.0 Q9HB90_C377 3.7 -- 3.5 -- P50851_C2675 -- 5.1 1.1 -- Q9NYL2_C22 1.8 -- 20.0 -- Q5T1V6_C414 -- 6.0 -- -- Q9HB90_C358 1.2 -- 1.5 -- P16455_C145 -- 20.0 -- 20.0 Q9Y5T5_C205 -- 8.8 -- 20.0 Q02556_C306 -- 5.6 -- -- Q15910_C503 -- 2.0 -- 1.5 Q96RU2_C171 -- 1.7 -- -- Q16877_C159 -- 12.9 1.4 -- P04150_C302 7.6 -- -- -- Q96JH7_C219 20.0 15.1 -- -- P48200_C137 -- -- -- -- O00622_C39 4.0 -- 2.8 -- Q5T1V6_C453 20.0 -- -- -- P51617_C608 -- 20.0 -- -- P42575_C370 -- 7.4 -- -- P09086_C346 1.7 5.5 -- -- Q09472_C1738 -- -- -- -- Q01201_C109 -- -- -- -- Q70CQ2_C741 -- 3.2 -- -- P41226_C599 -- -- -- 20.0 P14598_C378 -- 2.1 -- 1.4 Q9C0C9_C375 9.8 4.1 -- -- O00622_C134 20.0 -- -- -- O00541_C361 1.5 -- -- -- P43403_C117 -- -- -- -- Q96FA3_C282 -- -- -- -- Q9UPT9_C44 -- -- -- -- Q9Y4C1_C251 4.4 4.0 -- -- Q70CQ2_C1090 -- 19.7 -- -- O00622_C70 20.0 -- -- -- P04150_C622 20.0 -- -- --
TABLE-US-00003 TABLE 1B 5_500.mu.M.sub.-- 5_500.mu.M.sub.-- 6_500.mu.M.sub.-- 7_500.mu.M.sub.-- 7_500.mu.M.sub.-- 8_500.mu.M.sub.-- Identifier invitro_231 invitro_ramos invitro_231 invitro_231 invitro_ramos invitro_231 Q99873_C109 0.9 1.3 1.1 1.1 1.1 1.2 P24752_C119 1.8 2.4 1.1 2.3 2.0 1.8 P09211_C48 2.2 2.4 1.2 1.8 0.9 2.6 O14980_C34 1.1 1.2 1.2 1.7 1.8 2.4 P24752_C196 1.5 2.2 1.0 1.9 -- 2.7 Q15084_C55 0.9 1.1 1.0 1.0 -- 4.7 P24752_C413 1.3 -- 1.1 4.2 -- 1.4 P63244_C182 1.5 1.4 1.5 1.5 -- 0.9 P24752_C126 0.9 1.3 0.9 2.2 -- 2.1 Q15084_C190 -- 1.7 1.1 1.0 1.2 20.0 Q8TAQ2_C145 -- 2.8 -- 1.0 -- 3.1 P68036_C86 -- 4.8 1.8 0.9 -- 1.4 P15374_C95 1.5 1.1 1.1 0.8 -- 20.0 Q16763_C118 2.8 -- -- 1.5 -- -- Q16822_C306 1.7 -- -- 1.4 -- 2.2 O14980_C528 20.0 -- 0.7 0.7 -- 1.8 O00170_C122 -- -- -- -- -- -- O75874_C269 -- 0.8 -- 1.2 -- 12.4 O75362_C286 -- -- 0.9 1.3 -- 1.8 P40763_C259 -- -- 0.6 1.3 1.8 1.8 Q9Y3Z3_C522 -- 5.4 -- -- -- 1.8 P16455_C150 -- 9.6 -- -- 20.0 -- Q96GG9_C115 -- -- -- -- 1.6 -- P00813_C75 -- 2.5 -- -- 1.4 -- Q14790_C360 1.0 1.3 -- -- -- -- Q15306_C194 -- 2.2 -- -- -- -- Q6L8Q7_C108 -- -- -- -- -- -- P48735_C308 0.8 -- -- 0.9 -- 1.3 Q86UV5_C39 -- 1.2 -- -- -- -- P50851_C1704 -- 3.6 -- 1.8 1.6 -- O94953_C694 -- -- 1.1 -- -- 20.0 P19447_C342 -- 4.1 1.1 1.3 2.0 5.9 Q00535_C157 -- 20.0 -- -- -- -- Q9UPT9_C171 -- 20.0 -- -- 3.2 -- Q9HB90_C377 -- -- -- -- -- 5.1 P50851_C2675 -- 20.0 -- -- -- -- Q9NYL2_C22 20.0 -- -- 3.1 -- -- Q5T1V6_C414 -- -- -- -- -- -- Q9HB90_C358 -- -- 1.2 -- -- -- P16455_C145 -- 2.0 -- -- -- -- Q9Y5T5_C205 -- 1.1 -- -- -- -- O00541_C272 -- 12.3 -- -- -- -- Q02556_C306 -- 2.6 -- -- 20.0 -- Q15910_C503 -- 20.0 1.8 -- -- -- Q96RU2_C171 -- -- 0.8 -- -- 7.5 Q16877_C159 -- 20.0 -- -- -- -- P04150_C302 -- -- -- -- -- -- Q96JH7_C219 -- -- -- -- -- -- P48200_C137 -- -- -- -- 2.2 -- O00622_C39 -- -- -- -- -- -- Q5T1V6_C453 -- -- -- -- -- 20.0 P51617_C608 -- 10.1 -- -- -- -- P42575_C370 -- -- -- -- -- -- P09086_C346 -- 4.6 -- -- -- -- Q09472_C1738 -- 20.0 -- -- -- -- Q01201_C109 -- -- -- -- -- -- Q70CQ2_C1741 -- -- -- -- -- -- P41226_C599 -- -- -- -- -- -- P14598_C378 -- 4.0 -- -- -- -- Q9C0C9_C375 -- -- -- -- -- -- O00622_C134 -- -- -- -- -- -- O00541_C361 -- -- -- -- -- -- P43403_C117 -- -- -- -- -- 20.0 Q96FA3_C282 -- -- -- -- -- -- Q9UPT9_C44 -- 20.0 -- -- -- -- Q9Y4C1_C251 -- -- -- -- -- -- Q700Q2_C1090 -- 20.0 -- -- -- -- O00622_C70 -- -- -- -- -- 20.0 P04150_C622 -- -- -- -- -- -- 8_500.mu.M.sub.-- 9_500.mu.M.sub.-- 9_500.mu.M.sub.-- 10_500.mu.M.sub.-- Identifier invito_ramos invitro_231 invitro_ramos initro_231 Q99873_C109 1.2 1.0 1.5 2.3 P24752_C119 0.7 2.2 3.1 1.5 P09211_C48 2.0 2.3 1.4 1.4 O14980_C34 1.5 1.2 1.1 1.2 P24752_C196 1.5 3.1 2.1 2.5 Q15084_C55 2.2 1.4 1.0 20.0 P24752_C413 2.1 15.3 -- 13.0 P63244_C182 1.2 0.9 0.8 1.1 P24752_C126 12.6 1.4 -- 3.2 Q15084_C190 2.7 2.0 1.3 -- Q8TAQ2_C145 1.7 1.0 1.6 4.2 P68036_C86 1.0 0.8 1.5 1.1 P15374_C95 1.2 1.0 1.2 2.0 Q16763_C118 1.1 0.8 1.0 1.7 Q16822_C306 1.0 1.3 -- 1.9 O14980_C528 -- 0.8 0.7 0.8 O00170_C122 1.3 -- 1.1 -- O75874_C269 -- -- 1.4 20.0 O75362_C286 -- 1.2 -- 1.1 P40763_C259 1.7 1.5 1.2 3.4 Q9Y3Z3_C522 1.1 1.0 -- 1.8 P16455_C150 15.8 -- 4.0 -- Q96GG9_C115 1.4 -- 1.3 1.3 P00813_C75 1.3 1.3 1.1 -- Q14790_C360 1.4 -- -- 2.8 Q15306_C194 2.1 -- 1.1 -- Q6L8Q7_C108 -- 1.3 -- -- P48735_C308 -- 1.4 -- 1.1 Q86UV5_C39 -- 0.8 0.9 2.5 P50851_C1704 1.2 1.0 1.2 -- O94953_C694 1.1 1.2 1.0 -- P19447_C342 -- 2.5 -- 3.0 Q00535_C157 1.3 1.2 0.8 -- Q9UPT9_C171 2.3 -- -- -- Q9HB90_C377 -- 1.9 -- -- P50851_C2675 1.1 1.5 -- -- Q9NYL2_C22 -- 20.0 -- -- Q5T1V6_C414 2.0 -- -- -- Q9HB90_C358 -- 1.9 -- -- P16455_C145 20.0 -- 20.0 -- Q9Y5T5_C205 20.0 -- -- -- O00541_C272 1.6 -- -- -- Q02556_C306 2.2 -- 1.0 -- Q15910_C503 -- 1.1 -- -- Q96RU2_C171 -- 1.1 -- 1.4 Q16877_C159 1.0 1.5 -- 1.2 P04150_C302 -- -- -- 4.1 Q96JH7_C219 -- -- -- -- P48200_C137 1.6 -- -- -- O00622_C39 -- 2.0 -- -- Q5T1V6_C453 -- 2.1 -- -- P51617_C608 -- -- -- -- P42575_C370 2.0 -- 1.2 -- P09086_C346 -- -- -- -- Q09472_C1738 -- 1.5 -- -- Q01201_C109 -- -- -- -- Q70CQ2_C1741 -- -- -- -- P41226_C599 -- -- -- -- P14598_C378 1.3 -- -- -- Q9C0C9_C375 -- -- -- -- O00622_C134 -- 1.7 -- 20.0 O00541_C361 -- 20.0 -- -- P43403_C117 -- -- -- 20.0 Q96FA3_C282 -- -- -- -- Q9UPT9_C44 1.5 -- -- -- Q9Y4C1_C251 -- -- -- -- Q70EQ2_C1090 -- -- -- -- O00622_C70 -- -- -- -- P04150_C622 -- -- -- --
TABLE-US-00004 TABLE 1C 10_500.mu.M.sub.-- 11_500.mu.M.sub.-- 11_500.mu.M.sub.-- 12_500.mu.M.sub.-- 12_500.mu.M.sub.-- Identifier invitro_ramos invitro_231 invitro_ramos invitro_231 invitro_ramos Q99873_C109 1.3 20.0 20.0 1.4 1.3 P24752_C119 1.4 1.2 0.9 1.0 0.7 P09211_C48 0.8 1.7 1.1 2.9 1.1 O14980_C34 0.7 1.2 1.0 1.2 0.7 P24752_C196 1.7 1.5 1.3 1.2 0.9 Q15084_C55 -- 20.0 20.0 3.7 3.1 P24752_C413 1.6 1.9 1.7 1.5 1.1 P63244_C182 1.1 1.0 1.1 0.9 0.8 P24752_C126 1.5 1.5 -- 1.0 1.1 Q15084_C190 3.3 20.0 20.0 -- 20.0 Q8TAQ2_C145 -- 20.0 -- 2.8 1.6 P68036_C86 0.4 -- 1.0 -- -- P15374_C95 -- 0.8 0.8 -- 0.5 Q16763_C118 -- -- -- -- 0.6 Q16822_C306 1.1 3.2 1.2 1.5 0.7 O14980_C528 -- 1.0 0.7 1.1 -- O00170_C122 1.7 -- 2.8 -- 1.3 O75874_C269 -- 1.1 0.7 -- -- O75362_C286 -- 1.2 1.2 1.0 -- P40763_C259 -- 20.0 20.0 -- -- Q9Y3Z3_C522 -- 2.1 -- -- -- P16455_C150 20.0 -- 20.0 -- 20.0 Q96GG9_C115 1.0 -- 0.9 -- 0.6 P00813_C75 1.0 -- 7.5 -- 0.7 O14933_C98 -- -- 3.4 -- 1.3 Q14790_C360 1.5 20.0 20.0 -- Q15306_C194 2.5 -- 4.3 -- 3.7 Q6L8Q7_C108 -- 4.2 -- -- 1.5 P48735_C308 -- 1.1 -- 1.7 -- Q86UV5_C39 -- -- 1.0 -- -- P50851_C1704 0.8 -- 1.4 -- -- O94953_C694 1.4 -- 1.9 -- -- P19447_C342 -- 2.9 1.5 3.0 -- Q00535_C157 -- -- 1.3 -- 1.0 Q9UPT9_C171 3.8 -- 8.2 -- 4.1 Q9HB90_C377 -- -- 1.6 -- -- P50851_C2675 1.3 -- 1.3 -- 1.5 Q9NYL2_C22 -- -- -- -- -- Q5T1V6_C414 -- -- -- -- -- Q9HB90_C358 -- -- -- -- -- P16455_C145 -- -- 20.0 -- -- Q9Y5T5_C205 -- 20.0 -- 2.2 -- O00541_C272 2.2 -- 4.2 -- -- Q02556_C306 3.6 -- 3.1 -- -- Q15910_C503 -- -- 1.3 -- -- Q96RU2_C171 -- 20.0 -- -- -- Q16877_C159 -- -- 0.6 -- -- P04150_C302 -- -- 1.5 -- 1.3 Q96JH7_C219 -- -- -- -- -- P48200_C137 2.1 -- 3.7 -- -- O00622_C39 -- 9.6 -- -- -- Q5T1V6_C453 -- 20.0 -- -- -- P51617_C608 -- -- 1.3 -- -- P42575_C370 1.2 -- 6.0 -- -- P09086_C346 -- -- 1.7 -- -- Q09472_C1738 -- -- -- -- -- Q01201_C109 -- -- -- -- -- Q70CQ2_C741 0.9 -- -- -- -- P41226_C599 -- -- 1.9 -- -- P14598_C378 1.1 -- -- -- -- Q9C0C9_C375 0.8 -- -- -- -- O00622_C134 -- -- -- -- -- O00541_C361 -- -- -- -- -- P43403_C117 20.0 -- -- -- -- Q96FA3_C282 -- -- -- -- -- Q9UPT9_C44 -- -- -- -- -- Q9Y4C1_C251 -- -- -- -- -- Q70CQ2_C1090 -- -- -- -- -- O00622_C70 -- -- -- -- -- P04150_C622 -- 1.7 -- -- -- 13_500.mu.M.sub.-- 13_500.mu.M.sub.-- 14_500.mu.M.sub.-- 14_500.mu.M.sub.-- 15_500.mu.M.sub.-- Identifier invitro_231 invitro_ramos invito_231 invitro_ramos invito_231 Q99873_C109 1.0 0.9 0.8 1.4 0.9 P24752_C119 1.2 0.6 1.2 1.8 1.0 P09211_C48 1.5 1.2 3.2 2.1 2.5 O14980_C34 0.9 0.7 1.1 1.5 -- P24752_C196 1.6 1.7 1.1 1.4 0.9 Q15084_C55 1.9 1.1 1.0 1.4 0.8 P24752_C413 5.5 7.3 1.3 1.6 1.2 P63244_C182 1.4 0.8 1.2 1.5 -- P24752_C126 9.6 20.0 1.1 1.4 0.8 Q15084_C190 1.5 1.3 1.1 1.8 0.9 Q8TAQ2_C145 -- 0.8 1.1 2.3 -- P68036_C86 1.1 0.8 1.0 1.1 -- P15374_C95 -- 0.8 1.2 1.0 -- Q16763_C118 -- 0.7 0.7 1.2 -- Q16822_C306 0.9 1.0 -- 20.0 -- O14980_C528 -- 0.5 20.0 20.0 0.8 O00170_C122 -- 0.9 -- 2.5 -- O75874_C269 2.7 -- 0.9 1.3 -- O75362_C286 0.9 0.9 0.9 -- -- P40763_C259 -- 1.2 0.8 3.1 -- Q9Y3Z3_C522 0.8 -- 1.7 1.5 -- P16455_C150 -- 4.2 5.5 5.1 -- Q96GG9_C115 -- -- 1.1 1.6 -- P00813_C75 -- 0.9 1.5 1.7 -- O14933_C98 -- 1.0 -- 2.8 -- Q14790_C360 -- -- 1.4 1.9 -- Q15306_C194 -- 1.1 1.3 2.2 -- Q6L8Q7_C108 -- 1.0 0.5 2.2 1.1 P48735_C308 1.1 -- 4.7 -- -- Q86UV5_C39 0.8 0.6 0.8 -- -- P50851_C1704 0.7 0.8 1.9 1.7 -- O94953_C694 1.4 1.1 -- 2.0 -- P19447_C342 1.0 -- 4.7 -- -- Q00535_C157 -- -- 6.1 2.3 -- Q9UPT9_C171 -- 0.8 4.0 4.0 -- Q9HB90_C377 2.1 -- -- 20.0 -- P50851_C2675 -- -- 1.9 2.9 -- Q9NYL2_C22 5.2 -- 1.6 -- -- Q5T1V6_C414 -- 1.2 -- 4.0 -- Q9HB90_C358 1.5 -- 0.9 2.1 1.1 P16455_C145 -- 20.0 1.5 2.0 -- Q9Y5T5_C205 -- 3.3 -- -- O00541_C272 1.7 -- 2.6 -- -- Q02556_C306 -- 0.7 2.4 2.3 -- Q15910_C503 -- 0.8 0.6 1.9 0.8 Q96RU2_C171 -- 2.4 1.0 -- -- Q16877_C159 0.9 -- 2.3 -- -- P04150_C302 -- -- -- 2.2 -- Q96JH7_C219 -- -- 0.6 1.5 -- P48200_C137 -- -- 5.2 -- -- O00622_C39 1.2 -- 3.6 -- -- Q5T1V6_C453 -- 1.4 2.2 -- -- P51617_C608 -- -- -- -- -- P42575_C370 -- -- -- -- -- P09086_C346 -- 0.6 -- -- -- Q09472_C1738 -- 0.7 2.1 -- -- Q01201_C109 -- -- 1.7 2.4 -- Q70CQ2_C741 0.8 -- -- -- -- P41226_C599 -- -- -- -- -- P14598_C378 -- 0.7 -- -- -- Q9C0C9_C375 -- -- 2.8 -- -- O00622_C134 -- -- 0.7 -- -- O00541_C361 -- -- -- -- -- P43403_C117 -- -- -- 1.3 -- Q96FA3_C282 -- -- 1.4 -- -- Q9UPT9_C44 -- -- -- -- -- Q9Y4C1_C251 -- -- -- -- -- Q70CQ2_C1090 -- -- -- -- -- O00622_C70 -- -- -- -- -- P04150_C622 -- -- -- -- --
TABLE-US-00005 TABLE 1D 15_500.mu.M.sub.-- 27_500.mu.M.sub.-- 20_500.mu.M.sub.-- 20_500.mu.M.sub.-- 21_500.mu.M.sub.-- Identifier invitro_ramos invitro_231 invitro_231 invitro_ramos invitro_231 Q99873_C109 1.2 0.7 1.9 2.2 1.1 P24752_C119 1.2 0.9 3.6 2.6 0.9 P09211_C48 1.3 1.3 2.5 1.1 1.1 O14980_C34 1.2 0.9 1.0 1.2 0.9 P24752_C196 1.6 0.9 3.7 2.9 1.0 Q15084_C55 1.0 0.8 1.7 2.0 1.1 P24752_C413 0.9 0.9 20.0 14.0 1.4 P63244_C182 1.3 1.0 1.4 1.1 0.9 P24752_C126 -- 0.9 10.6 20.0 1.2 Q15084_C190 1.2 0.9 2.1 2.1 1.1 Q8TAQ2_C145 1.2 0.9 2.9 3.9 1.2 P68036_C86 1.3 1.2 1.1 1.2 1.2 P15374_C95 0.7 0.4 1.0 0.9 1.1 Q16763_C118 1.2 0.8 -- 2.9 0.9 Q16822_C306 -- 1.4 2.5 1.6 1.2 O14980_C528 1.0 1.2 1.4 4.0 0.9 O00170_C122 1.4 0.4 -- 2.2 1.4 O75874_C269 0.6 0.4 -- 0.6 1.5 O75362_C286 -- 20.0 1.3 -- 0.9 P40763_C259 1.9 0.7 -- 3.0 -- Q9Y3Z3_C522 1.6 0.8 2.1 1.3 -- P16455_C150 1.9 1.0 -- 18.3 -- Q96GG9_C115 0.8 1.1 1.2 1.2 1.0 P00813_C75 1.1 1.1 -- 1.5 -- O14933_C98 1.3 1.2 -- 3.9 -- Q14790_C360 -- 0.8 1.9 1.5 1.9 Q15306_C194 2.2 -- -- 3.0 -- Q6L8Q7_C108 -- 0.5 1.6 -- -- P48735_C308 -- 1.9 0.5 -- 0.6 Q86UV5_C39 0.7 0.8 1.1 1.8 20.0 P50851_C1704 1.9 -- 2.0 1.7 -- O94953_C694 1.1 1.4 2.8 2.0 -- P19447_C342 -- 0.8 2.6 -- 6.1 Q00535_C157 -- -- -- 2.0 -- Q9UPT9_C171 -- -- -- 20.0 -- Q9HB90_C377 -- 1.0 -- -- 1.4 P50851_C2675 1.6 -- -- -- 0.9 Q9NYL2_C22 -- 1.1 20.0 -- 2.0 Q5T1V6_C414 1.0 0.8 -- 3.1 1.2 Q9HB90_C358 -- 0.3 -- -- 1.2 P16455_C145 1.3 -- -- 20.0 -- Q9Y5T5_C205 -- 1.6 -- -- 1.6 O00541_C272 -- 1.1 -- -- -- Q02556_C306 2.1 -- -- 2.1 -- Q15910_C503 -- -- -- -- -- Q96RU2_C171 -- 0.9 -- 2.4 1.1 Q16877_C159 -- -- -- 1.0 -- P04150_C302 0.9 1.3 -- -- -- Q96JH7_C219 1.3 1.7 -- 1.2 -- P48200_C137 2.0 1.4 -- 5.2 -- O00622_C39 -- 1.0 -- -- -- Q5T1V6_C453 -- -- -- -- -- P51617_C608 -- -- -- -- -- P42575_C370 -- 0.9 -- -- -- P09086_C346 0.8 -- -- 2.3 0.9 Q09472_C1738 -- -- -- 3.2 -- Q01201_C109 -- 0.8 -- -- -- Q70CQ2_C741 -- -- 2.8 1.7 -- P41226_C599 -- 0.7 1.1 12.2 -- P14598_C378 -- -- -- -- -- Q9C0C9_C375 -- 0.7 -- -- -- O00622_C134 -- 1.5 20.0 -- -- O00541_C361 -- 0.6 20.0 20.0 -- P43403_C117 20.0 -- -- 20.0 -- Q96FA3_C282 -- -- -- -- -- Q9UPT9_C44 -- 20.0 -- -- -- Q9Y4C1_C251 -- -- -- -- 0.7 Q70CQ2_C1090 -- -- -- 2.7 -- O00622_C70 -- 1.1 -- -- -- P04150_C622 -- -- -- -- -- 21_500.mu.M.sub.-- 22_500.mu.M.sub.-- 22_500.mu.M.sub.-- 23_500.mu.M.sub.-- 23_500.mu.M.sub.-- Identifier invitro_ramos invitro_231 invitro_ramos invitro_231 invitro_ramos Q99873_C109 1.5 1.0 1.0 0.8 1.0 P24752_C119 1.1 1.0 0.8 1.7 2.0 P09211_C48 1.3 0.9 0.7 0.8 0.9 O14980_C34 1.2 1.1 -- 3.4 5.3 P24752_C196 1.1 1.4 0.7 -- 1.8 Q15084_C55 2.1 2.1 1.6 0.8 1.0 P24752_C413 1.4 1.1 1.1 1.1 1.2 P63244_C182 0.9 1.6 -- 4.6 -- P24752_C126 1.4 2.4 1.7 0.8 0.8 Q15084_C190 2.3 2.2 4.2 0.8 1.1 Q8TAQ2_C145 -- -- 1.2 1.3 -- P68036_C86 2.0 -- 0.8 7.4 13.2 P15374_C95 -- -- 0.7 0.8 1.1 Q16763_C118 -- -- 0.9 1.6 2.4 Q16822_C306 2.0 -- -- 3.0 -- 014980_C528 0.8 -- -- -- 2.9 000170_C122 -- 1.3 1.1 0.7 1.4 075874_C269 -- 1.0 -- 0.7 1.1 075362_C286 -- -- -- 20.0 -- P40763_C259 2.9 2.0 -- 1.1 0.8 Q9Y3Z3_C522 1.4 -- 0.8 -- -- P16455_C150 17.2 -- 2.9 -- 1.0 Q96GG9_C115 1.0 1.0 0.8 -- 20.0 POOS13_C75 1.3 -- 1.3 -- 1.1 O14933_C98 -- -- 2.0 0.7 1.2 Q14790_C360 1.9 -- -- 1.4 1.4 Q15306_C194 1.7 -- 1.1 -- 1.9 Q6L8Q7_C108 -- -- -- 1.0 -- P48735_C308 -- -- -- -- -- Q86UV5_C39 1.2 -- -- -- 1.3 P50851_C1704 1.6 -- -- -- 3.4 094953_C694 1.8 -- -- -- 3.2 P19447_C342 -- -- -- 1.6 -- Q00535_C157 -- -- 0.9 -- 2.8 Q9UPT9_C171 -- -- 1.1 -- 5.1 Q9HB90_C377 -- 1.7 0.9 1.5 -- P50851_C2675 1.5 1.1 1.1 -- 2.6 Q9NYL_2 C22 -- 1.0 -- 1.4 -- Q5T1V6_C414 -- -- 1.3 -- 1.4 Q9HB90_C358 -- 1.7 -- -- -- P16455_C145 -- -- -- -- 1.0 Q9Y5T5_C205 -- -- 1.6 -- 1.2 O00541_C272 5.5 1.6 -- -- 2.0 Q02556_C306 -- -- -- -- -- Q15910_C503 -- -- -- -- 2.0 Q96RU2_C171 -- -- -- -- 1.2 Q16877_C159 1.2 -- -- -- 1.0 P04150_C302 -- -- 0.9 -- 4.7 Q96JH7_C219 -- -- -- -- -- P48200_C137 -- -- -- -- 1.6 O00622_C39 -- -- -- 4.4 -- Q5T1V6_C453 -- -- -- -- -- P51617_C608 -- -- -- -- 1.5 P42575_C370 -- -- -- -- 2.1 P09086_C346 -- -- -- -- 1.1 Q09472_C1738 -- -- -- -- -- Q01201_C109 -- -- -- -- 1.3 Q70CQ2_C741 1.7 -- -- -- -- P41226_C599 2.5 -- -- -- 3.2 P14598_C378 -- -- -- -- -- Q9C0C9_C375 2.4 -- -- -- 1.4 O00622_C134 -- -- -- -- -- O00541_C361 -- -- -- -- 1.1 P43403_C117 -- -- -- -- -- Q96FA3_C282 -- -- -- -- -- Q9UPT9_C44 -- -- -- -- -- Q9Y4C1_C251 -- -- -- -- -- Q70CQ2_C1090 0.9 -- -- -- -- O00622_C70 -- -- -- -- -- P04150_C622 -- -- -- -- --
TABLE-US-00006 TABLE 1E 24_500.mu.M.sub.-- 25_500.mu.M.sub.-- 26_500.mu.M.sub.-- 27_500.mu.M.sub.-- 27_500.mu.M.sub.-- Identifier invitro_ramos invitro_231 invitro_ramos invitro_231 invitro_ramos Q99873_C109 1.0 1.0 0.9 0.9 1.0 P24752_C119 1.3 1.0 1.0 1.0 1.0 P09211_C48 1.6 1.8 0.9 1.0 1.0 O14980_C34 1.1 0.9 0.9 1.0 1.0 P24752_C196 1.1 0.9 1.0 1.0 1.1 Q15084_C55 1.3 1.0 0.9 1.1 1.2 P24752_C413 1.2 1.0 0.9 1.1 1.3 P63244_C182 2.6 1.2 1.0 0.8 1.1 P24752_C126 1.1 0.9 0.9 1.0 1.3 Q15084_C190 1.7 1.0 0.9 -- 1.1 Q8TAQ2_C145 5.4 0.9 0.9 -- 1.2 P68036_C86 -- 1.5 0.7 0.9 -- P15374_C95 1.1 0.8 0.8 -- 1.0 Q16763_C118 1.2 1.0 0.9 -- 0.9 Q16822_C306 -- 0.8 -- 1.5 1.1 O14980_C528 20.0 -- 0.9 0.9 0.8 O00170_C122 1.3 -- 0.9 -- 1.0 O75874_C269 0.9 1.0 -- 1.0 -- O75362_C286 -- 20.0 -- 0.9 1.0 P40763_C259 1.7 0.9 -- -- -- Q9Y3Z3_C522 1.1 -- 0.9 1.0 0.9 P16455_C150 4.0 -- 0.9 -- 2.3 Q96GG9_C115 1.7 -- 0.9 -- 1.0 P00813_C75 1.2 -- 0.8 -- 1.0 O14933_C98 -- -- 1.0 -- 1.0 Q14790_C360 1.4 1.0 -- -- 1.2 Q15306_C194 1.9 -- 1.1 -- 1.0 Q6L8Q7_C108 1.4 0.8 1.1 -- 1.3 P48735_C308 -- 14.5 -- 1.0 -- Q86UV5_C39 1.0 1.0 -- -- -- P50851_C1704 1.3 0.8 -- 1.1 -- O94953_C694 -- -- -- 1.0 -- P19447_C342 -- 0.9 -- 1.1 -- Q00535_C157 -- -- 0.9 -- 1.0 Q9UPT9_C171 3.1 1.1 1.0 -- 1.2 Q9HB90_C377 1.5 1.0 -- -- -- P50851_C2675 1.0 -- 0.9 -- -- Q9NYL2_C22 -- 0.8 -- -- -- Q5T1V6_C414 1.7 -- -- -- 1.0 Q9HB90_C358 0.9 0.9 -- -- -- P16455_C145 20.0 -- -- -- 20.0 Q9Y5T5_C205 -- -- -- -- 0.9 O00541_C272 2.6 -- -- -- -- Q02556_C306 1.2 -- -- -- 1.1 Q15910_C503 1.3 0.8 -- -- -- Q96RU2_C171 -- -- -- 1.2 -- Q16877_C159 -- -- -- -- -- P04150_C302 -- -- -- 1.0 -- Q96JH7_C219 1.2 -- -- -- -- P48200_C137 -- -- -- -- -- O00622_C39 -- 1.0 -- -- -- Q5T1V6_C453 1.7 0.9 -- 1.0 -- P51617_C608 1.3 -- -- -- -- P42575_C370 2.2 -- -- -- -- P09086_C346 1.1 0.7 -- -- -- Q09472_C1738 -- 1.6 -- 1.0 -- Q01201_C109 1.5 -- -- -- -- Q70CQ2_C741 -- -- -- -- -- P41226_C599 -- -- -- -- 1.3 P14598_C378 -- -- -- -- -- Q9C0C9_C375 -- -- -- -- -- O00622_C134 -- 1.0 -- -- -- O00541_C361 -- -- -- 2.0 -- P43403_C117 -- -- -- -- -- Q96FA3_C282 -- -- 0.5 -- -- Q9UPT9_C44 -- -- -- -- 1.2 Q9Y4C1_C251 1.3 -- -- -- -- Q70CQ2_C1090 -- -- -- -- -- O00622_C70 -- -- -- -- -- P04150_C622 -- -- -- -- -- 28_500.mu.M.sub.-- 28_500.mu.M.sub.-- 29_500.mu.M.sub.-- 29_500.mu.M.sub.-- 30_500.mu.M.sub.-- Identifier invitro_231 invitro_ramos invitro_231 invitro_ramos invitro_231 Q99873_C109 0.8 1.1 1.0 0.9 1.1 P24752_C119 1.1 1.1 1.3 0.6 0.7 P09211_C48 1.3 1.5 1.0 0.4 1.4 O14980_C34 0.8 0.9 1.3 0.9 0.7 P24752_C196 1.0 1.1 1.3 1.0 0.8 Q15084_C55 1.1 1.5 1.0 1.3 2.2 P24752_C413 1.2 1.4 1.3 0.7 1.1 P63244_C182 1.0 1.1 1.3 1.2 1.2 P24752_C126 1.7 1.6 1.1 0.8 1.1 Q15084_C190 1.1 1.6 1.2 -- 2.3 Q8TAQ2_C145 1.1 1.2 1.0 -- 1.1 P68036_C86 1.4 -- 0.9 1.5 -- P15374_C95 1.1 0.9 0.9 1.0 0.7 Q16763_C118 1.0 0.8 -- -- 0.7 Q16822_C306 1.1 -- 1.1 0.5 1.2 O14980_C528 -- -- 1.0 -- -- O00170_C122 0.9 1.2 -- 1.3 1.1 O75874_C269 1.1 -- 0.8 -- 0.7 O75362_C286 1.0 1.0 0.9 -- -- P40763_C259 1.0 1.1 1.5 -- -- Q9Y3Z3_C522 1.0 -- 1.5 -- 0.7 P16455_C150 -- 1.7 -- 1.3 -- Q96GG9_C115 -- -- -- -- 0.7 P00813_C75 -- 0.9 -- -- -- O14933_C98 -- 1.2 -- -- 1.3 Q14790_C360 1.1 -- -- -- 2.0 Q15306_C194 -- 1.1 -- 1.2 -- Q6L8Q7_C108 1.0 1.2 -- -- 1.1 P48735_C308 0.9 -- 1.0 -- 0.7 Q86UV5_C39 -- -- 1.2 -- -- P50851_C1704 -- -- -- -- -- O94953_C694 -- -- 1.2 -- -- P19447_C342 -- -- 0.8 -- 1.3 Q00535_C157 0.9 1.0 -- -- -- Q9UPT9_C171 -- 1.3 -- -- -- Q9HB90_C377 1.3 -- 1.3 -- 1.4 P50851_C2675 -- -- 1.1 -- 0.9 Q9NYL2_C22 1.4 -- 1.2 -- 1.1 Q5T1V6_C414 -- -- -- -- -- Q9HB90_C358 1.1 -- -- -- 1.0 P16455_C145 -- 20.0 -- 20.0 -- Q9Y5T5_C205 -- 1.6 -- -- -- O00541_C272 -- -- 0.9 -- -- Q02556_C306 -- -- -- -- -- Q15910_C503 -- -- 1.4 -- -- Q96RU2_C171 -- -- -- -- -- Q16877_C159 -- -- -- -- -- P04150_C302 -- -- -- -- -- Q96JH7_C219 -- -- -- -- -- P48200_C137 -- -- -- -- -- O00622_C39 1.0 -- -- -- -- Q5T1V6_C453 -- -- -- -- -- P51617_C608 -- -- -- -- -- P42575_C370 -- -- -- -- -- P09086_C346 -- -- -- -- -- Q09472_C1738 -- -- 1.3 -- -- Q01201_C109 -- 1.1 -- -- -- Q70CQ2_C741 -- -- -- -- -- P41226_C599 -- -- -- -- -- P14598_C378 -- -- -- -- -- Q9C0C9_C375 -- -- -- -- -- O00622_C134 -- -- -- -- -- O00541_C361 -- -- -- -- -- P43403_C117 -- 1.1 -- -- -- Q96FA3_C282 -- 0.7 -- -- -- Q9UPT9_C44 -- -- -- -- -- Q9Y4C1_C251 -- -- -- -- -- Q70CQ2_C1090 -- -- -- -- -- O00622_C70 -- -- -- -- -- P04150_C622 -- -- -- -- --
TABLE-US-00007 TABLE 1F 30_500.mu.M.sub.-- 31_500.mu.M.sub.-- 31_500.mu.M.sub.-- 32_500.mu.M.sub.-- 32_500.mu.M.sub.-- Identifier invitro_ramos invitro_231 invitro_ramos invitro_231 invitro_ramos Q99873_C109 1.8 1.0 1.3 1.7 1.8 P24752_C119 0.9 1.3 0.7 1.7 1.3 P09211_C48 1.2 2.0 1.3 1.9 1.4 O14980_C34 1.1 1.0 0.8 1.2 1.0 P24752_C196 1.1 1.2 0.9 2.3 2.0 Q15084_C55 -- 1.2 1.1 20.0 20.0 P24752_C413 1.9 1.6 1.0 6.8 4.1 P63244_C182 1.6 1.3 -- 1.9 -- P24752_C126 1.6 1.1 -- 7.3 8.6 Q15084_C190 -- 1.3 1.2 20.0 20.0 Q8TAQ2_C145 1.5 1.1 0.9 20.0 20.0 P68036_C86 1.5 1.7 1.3 1.1 0.9 P15374_C95 1.0 0.9 0.7 1.2 1.0 Q16763_C118 1.1 0.9 0.8 1.1 1.1 Q16822_C306 1.1 1.9 1.2 3.2 -- O14980_C528 -- 5.2 3.2 1.7 1.2 O00170_C122 2.0 -- 0.6 3.7 1.9 O75874_C269 -- 0.4 0.7 -- -- O75362_C286 2.1 -- -- -- -- P40763_C259 -- 1.7 -- 10.2 -- Q9Y3Z3_C522 -- 1.5 -- -- 1.8 P16455_C150 5.1 -- 2.8 -- 20.0 Q96GG9_C115 1.1 -- 0.9 -- 0.9 P00813_C75 3.1 -- 0.8 -- 1.7 O14933_C98 2.1 -- -- 13.8 4.6 Q14790_C360 5.2 -- -- -- -- Q15306_C194 1.7 -- 1.1 -- 3.7 Q6L8Q7_C108 1.6 2.0 -- 2.5 1.9 P48735_C308 -- 1.3 1.1 -- -- Q86UV5_C39 -- -- -- -- -- P50851_C1704 -- -- 1.4 -- 2.0 O94953_C694 -- -- -- -- -- P19447_C342 -- -- -- -- -- Q00535_C157 -- -- 1.1 1.7 1.7 Q9UPT9_C171 2.3 2.1 -- -- 9.3 Q9HB90_C377 -- 2.2 -- -- 1.5 P50851_C2675 -- -- -- 1.8 1.1 Q9NYL2_C22 -- -- -- 2.1 -- Q5T1V6_C414 2.0 -- -- -- -- Q9HB90_C358 -- 2.3 -- 1.9 1.2 P16455_C145 -- -- 1.0 -- 20.0 Q9Y5T5_C205 -- -- -- -- -- O00541_C272 -- -- -- -- -- Q02556_C306 2.0 -- -- -- -- Q15910_C503 -- -- -- -- 1.7 Q96RU2_C171 -- -- -- -- -- Q16877_C159 -- -- -- -- 1.5 P04150_C302 -- -- -- -- -- Q96JH7_C219 -- -- 1.0 -- -- P48200_C137 -- -- -- -- -- O00622_C39 -- 1.2 -- -- -- Q5T1V6_C453 -- -- -- -- -- P51617_C608 -- -- -- 5.8 2.9 P42575_C370 4.1 -- -- -- -- P09086_C346 -- -- 0.6 -- -- Q09472_C1738 -- -- -- -- -- Q01201_C109 -- -- -- -- 20.0 Q70CQ2_C741 -- 4.2 -- -- -- P41226_C599 -- -- -- -- -- P14598_C378 -- -- -- -- -- Q9C0C9_C375 -- -- -- -- -- O00622_C134 -- -- -- -- -- O00541_C361 -- -- -- -- -- P43403_C117 -- -- -- -- -- Q96FA3_C282 1.5 -- -- -- -- Q9UPT9_C44 1.7 -- -- -- -- Q9Y4C1_C251 -- -- -- -- -- Q70CQ2_C1090 -- -- -- -- -- O00622_C70 -- -- -- -- -- P04150_C622 -- 4.8 -- -- -- 33_500.mu.M.sub.-- 33_500.mu.M.sub.-- 34_500.mu.M.sub.-- 34_500.mu.M.sub.-- 35_500.mu.M.sub.-- Identifier invitro_231 invitro_ramos invitro_231 invitro_ramos invitro_231 Q99873_C109 1.0 0.9 0.8 1.1 0.8 P24752_C119 0.7 0.7 0.8 -- 0.9 P09211_C48 2.0 0.7 0.8 1.0 0.7 O14980_C34 0.8 0.9 0.8 1.1 0.8 P24752_C196 0.9 1.3 0.8 1.0 0.9 Q15084_C55 3.3 -- 0.9 -- 0.9 P24752_C413 1.3 -- 0.9 1.3 -- P63244_C182 -- 1.1 -- -- 0.9 P24752_C126 4.3 -- -- 1.1 -- Q15084_C190 3.3 -- 0.9 -- 0.9 Q8TAQ2_C145 -- 1.5 -- -- 0.9 P68036_C86 -- 0.9 -- -- -- P15374_C95 -- -- -- -- -- Q16763_C118 -- -- -- -- -- Q16822_C306 -- -- -- -- 0.9 O14980_C528 -- -- -- 1.1 -- O00170_C122 -- -- 1.0 -- -- O75874_C269 1.2 -- -- -- -- O75362_C286 -- -- -- -- -- P40763_C259 -- -- -- -- -- Q9Y3Z3_C522 0.8 -- -- -- -- P16455_C150 -- 1.9 -- 1.4 -- Q96GG9_C115 -- -- -- -- -- P00813_C75 -- 0.8 -- 1.1 -- O14933_C98 -- 1.2 0.8 -- -- Q14790_C360 3.1 -- -- -- -- Q15306_C194 -- 1.1 -- -- -- Q6L8Q7_C108 -- -- -- -- 0.8 P48735_C308 -- -- -- -- -- Q861UV5_C39 -- -- -- -- -- P50851_C1704 -- -- -- -- -- O94953_C694 -- -- -- -- -- P19447_C342 -- -- -- -- -- Q00535_C157 -- -- 0.7 -- -- Q9UPT9_C171 -- -- -- -- -- Q9HB90_C377 1.1 -- -- -- -- P50851_C2675 -- -- -- 1.1 -- Q9NYL2_C22 3.7 -- 0.8 -- -- Q5T1V6_C414 -- -- 0.6 -- -- Q9HB90_C358 -- -- -- -- -- P16455_C145 -- -- -- -- -- Q9Y5T5_C205 -- -- -- -- -- O00541_C272 1.4 -- -- -- 0.8 Q02556_C306 -- -- -- -- -- Q15910_C503 -- -- -- -- -- Q96RU2_C171 -- -- -- -- -- Q16877_C159 -- -- -- 1.4 -- P04150_C302 -- -- -- -- -- Q96JH7_C219 -- -- -- -- -- P48200_C137 -- -- -- -- -- O00622_C39 -- -- -- -- -- Q5T1V6_C453 -- -- -- -- -- P51617_C608 -- -- -- -- -- P42575_C370 -- -- -- -- -- P09086_C346 -- -- -- -- -- Q09472_C1738 -- -- -- -- -- Q01201_C109 -- -- -- -- -- Q70CQ2_C741 -- -- -- -- -- P41226_C599 -- -- -- -- -- P14598_C378 -- -- -- -- -- Q9C0C9_C375 -- -- -- -- -- O00622_C134 -- -- -- -- -- O00541_C361 -- -- -- -- -- P43403_C117 -- -- -- -- -- Q96FA3_C282 -- -- -- -- -- Q9UPT9_C44 -- -- -- -- -- Q9Y4C1_C251 -- -- -- -- -- Q70CQ2_C1090 -- -- -- -- -- O00622_C70 -- -- -- -- -- P04150_C622 -- -- -- -- --
TABLE-US-00008 TABLE 1G 35_500.mu.M.sub.-- 36_500.mu.M.sub.-- 37_500.mu.M.sub.-- 38_500.mu.M.sub.-- 38_500.mu.M.sub.-- Identifier invitro_ramos invitro_231 invitro_ramos invitro_231 invitro_ramos Q99873_C109 1.0 1.0 1.0 0.9 1.4 P24752_C119 1.0 1.2 0.8 1.0 1.7 P09211_C48 1.3 1.6 1.2 1.9 -- O14980_C34 1.3 1.3 0.9 1.0 1.0 P24752_C196 1.4 1.5 1.0 1.0 1.3 Q15084_C55 -- -- 1.0 1.0 1.3 P24752_C413 1.3 2.7 0.9 1.1 20.0 P63244_C182 0.9 1.3 0.9 1.1 -- P24752_C126 10.4 -- 0.9 1.0 1.4 Q15084_C190 -- -- 1.1 1.1 -- Q8TAQ2_C145 -- -- -- 1.1 1.9 P68036_C86 -- -- -- -- 1.5 P15374_C95 -- -- -- 2.7 12.5 Q16763_C118 -- -- -- 1.0 1.0 Q16822_C306 1.0 -- 1.0 1.0 -- O14980_C528 0.8 -- 0.9 1.0 -- O00170_C122 -- -- -- 1.5 1.5 O75874_C269 -- 1.7 -- 0.7 -- O75362_C286 -- -- 0.9 0.9 1.2 P40763_C259 1.1 -- 0.9 -- 1.5 Q9Y3Z3_C522 -- -- 0.7 -- -- P16455_C150 1.3 -- 1.0 -- 1.8 Q96GG9_C115 0.9 -- 0.8 1.2 1.0 P00813_C75 1.1 -- 0.9 -- 2.3 O14933_C98 -- -- -- 1.6 1.7 Q14790_C360 1.3 -- 1.0 1.0 4.1 Q15306_C194 1.0 -- 0.9 -- 2.1 Q6L8Q7_C108 -- -- -- 0.9 6.8 P48735_C308 -- 1.3 -- 1.2 -- Q86UV5_C39 1.0 -- 0.7 3.7 -- P50851_C1704 1.1 -- 1.0 -- 2.5 O94953_C694 1.1 -- 1.1 -- 2.4 P19447_C342 -- -- -- 1.3 -- Q00535_C157 -- -- -- 0.9 -- Q9UPT9_C171 1.5 -- -- 1.4 -- Q9HB90_C377 -- -- -- 1.0 -- P50851_C2675 1.0 -- 1.0 -- -- Q9NYL2_C22 -- -- -- 1.2 -- Q5T1V6_C414 1.3 -- 1.0 0.9 1.8 Q9HB90_C358 -- -- -- 1.1 -- P16455_C145 -- -- -- -- -- Q9Y5T5_C205 -- -- -- -- 1.9 O00541_C272 1.2 -- 1.0 1.0 -- Q02556_C306 1.2 -- -- -- -- Q15910_C503 -- -- -- -- -- Q96RU2_C171 -- -- -- 3.3 -- Q16877_C159 -- -- -- -- -- P04150_C302 0.9 -- 0.9 -- -- Q96JH7_C219 -- -- -- -- 1.8 P48200_C137 1.1 -- 0.9 -- -- O00622_C39 -- -- -- 0.8 -- Q5T1V6_C453 -- -- -- -- -- P51617_C608 -- -- -- -- 2.4 P42575_C370 -- -- 0.7 -- -- P09086_C346 -- -- -- -- -- Q09472_C1738 -- -- -- -- -- Q01201_C109 -- -- -- -- -- Q70CQ2_C741 -- -- 1.1 -- 4.1 P41226_C599 1.2 -- -- -- -- P14598_C378 -- -- -- -- -- Q9C0C9_C375 -- -- -- -- -- O00622_C134 -- -- -- -- -- O00541_C361 -- -- -- -- -- P43403_C117 -- -- -- -- -- Q96FA3_C282 -- -- -- -- 7.0 Q9UPT9_C44 -- -- -- -- -- Q9Y4C1_C251 -- -- -- -- -- Q70CQ2_C1090 -- -- -- -- -- O00622_C70 -- -- -- -- -- P04150_C622 -- -- -- -- -- 39_500.mu.M.sub.-- 40_500.mu.M.sub.-- 40_500.mu.M.sub.-- 41_500.mu.M.sub.-- 41_500.mu.M.sub.-- Identifier invitro_231 invitro_231 invitro_ramos invitro_231 invitro_ramos Q99873_C109 1.5 0.8 1.3 0.9 0.9 P24752_C119 1.7 0.8 0.9 1.0 0.8 P09211_C48 2.0 1.6 1.6 1.3 0.9 O14980_C34 1.3 0.8 1.1 3.1 2.0 P24752_C196 3.0 0.8 -- 1.3 1.0 Q15084_C55 3.9 0.8 -- 0.9 0.8 P24752_C413 2.7 0.9 -- 0.8 0.9 P63244_C182 1.0 1.0 0.6 -- 4.7 P24752_C126 -- 0.9 -- 1.0 0.9 Q15084_C190 7.0 0.9 1.7 1.0 -- Q8TAQ2_C145 1.2 0.9 1.1 1.8 2.8 P68036_C86 1.9 0.8 0.9 9.8 7.9 P15374_C95 -- -- 0.9 1.0 0.8 Q16763_C118 1.4 0.9 0.8 -- 3.4 Q16822_C306 -- -- -- 1.2 1.0 O14980_C528 -- -- -- 0.6 -- O00170_C122 -- -- -- 1.1 1.0 O75874_C269 -- -- 0.8 0.8 -- O75362_C286 20.0 -- -- -- 0.6 P40763_C259 -- -- 1.4 0.9 -- Q9Y3Z3_C522 1.1 0.8 -- 0.9 -- P16455_C150 -- -- 1.9 -- 0.9 Q96GG9_C115 -- -- 0.9 -- -- P00813_C75 -- -- 0.9 -- 1.1 O14933_C98 -- -- 1.3 1.2 1.1 Q14790_C360 -- -- -- 1.0 0.8 Q15306_C194 -- -- 1.1 -- 1.2 Q6L8Q7_C108 3.2 0.8 -- 1.3 -- P48735_C308 -- -- -- -- -- Q86UV5_C39 -- -- 1.6 -- -- P50851_C1704 -- -- -- -- -- O94953_C694 -- -- -- -- -- P19447_C342 -- -- -- 1.1 -- Q00535_C157 -- -- -- 1.5 -- Q9UPT9_C171 -- -- -- 3.0 1.4 Q9HB90_C377 -- -- -- 1.1 -- P50851_C2675 -- -- -- 1.9 -- Q9NYL2_C22 -- -- -- 1.5 -- Q5T1V6_C414 -- -- 1.4 1.2 1.0 Q9HB90_C358 -- -- -- 1.3 -- P16455_C145 -- -- 1.0 1.9 -- Q9Y5T5_C205 -- -- 0.9 0.8 -- O00541_C272 -- -- -- -- -- Q02556_C306 -- -- 0.9 -- 1.4 Q15910_C503 -- -- -- -- -- Q96RU2_C171 -- -- -- -- -- Q16877_C159 -- -- 1.2 -- -- P04150_C302 -- -- -- -- -- Q96JH7_C219 -- -- 1.9 -- -- P48200_C137 -- -- 1.1 1.2 -- O00622_C39 -- -- -- -- -- Q5T1V6_C453 -- -- -- -- -- P51617_C608 -- -- -- 1.1 -- P42575_C370 -- -- 2.7 -- 1.4 P09086_C346 -- -- -- -- -- Q09472_C1738 -- -- -- -- -- Q01201_C109 -- -- -- -- -- Q70CQ2_C741 -- -- -- -- -- P41226_C599 -- -- -- -- -- P14598_C378 -- -- -- -- -- Q9C0C9_C375 -- -- -- -- -- O00622_C134 -- -- -- -- -- O00541_C361 -- -- -- -- -- P43403_C117 -- -- -- -- -- Q96FA3_C282 -- -- -- -- -- Q9UPT9_C44 -- -- -- -- -- Q9Y4C1_C251 -- -- -- -- -- Q70CQ2_C1090 -- -- -- -- -- O00622_C70 -- -- -- -- -- P04150_C622 -- -- -- -- --
TABLE-US-00009 TABLE 1H 42_500.mu.M.sub.-- 43_500.mu.M.sub.-- 43_500.mu.M.sub.-- 44_500.mu.M.sub.-- 45_500.mu.M.sub.-- Identifier invitro_ramos invitro_231 invitro_ramos invitro_231 invitro_231 Q99873_C109 0.9 1.4 1.2 0.8 2.2 P24752_C119 0.8 1.8 1.1 0.8 12.4 P09211_C48 0.7 4.1 2.2 0.9 19.7 O14980_C34 0.9 1.1 -- 0.8 1.8 P24752_C196 0.8 2.4 2.3 0.8 20.0 Q15084_C55 0.9 20.0 20.0 1.0 -- P24752_C413 0.7 15.9 -- 0.8 20.0 P63244_C182 0.8 0.9 0.9 -- 0.9 P24752_C126 0.8 -- 20.0 0.8 20.0 Q15084_C190 -- 20.0 -- 1.0 -- Q8TAQ2_C145 1.0 20.0 -- 1.0 -- P68036_C86 0.8 1.1 1.7 -- -- P15374_C95 0.9 -- 20.0 0.8 -- Q16763_C118 0.8 -- 1.0 0.8 -- Q16822_C306 0.8 3.3 -- -- 3.3 O14980_C528 -- 4.6 -- -- -- O00170_C122 0.8 -- 2.1 -- -- O75874_C269 -- 0.8 -- 0.8 20.0 O75362_C286 1.0 1.9 1.2 -- 20.0 P40763_C259 -- -- -- -- -- Q9Y3Z3_C522 -- 2.2 -- 0.8 -- P16455_C150 0.8 -- 20.0 -- -- Q96GG9_C115 0.9 -- -- -- -- P00813_C75 0.8 -- 1.2 -- -- O14933_C98 1.0 -- -- 2.0 -- Q14790_C360 -- -- -- -- -- Q15306_C194 1.2 -- 3.0 -- -- Q6L8Q7_C108 -- -- -- 0.8 -- P48735_C308 -- 0.8 -- -- 1.0 Q86UV5_C39 -- 1.5 -- -- -- P50851_C1704 -- -- -- -- -- O94953_C694 0.9 20.0 -- -- -- P19447_C342 -- -- -- -- -- Q00535_C157 -- -- 1.2 -- -- Q9UPT9_C171 1.0 -- 4.5 -- -- Q9HB90_C377 -- -- -- 0.7 -- P50851_C2675 -- 1 -- -- -- Q9NYL2_C22 -- 2.9 -- 0.8 20.0 Q5T1V6_C414 -- -- -- 0.8 -- Q9HB90_C358 -- -- -- -- -- P16455_C145 0.9 -- -- -- -- Q9Y5T5_C205 1.5 -- -- -- -- O00541_C272 0.9 -- -- -- -- Q02556_C306 0.9 -- 2.7 -- -- Q15910_C503 -- 20.0 -- -- -- Q96RU2_C171 -- -- -- -- -- Q16877_C159 -- -- -- -- -- P04150_C302 -- 1.5 -- -- -- Q96JH7_C219 -- -- -- -- -- P48200_C137 -- -- -- -- -- O00622_C39 -- -- -- -- -- Q5T1V6_C453 1.0 20.0 -- -- -- P51617_C608 -- -- -- -- -- P42575_C370 -- -- -- -- -- P09086_C346 -- -- -- -- -- Q09472_C1738 -- 2.2 -- -- -- Q01201_C109 -- -- -- -- -- Q70CQ2_C741 -- -- -- -- -- P41226_C599 -- -- -- -- -- P14598_C378 -- -- 1.2 -- -- Q9C0C9_C375 -- -- -- -- -- O00622_C134 -- -- -- -- -- O00541_C361 -- -- -- -- -- P43403_C117 -- -- -- -- -- Q96FA3_C282 1.2 -- -- -- -- Q9UPT9_C44 -- -- 2.1 -- -- Q9Y4C1_C251 -- -- -- -- -- Q70CQ2_C1090 -- -- -- -- -- O00622_C70 -- -- -- -- -- P04150_C622 -- -- -- -- -- 46_500.mu.M.sub.-- 47_500.mu.M.sub.-- 48_500.mu.M.sub.-- 49_500.mu.M.sub.-- 50_500.mu.M.sub.-- Identifier invitro_231 invitro_231 invitro_231 invitro_231 invitro_231 Q99873_C109 0.8 0.9 1.0 2.0 1.0 P24752_C119 0.8 1.0 1.3 4.9 2.1 P09211_C48 1.0 2.5 1.1 -- 2.3 O14980_C34 1.0 1.0 1.1 1.9 1.2 P24752_C196 0.9 1.1 1.3 4.7 3.9 Q15084_C55 0.8 0.9 1.0 12.8 1.3 P24752_C413 1.1 0.9 1.1 2.6 9.2 P63244_C182 0.8 1.1 1.1 -- 0.8 P24752_C126 0.9 1.0 -- 20.0 20.0 Q15084_C190 0.9 1.0 -- 7.7 -- Q8TAQ2_C145 1.0 1.5 1.1 -- 2.8 P68036_C86 1.2 2.0 -- 1.9 -- P15374_C95 1.0 1.0 -- -- 1.3 Q16763_C118 1.0 -- -- -- 0.8 Q16822_C306 0.9 1.0 -- -- 2.7 O14980_C528 0.7 2.2 -- -- -- O00170_C122 -- -- -- 4.8 2.3 O75874_C269 0.6 0.9 1.0 -- 20.0 O75362_C286 0.9 -- -- 1.0 -- P40763_C259 1.2 -- -- -- -- Q9Y3Z3_C522 -- -- 1.4 3.0 -- P16455_C150 -- -- -- -- -- Q96GG9_C115 -- -- -- -- -- P00813_C75 -- -- -- -- -- O14933_C98 -- -- -- -- 1.5 Q14790_C360 -- -- -- -- -- Q15306_C194 -- -- -- -- -- Q6L8Q7_C108 -- -- -- -- 1.8 P48735_C308 0.2 1.2 1.1 -- 2.2 Q86UV5_C39 -- -- -- -- -- P50851_C1704 -- -- -- 4.0 -- O94953_C694 -- -- -- -- -- P19447_C342 1.0 -- -- -- -- Q00535_C157 -- -- -- -- -- Q9UPT9_C171 -- -- -- -- -- Q9HB90_C377 -- 1.6 1.4 -- -- P50851_C2675 -- -- -- -- -- Q9NYL2_C22 -- -- -- -- -- Q5T1V6_C414 -- -- -- -- -- Q9HB90_C358 1.1 -- -- -- -- P16455_C145 -- -- -- -- -- Q9Y5T5_C205 1.0 -- -- -- -- O00541_C272 -- -- -- -- -- Q02556_C306 -- -- -- -- -- Q15910_C503 -- -- -- -- -- Q96RU2_C171 0.9 1.5 -- -- -- Q16877_C159 -- -- -- -- -- P04150_C302 -- -- -- -- -- Q96JH7_C219 -- -- -- -- -- P48200_C137 -- -- -- -- -- O00622_C39 1.9 -- -- -- 2.3 Q5T1V6_C453 -- -- -- -- -- P51617_C608 -- -- -- -- -- P42575_C370 -- -- -- -- -- P09086_C346 -- -- -- -- -- Q09472_C1738 -- -- -- -- -- Q01201_C109 -- -- -- -- -- Q70CQ2_C741 -- -- -- -- -- P41226_C599 -- -- -- -- -- P14598_C378 -- -- -- -- -- Q9C0C9_C375 -- -- -- -- -- O00622_C134 -- -- -- -- -- O00541_C361 -- -- -- -- -- P43403_C117 -- -- -- -- -- Q96FA3_C282 -- -- -- -- -- Q9UPT9_C44 -- -- -- -- -- Q9Y4C1_C251 1.0 -- -- -- -- Q70CQ2_C1090 -- -- -- -- -- O00622_C70 -- -- -- -- -- P04150_C622 -- -- -- -- --
TABLE-US-00010 TABLE 1I 51_500.mu.M.sub.-- 51_500.mu.M.sub.-- 52_500.mu.M.sub.-- 52_500.mu.M.sub.-- 53_500.mu.M.sub.-- Identifier invitro_231 invitro_ramos invitro_231 invitro_ramos invitro_231 Q99873_C109 0.9 9.5 0.9 1.3 0.9 P24752_C119 0.8 0.9 1.4 0.8 0.9 P09211_C48 1.1 1.0 1.0 1.9 2.3 O14980_C34 0.9 1.0 1.0 1.1 0.9 P24752_C196 1.1 -- 1.3 1.5 1.2 Q15084_C55 2.5 2.7 1.1 1.8 0.9 P24752_C413 1.0 1.0 1.9 1.5 1.0 P63244_C182 0.7 0.9 1.0 -- 0.9 P24752_C126 1.0 1.2 1.3 -- -- Q15084_C190 -- 5.6 1.0 1.9 1.0 Q8TAQ2_C145 -- 1.1 0.9 1.8 -- P68036_C86 -- 1.0 0.8 1.3 1.0 P15374_C95 -- 0.9 1.2 -- -- Q16763_C118 -- 0.9 1.2 -- 1.0 Q16822_C306 -- 0.8 1.3 1.6 1.3 O14980_C528 -- 0.9 1.3 -- -- O00170_C122 1.4 -- -- 1.4 -- O75874_C269 -- -- -- -- -- O75362_C286 0.9 20.0 1.0 -- 1.1 P40763_C259 -- -- 2.2 -- -- Q9Y3Z3_C522 -- -- 1.2 -- -- P16455_C150 -- -- -- 2.2 -- Q96GG9_C115 -- 0.8 -- 1.0 1.0 P00813_C75 -- 1.0 -- 1.2 -- O14933_C98 -- -- 1.4 1.4 -- Q14790_C360 -- -- -- -- -- Q15306_C194 -- 1.4 -- 1.4 -- Q6L8Q7_C108 -- -- 1.4 6.8 -- P48735_C308 -- -- 1.1 -- 0.5 Q86UV5_C39 -- 1.8 0.9 -- -- P50851_C1704 -- 1.1 -- 1.1 -- O94953_C694 -- 1.4 -- 1.4 -- P19447_C342 1.4 -- 1.0 -- -- Q00535_C157 -- 1.0 -- -- 1.2 Q9UPT9_C171 -- -- -- -- -- Q9HB90_C377 0.6 -- -- -- -- P50851_C2675 -- 0.9 -- -- -- Q9NYL2_C22 -- -- -- -- -- Q5T1V6_C414 -- -- -- -- 0.8 Q9HB90_C358 -- -- -- -- -- P16455_C145 -- 20.0 -- -- -- Q9Y5T5_C205 1.7 3.2 -- -- 1.1 O00541_C272 -- -- -- -- -- Q02556_C306 -- 1.5 -- -- -- Q15910_C503 -- -- 0.9 -- -- Q96RU2_C171 -- -- -- -- -- Q16877_C159 -- 1.1 -- -- -- P04150_C302 -- -- -- 1.8 -- Q96JH7_C219 -- 2.3 -- 1.6 -- P48200_C137 -- -- -- 1.3 -- O00622_C39 -- -- -- -- -- Q5T1V6_C453 -- 1.8 -- -- -- P51617_C608 -- 1.1 -- 17.6 -- P42575_C370 -- -- -- -- -- P09086_C346 -- -- -- -- -- Q09472_C1738 -- -- 1.1 -- -- Q01201_C109 -- 1.6 -- -- -- Q70CQ2_C741 -- 0.9 -- -- -- P41226_C599 -- -- -- -- -- P14598_C378 -- -- -- -- -- Q9C0C9_C375 -- 0.8 -- -- -- O00622_C134 -- -- -- -- -- O00541_C361 -- -- -- -- -- P43403_C117 -- -- -- -- -- Q96FA3_C282 -- -- -- 20.0 -- Q9UPT9_C44 -- -- -- -- -- Q9Y4C1_C251 -- -- -- -- -- Q70CQ2_C1090 -- -- -- -- -- O00622_C70 -- -- -- -- -- P04150_C622 -- -- -- -- -- 53_500.mu.M.sub.-- 54_500.mu.M.sub.-- 55_500.mu.M.sub.-- 56_500.mu.M.sub.-- 56_500.mu.M.sub.-- Identifier invitro_ramos invitro_231 invitro_2 invitro_231 invitro_ramos Q99873_C109 1.1 1.2 1.0 1.0 1.1 P24752_C119 0.7 1.0 0.9 1.0 1.2 P09211_C48 1.2 1.3 1.4 1.9 1.2 O14980_C34 1.0 1.1 0.9 0.9 0.9 P24752_C196 0.9 1.1 1.0 1.1 1.0 Q15084_C55 1.0 1.2 0.9 1.0 1.1 P24752_C413 -- 1.1 1.0 1.0 0.9 P63244_C182 0.8 -- 1.0 1.1 -- P24752_C126 -- 1.1 0.9 0.9 -- Q15084_C190 1.0 1.2 -- 0.9 1.1 Q8TAQ2_C145 1.2 -- -- 1.4 1.2 P68036_C86 1.0 -- 1.0 1.5 1.3 P15374_C95 0.7 -- -- 1.0 1.1 Q16763_C118 0.8 -- -- 1.2 1.1 Q16822_C306 -- -- -- 1.4 -- O14980_C528 0.8 -- -- 20.0 -- O00170_C122 0.9 -- -- 0.8 0.9 O75874_C269 -- 1.1 -- 0.9 -- O75362_C286 -- -- -- 1.0 -- P40763_C259 1.9 1.3 -- -- -- Q9Y3Z3_C522 0.7 -- -- 1.0 1.0 P16455_C150 1.1 -- -- -- 1.4 Q96GG9_C115 0.9 1.1 -- -- 1.0 P00813_C75 -- -- -- -- 0.9 O14933_C98 1.0 -- -- -- 1.1 Q14790_C360 -- 1.2 -- 1.0 -- Q15306_C194 1.0 -- -- -- 1.5 Q6L8Q7_C108 1.1 -- -- -- -- P48735_C308 -- -- -- 1.0 -- Q86UV5_C39 -- -- -- 1.0 -- P50851_C1704 -- -- -- -- -- O94953_C694 -- -- -- -- -- P19447_C342 -- -- -- 1.2 -- Q00535_C157 0.8 -- -- 1.1 -- Q9UPT9_C171 -- -- -- -- 1.9 Q9HB90_C377 1.1 -- -- 1.2 -- P50851_C2675 -- -- -- -- -- Q9NYL2_C22 -- 1.1 1.2 1.1 -- Q5T1V6_C414 -- -- -- 1.0 1.0 Q9HB90_C358 0.6 -- -- 1.0 1.0 P16455_C145 1.0 -- -- -- -- Q9Y5T5_C205 -- -- -- 1.1 0.9 O00541_C272 -- -- 1.0 1.1 -- Q02556_C306 -- -- -- -- -- Q15910_C503 -- -- -- -- -- Q96RU2_C171 -- -- -- -- -- Q16877_C159 -- -- -- -- -- P04150_C302 -- -- -- -- -- Q96JH7_C219 -- -- -- -- 1.0 P48200_C137 -- -- -- -- -- O00622_C39 -- -- -- -- -- Q5T1V6_C453 -- -- -- -- -- P51617_C608 -- -- -- -- -- P42575_C370 -- -- -- -- -- P09086_C346 -- -- -- -- -- Q09472_C1738 -- -- -- -- -- Q01201_C109 -- -- -- -- -- Q70CQ2_C741 -- -- -- -- -- P41226_C599 -- -- -- -- -- P14598_C378 -- -- -- -- -- Q9C0C9_C375 -- -- -- -- -- O00622_C134 -- -- -- -- -- O00541_C361 -- -- -- -- -- P43403_C117 -- -- -- -- -- Q96FA3_C282 -- -- -- -- -- Q9UPT9_C44 -- -- -- -- -- Q9Y4C1_C251 -- -- -- -- -- Q70CQ2_C1090 -- -- -- -- -- O00622_C70 -- -- -- -- -- P04150_C622 -- -- -- -- --
[0506] Table 2 illustrates a list of liganded cysteines and their reactivity profiles with the fragment electrophile library from isoTOP-ABPP experiments performed in situ. Table 2 further shows the accession number (or the protein identifier) of the protein.
TABLE-US-00011 TABLE 2A SEQ ID 2_200.mu.M.sub.-- 4_100.mu.M.sub.-- 8_200.mu.M.sub.-- 9_200.mu.M.sub.-- Identifier Protein NO: insitu_231 insitu_231 insitu_231 insitu_231 P04406_C152 GAPDH Glyceraldehyde-3-phosphate 16 20.0 1.6 1.1 1.7 dehydrogenase P61978_C132 HNRNPK Heterogeneous nuclear 768 5.0 3.8 4.2 1.4 ribonucleoprotein K Q13526_C113 PIN1 Peptidyl-prolyl cis-trans isomerase 1.9 20.0 3.4 2.6 3.3 NIMA-interacting 1 P24752_C119 ACAT1 Acetyl-CoA acetyltransferase, 22 2.6 3.0 5.5 3.8 mitochondrial P24752_C413 ACAT1 Acetyl-CoA acetyltransferase, 56 20.0 4.5 20.0 19.5 mitochondrial Q9NUY8_C283 TBC1D23 TBC1 domain family member 23 101 4.1 2.1 4.9 2.1 P13667_C206 PDIA4 Protein disulfide-isomerase A4 36 2.6 6.8 15.2 1.1 P12268_C140 IMPDH2 Inosine-5-monophosphate 45 1.3 1.0 0.9 1.2 dehydrogenase 2 Q15365_C194 PCBP1 Poly(rC)-binding protein 1 29 5.1 1.4 1.4 10.1 Q9NVC6_C649 MED17 Mediator of RNA polymerase II 211 7.7 1.7 3.2 2.2 transcription subunit 17 P42166_C561 TMPO Lamina-associated polypeptide 2, 88 8.3 20.0 4.6 3.7 isoform alpha Q9Y696_C35 CLIC4 Chloride intracellular channel protein 21 1.7 20.0 4.7 2.6 4 P10599_C32 TXN Thioredoxin 34 7.9 3.3 5.9 20.0 P31943_C267 HNRNPH1 Heterogeneous nuclear 769 3.8 6.0 5.3 2.1 ribonucleoprotein H Q86SX6_C67 GLRX5 Glutaredoxin-related protein 5, 26 1.1 1.4 1.0 5.1 mitochondrial P15121_C299 AKR1B1 Aldose reductase 48 0.9 1.0 1.7 1.4 P52597_C267 HNRNPF Heterogeneous nuclear 108 4.2 20.0 -- 3.2 ribonucleoprotein F Q9ULV4_C420 CORO1C Coronin-1C 770 3.0 1.3 4.6 2.0 P62888_C92 RPL30 60S ribosomal protein L30 100 1.0 2.7 1.5 1.3 Q9NQR4_C153 NIT2 Omega-amidase NIT2 47 13.1 0.7 20.0 20.0 P42765_C92 ACAA2 3-ketoacyl-CoA thiolase, 71 20.0 3.7 20.0 4.3 mitochondrial Q15084_C55 PDIA6 Protein disulfide-isomerase A6 51 3.0 4.9 4.4 2.1 Q96HE7_C241 ERO1L ERO1-like protein alpha 61 2.4 3.0 3.7 -- Q99439_C164 CNN2 Calponin-2 41 -- 1.5 1.1 4.7 P25205_C119 MCM3 DNA replication licensing factor 74 3.5 4.3 3.5 1.9 MCM3 Q9NS86_C187 LANCL2 LanC-like protein 2 203 -- 1.6 6.1 2.5 Q15233_C145 NONO Non-POU domain-containing 72 -- 1.6 12.0 1.4 octamer-binding protein Q9BRA2_C43 TXNDC17 Thioredoxin domain-containing 62 15.5 3.5 20.0 20.0 protein 17 P35611_C68 ADD1 Alpha-adducin 771 5.8 4.3 2.4 4.3 O75521_C380 ECI2 Enoyl-CoA delta isomerase 2, 155 0.6 1.6 0.8 0.7 mitochondrial Q9BXW7_C392 CECR5 Cat eye syndrome critical region 772 3.6 2.2 3.2 1.5 protein 5 P30101_C406 PDIA3 Protein disulfide-isomerase A3 773 3.2 9.0 20.0 1.4 Q96AB3_C114 ISOC2 Isochorismatase domain-containing 159 1.6 0.8 4.7 -- protein 2, mitochondria P13667_C555 PDIA4 Protein disulfide-isomerase A4 774 3.2 9.0 20.0 1.4 Q09161_C44 NCBP1 Nuclear cap-binding protein 102 2.1 1.8 2.0 -- subunit 1 P78417_C32 GSTO1 Glutathione S-transferase omega-1 32 -- 19.9 20.0 -- Q9ULW0_C536 TPX2 Targeting protein for Xklp2 437 20.0 20.0 20.0 5.7 Q9NRG0_C55 CHRAC1 Chromatin accessibility complex 252 20.0 20.0 7.4 2.9 protein 1 Q96T76_C848 MMS19 nucleotide excision repair protein 114 3.9 20.0 1.7 20.0 homolog Q8TAQ2_C145 SMARCC2 SWI/SNF complex subunit 775 4.6 3.1 -- -- SMARCC2 Q9BVC5_C10 C2orf49 Ashwin 168 2.7 3.3 4.5 -- Q7Z2W4_C645 ZC3HAV1 Zinc finger CCCH-type antiviral 776 -- 4.4 5.1 3.0 protein 1 Q9BQ69_C186 MACROD1 O-acetyl-ADP-ribose 777 4.8 1.8 5.0 1.2 deacetylase MACROD1 Q16831_C162 UPP1 Uridine phosphorylase 1 364 1.2 1.0 20.0 7.7 P30101_C57 PDIA3 Protein disulfide-isomerase A3 133 2.2 20.0 20.0 -- P12268_C331 IMPDH2 Inosine-5-monophosphate 144 12.1 4.6 -- 1.3 dehydrogenase 2 O95571_C170 ETHE1 Protein ETHE1, mitochondrial 176 3.8 2.3 6.8 2.0 O00299_C24 CLIC1 Chloride intracellular channel 69 9.5 20.0 10.3 3.0 protein 1 O14879_C343 IFIT3 Interferon-induced protein with 308 7.6 6.6 2.1 17.2 tetratricopeptide Q96CM8_C64 ACSF2 Acyl-CoA synthetase family member 194 20.0 20.0 20.0 -- 2, mitochondrial P51946_C244 CCNH Cyclin-H 778 1.9 5.3 -- -- P49588_C773 AARS Alanine--tRNA ligase, cytoplasmic 122 -- 1.3 1.5 1.5 Q96RN5_C618 MED15 Mediator of RNA polymerase II 171 6.4 4.5 20.0 -- transcription subuni O15294_C758 OGT UDP-N-acetylglucosamine--peptide N- 104 2.6 6.4 2.4 2.1 acetylglucosami P46734_C207 MAP2K3 Dual specificity mitogen-activated 184 0.8 0.6 0.8 3.3 protein kinase Q96S55_C272 WRNIP1 ATPase WRNIP1 215 2.2 3.7 1.2 4.5 O95229_C54 ZWINT ZW10 interactor 779 3.4 12.4 3.1 3.7 O60610_C1227 DIAPH1 Protein diaphanous homolog 1 780 2.2 1.6 -- 1.5 Q13428_C38 TCOF1 Treacle protein 150 3.3 2.8 2.0 -- Q9Y277_C65 VDAC3 Voltage-dependent anion-selective 512 3.3 4.7 6.2 -- channel protein P57764_C268 GSDMD Gasdermin-D 110 20.0 20.0 8.4 -- Q9Y3A3_C134 MOB4 MOB-like protein phocein 121 -- 1.2 1.6 20.0 Q02252_C317 ALDH6A1 Methylmalonate-semialdehyde 265 10.3 14.4 -- -- dehydrogenase Q9NYL9_C132 TMOD3 Tropomodulin-3 130 2.2 0.8 0.9 7.6 P83731_C6 RPL24 60S ribosomal protein L24 147 2.4 0.7 -- 4.3 O95336_C32 PGLS 6-phosphogluconolactonase 55 14.9 -- 20.0 6.7 Q13155_C291 AIMP2 Aminoacyl tRNA synthase complex- 83 20.0 20.0 2.4 1.9 interacting multif Q13418_C346 ILK Integrin-linked protein kinase 149 1.4 0.8 -- 2.7 A6NDU8_C179 C5orf51 UPF0600 protein C5orf51 298 4.2 3.0 -- 20.0 Q9UKF6_C498 CPSF3 Cleavage and polyadenylation 118 6.2 3.5 20.0 -- specificity factor su Q96F86_C413 EDC3 Enhancer of mRNA-decapping protein 141 20.0 20.0 -- -- 3 P42224_C492 STAT1 Signal transducer and activator of 228 16.9 -- -- 3.1 transcription 1 P11216_C326 PYGB Glycogen phosphorylase, brain form 366 -- 1.7 20.0 2.6 P21980_C277 TGM2 Protein-glutamine gamma- 356 0.7 0.5 0.7 2.3 glutamyltransferase 2 Q9HAV7_C124 GRPEL1 GrpE protein homolog 1, 206 3.2 1.7 2.2 4.7 mitochondrial P24752_C126 ACAT1 Acetyl-CoA acetyltransferase, 89 9.1 2.8 4.5 14.1 mitochondrial Q9NQ88_C161 TIGAR Fructose-2,6-bisphosphatase TIGAR 117 6.0 -- -- 2.6 Q13155_C23 AIMP2 Aminoacyl tRNA synthase complex- 132 -- 2.8 2.7 interacting multifunctional protein 2 Q9NQW6_C712 ANLN Actin-binding protein anillin 281 -- 4.1 14.1 3.1 P51649_C340 ALDH5A1 Succinate-semialdehyde 189 1.9 1.0 2.7 -- dehydrogenase, mitochondria Q15021_C439 NCAPD2 Condensin complex subunit 1 139 -- 2.2 0.6 6.1 Q5T0N5_C69 FNBP1L Formin-binding protein 1-like 418 20.0 20.0 3.6 -- P38606_C138 ATP6V1A V-type proton ATPase catalytic 201 -- 20.0 20.0 20.0 subunit A Q9HCC0_C216 MCCC2 Methylcrotonoyl-CoA carboxylase 363 4.6 5.9 3.9 -- beta chain, mitoch Q9NQC3_C1101 RTN4 Reticulon-4 247 12.0 20.0 -- 20.0 P35754_C23 GLRX Glutaredoxin-1 142 -- 20.0 -- -- Q99757_C90 TXN2 Thioredoxin, mitochondrial 208 10.1 3.6 4.5 20.0 Q9Y3D0_C93 FAM96B Mitotic spindle-associated MMXD 179 20.0 20.0 -- -- complex subunit MIP18 Q9UMS0_C213 NFU1 iron-sulfur cluster scaffold homolog, 394 7.4 4.4 -- -- mitochondrial Q9NXV6_C516 CDKN2AIP CDKN2A-interacting protein 255 20.0 -- -- 6.6 Q96RS6_C376 NUDCD1 NudC domain-containing 79 -- 1.6 -- 2.2 protein 1 Q14997_C1840 PSME4 Proteasome activator complex subunit 257 2.8 20.0 20.0 -- 4 P50570_C27 DNM2 Dynamin-2 73 4.6 2.2 -- -- Q86YH6_C71 PDSS2 Decapreiryl-diphosphate synthase 401 20.0 20.0 -- -- subunit 2 Q99497_C106 PARK7 Protein DJ-1 109 -- 0.9 -- -- Q9UJW0_C258 DCTN4 Dynactin subunit 4 103 4.0 -- 20.0 -- Q9BUH6_C180 C9orf142 Uncharacterized protein C9orf142 348 -- 1.2 6.6 2.8 P24752_C196 ACAT1 Acetyl-CoA acetyltransferase, 33 20.0 3.5 -- 5.8 mitochondrial Q13162_C51 PRDX4 Peroxiredoxin-4 781 4.6 1.2 1.4 3.3 Q9BTA9_C553 WAC WW domain-containing adapter protein 153 17.6 17.8 9.6 -- with coiled-coil P48643_C253 CCT5 T-complex protein 1 subunit epsilon 126 -- 0.8 0.8 -- O75362_C286 ZNF217 Zinc finger protein 217 268 8.9 -- -- -- O60825_C158 PFKFB2 6-phosphofructo-2-kinase/fructose- 272 0.7 -- -- -- 2,6-bisphosphata Q8NBS9_C350 TXNDC5 Thioredoxin domain-containing 136 -- 2.4 -- 1.7 protein 5 Q9NYL2_C22 MLTK Mitogen-activated protein kinase 430 5.5 20.0 1.4 10.3 MLTK P27707_C9 DCK Deoxy cytidine kinase 93 -- 1.4 -- 4.9 Q93009_C223 USP7 Ubiquitin carboxyl-terminal hydrolase 782 -- 6.8 20.0 -- 7 O14929_C101 HAT1 Histone acetyltransferase type B 154 -- -- -- 9.0 catalytic subunit Q9UPQ0_C140 LIMCH1 LIM and calponin homology 783 20.0 -- 20.0 -- domains-containing protein Q96NY7_C487 CLIC6 Chloride intracellular channel 447 -- 20.0 5.1 3.6 protein 6 Q9NQ88_C114 TIGAR Fructose-2,6-bisphosphatase TIGAR 143 2.8 -- -- 1.9 Q14790_C360 CASP8 Caspase-8 335 20.0 20.0 -- -- P04183_C230 TK1 Thymidine kinase, cytosolic 784 1.9 0.8 -- 10.0 P68366_C54 TUBA4A Tubulin alpha-4A chain 137 9.7 0.8 0.9 -- Q13428_C1298 TCOF1 Treacle protein 785 4.7 5.6 20.0 -- Q5MNZ6_C63 WDR45L WD repeat domain 434 -- 2.0 20.0 -- phosphoinositide-interacting protein O14980_C528 XPO1 Exportin-1 786 20.0 1.9 -- -- Q86W42_C35 THOC6 THO complex subunit 6 homolog 217 -- -- -- 20.0 Q9Y6G9_C51 DYNC1LI1 Cytoplasmic dynein 1 light 107 -- 3.5 4.1 -- intermediate chain 1 Q9NY27_C22 PPP4R2 Serine/threonine-protein phosphatase 282 20.0 20.0 -- 4 regulatory Q8NFH5_C255 NUP35 Nucleoporin NUP53 386 5.1 3.9 -- -- Q9Y676_C128 MRPS18B 28S ribosomal protein S18b, 495 10.2 2.3 1.0 -- mitochondrial P35658_C728 NUP214 Nuclear pore complex protein 311 3.9 1.7 -- -- Nup214 Q9NTX5_C133 ECHDC1 Ethylmalonyl-CoA decarboxylase 532 1.5 1.7 2.0 -- Q15118_C71 PDK1 623 20.0 20.0 20.0 -- Q00765_C18 REEP5 Receptor expression-enhancing 344 -- 20.0 2.7 -- protein 5 P22307_C71 SCP2 Non-specific lipid-transfer protein 787 3.2 2.7 20.0 -- O75521_C312 ECI2 Enoyl-CoA delta isomerase 2, 788 0.7 1.0 0.5 0.5 mitochondrial P49189_C288 ALDH9A1 4-trimethylaminobutyraldehyde 236 -- -- -- 20.0 dehydrogenase Q5T440_C170 IBA57 Putative transferase CAF17, 608 14.0 1.5 -- -- mitochondrial Q15084_C190 PDIA6 Protein disulfide-isomerase A6 96 -- 20.0 5.2 -- Q96C19_C172 EFHD2 EF-hand domain-containing protein 246 -- 5.3 -- 2.8 D2 P22061_C102 PCMT1 Protein-L-isoaspartate(D-aspartate) 163 -- 2.8 4.5 1.2 O-methyltransferase Q9NP73_C86 ALG13 UDP-N-acetylglucosamine 528 20.0 20.0 -- -- transferase subunit ALG13 Q9BRF8_C54 CPPED1 Calcineurin-like phosphoesterase 239 -- 20.0 -- 2.4 domain-containing Q6ICB0_C108 DESI1 Desumoylating isopeptidase 1 197 20.0 -- 20.0 -- P29590_C389 PML Protein PML 304 15.6 -- -- -- P07858_C211 CTSB Cathepsin B 323 -- 2.3 -- 5.0 Q9NX18_C83 SDHAF2 Succinate dehydrogenase assembly 658 3.1 3.0 -- -- factor 2, mitochondrial P46109_C249 CRKL Crk-like protein VPCAYDK 99 20.0 -- -- 6.7 K.RVPC*AYDK.T P45984_C177 MAPK9 Mitogen-activated protein kinase 9 302 -- 12.0 20.0 -- P19447_C342 ERCC3 TFIIH basal transcription factor 402 -- -- 12.4 -- complex helicase P42166_C341 TMPO Lamina-associated polypeptide 2, 175 10.4 -- 4.4 -- isoform alpha Q8N1F7_C522 NUP93 Nuclear pore complex protein Nup93 275 -- -- 20.0 -- Q86UY8_C276 NT5DC3 5-nucleotidase domain-containing 789 3.8 -- -- -- protein 3 Q8WWI1_C228 LMO7 LIM domain only protein 7 504 -- -- -- -- Q9NWA0_C139 MED9 Mediator of RNA polymerase II 654 4.3 3.4 -- -- transcription subunit P09110_C381 ACAA1 3-ketoacyl-CoA thiolase, 205 -- -- 0.8 -- peroxisomal Q2NL82_C126 TSR1 Pre-rRNA-processing protein TSR1 233 20.0 13.8 -- -- homolog Q5JPI3_C308 C3orf38 Uncharacterized protein C3orf38 544 2.9 -- -- -- P23919_C163 DTYMK Thymidylate kinase 173 -- 0.4 0.6 -- Q96EB1_C218 ELP4 Elongator complex protein 4 466 -- 20.0 -- 8.4 Q96FX7_C209 TRMT61A tRNA (adenine(58)-N(1))- 313 20.0 2.4 -- -- methyltransferase catalytic
O14933_C98 UBE2L6 Ubiquitin/ISG15-conjugating 331 -- 3.0 -- -- enzyme E2 L6 Q29RF7_C242 PDS5A Sister chromatid cohesion protein 232 -- 3.0 1.0 -- PDS5 homolog A Q96T76_C819 MMS19 MMS19 nucleotide excision repair 181 -- -- -- -- protein homolog P23919_C117 DTYMK Thymidylate kinase 267 -- 0.4 7.3 Q15149_C4574 PLEC Plectin 336 -- 4.2 -- 0.8 Q96RP9_C153 GFM1 Elongation factor G, mitochondrial 524 -- 1.0 -- -- P04818_C199 TYMS Thymidylate synthase 406 20.0 20.0 -- -- P27708_C73 CAD CAD protein 191 3.1 -- 20.0 -- P55265_C1224 ADAR Double-stranded RNA-specific 359 9.1 -- -- -- adenosine deaminase Q9Y3D2_C105 MSRB2 Methionine-R-sulfoxide reductase 403 20.0 20.0 -- B2, mitochondrial O00244_C12 ATOX1 Copper transport protein ATOX1 365 -- -- -- 4.7 Q8WV74_C207 NUDT8 Nucleoside diphosphate-linked 468 20.0 20.0 20.0 -- moiety X motif 8, mitochondrial Q9NRW3_C130 APOBEC3C Probable DNA dC- dU-editing 358 20.0 20.0 -- -- enzyme APOBEC-3C P24468_C326 NR2F2 COUP transcription factor 2 790 20.0 20.0 20.0 -- P42166_C684 TMPO Lamina-associated polypeptide 2, 312 -- -- 4.6 -- isoform alpha Q96EY5_C231 FAM125A Multivesicular body subunit 12A 540 4.1 1.7 -- 1.9 P14635_C238 CCNB1 G2/mitotic-specific cyclin-B1 448 -- -- -- 3.7 Q8NDH3_C81 NPEPL1 Probable aminopeptidase NPEPL1 791 20.0 20.0 20.0 -- Q9P0J1_C149 PDP1 276 20.0 -- -- -- Q96P48_C900 ARAP1 Arf-GAP with Rho-GAP domain, 433 5.5 -- -- -- ANK repeat and PH domain Q96HE7_C37 ERO1L ERO1-like protein alpha 347 -- 5.4 -- 20.0 Q07065_C100 CKAP4 Cytoskeleton-associated protein 4 733 -- -- -- 15.4 Q9BRJ7_C88 NUDT16L1 Protein syndesmos 432 20.0 20.0 -- -- O75439_C265 PMPCB Mitochondrial-processing peptidase 320 -- -- -- -- subunit beta O43175_C369 PHGDH D-3-phosphoglycerate 248 20.0 -- -- -- dehydrogenase Q9UNI6_C265 DUSP12 Dual specificity protein 241 -- 0.8 -- -- phosphatase 12 Q06203_C100 PPAT Amidophosphoribosyltransferase 188 -- 1.7 -- -- A0AVT1_C347 UBA6 Ubiquitin-like modifier-activating 158 -- 20.0 -- 3.3 enzyme 6 Q86X76_C203 NIT1 Nitrilase homolog 1 471 -- 0.7 -- -- Q6XZF7_C691 DNMBP Dynamin-binding protein 353 -- 1.2 -- 3.4 Q15398_C129 DLGAP5 Disks large-associated protein 5 167 20.0 -- -- -- O75717_C773 WDHD1 WD repeat and HMG-box DNA- 289 -- -- 4.2 -- binding protein 1 Q01433_C107 AMPD2 AMP deaminase 2 259 4.4 2.3 6.2 3.1 Q8WVV9_C464 HNRPLL Heterogeneous nuclear 487 -- -- -- -- ribonucleoprotein L-like O14733_C131 MAP2K7 Dual specificity mitogen-activated 427 -- -- -- -- protein kinase Q14137_C404 BOP1 Ribosome biogenesis protein BOP1 535 20.0 1.2 -- -- Q96RU2_C171 USP28 Ubiquitin carboxyl-terminal 569 -- 20.0 1.2 -- hydrolase 28 Q9Y679_C391 AUP1 Ancient ubiquitous protein 1 564 -- 20.0 20.0 -- P51610_C1872 HCFC1 Host cell factor 1 270 4.1 -- -- -- P22307_C307 SCP2 Non-specific lipid-transfer protein 541 20.0 20.0 20.0 -- Q9BTE3_C325 MCMBP Mini-chromosome maintenance 792 -- -- -- -- complex-binding protein Q9HA64_C24 FN3KRP Ketosamine-3-kinase 106 6.3 20.0 -- 20.0 Q5TFE4_C119 NT5DC1 5-nucleotidase domain-containing 75 -- -- -- 20.0 protein 1 Q96N67_C2125 DOCK7 Dedicator of cytokinesis protein 7 408 -- 4.1 5.4 -- P52948_C1312 NUP98 Nuclear pore complex protein Nup98- 575 -- -- -- -- Nup96 Q5UIP0_C2298 RIF1 Telomere-associated protein RIF1 607 -- 20.0 -- -- P51812_C436 RPS6KA3 Ribosomal protein S6 kinase 662 -- -- -- 2.1 alpha-3 Q92616_C1692 GCN1L1 Translational activator GCN1 174 1.4 -- -- -- Q15345_C297 LRRC41 Leucine-rich repeat-containing 536 -- -- -- -- protein 41 Q9NPH0_C267 ACP6 Lysophosphatidic acid phosphatase 329 -- 20.0 20.0 -- type 6 P04183_C66 TK1 Thymidine kinase, cytosolic 138 -- -- 2.3 7.0 P42166_C629 TMPO Lamina-associated polypeptide 2, 793 4.4 -- 1.7 -- isoform alpha Q15013_C124 MAD2L1BP MAD2L1-binding protein 794 -- 13.0 5.4 -- Q9Y5Y2_C72 NUBP2 Cytosolic Fe--S cluster assembly 795 5.3 -- -- 4.0 factor NUBP2 O15446_C86 CD3EAP DNA-directed RNA polymerase I 796 3.9 -- -- -- subunit RPA34 Q13630_C116 TSTA3 GDP-L-fucose synthase 797 20.0 -- -- 1.9 Q8IYQ7_C324 THNSL1 Threonine synthase-like 1 628 -- -- 20.0 1.6 P05091_C319 ALDH2 Aldehyde dehydrogenase, 318 -- 20.0 -- -- mitochondrial Q29RF7_C532 PDS5A Sister chromatid cohesion protein 261 -- 20.0 -- -- PDS5 homolog A Q9Y570_C381 PPME1 Protein phosphatase methylesterase 1 578 12.6 1.1 -- 20.0 Q14980_C961 NUMA1 Nuclear mitotic apparatus protein 1 429 -- -- -- -- P53384_C235 NUBP1 Cytosolic Fe--S cluster assembly 552 -- 0.6 0.6 6.8 factor NUBP1 Q15003_C418 NCAPH Condensin complex subunit 2 258 20.0 2.0.0 -- -- P53634_C258 CTSC Dipeptidyl peptidase 1 798 0.9 1.2 -- -- Q8NFF5_C499 FLAD1 FAD synthase 750 6.2 -- -- -- Q9ULA0_C144 DNPEP Aspartyl aminopeptidase 134 -- -- -- 2.1 P22307_C94 SCP2 Non-specific lipid-transfer protein 559 -- 20.0 -- -- O15294_C620 OGT UDP-N-acetylglucosamine-peptide N- 279 -- 20.0 -- -- acetylglucosamine Q9Y5S2_C1517 CDC42BPB Serine/threonine-protein kinase 554 20.0 20.0 -- -- MRCK beta Q8TD19_C623 NEK9 Serine/threonine-protein kinase Nek9 393 -- -- -- -- Q8N2W9_C326 PIAS4 E3 SUMO-protein ligase PIAS4 625 -- -- -- -- Q13158_C98 FADD Protein FADD 799 20.0 -- -- -- Q9UKX7_C151 NUP50 Nuclear pore complex protein Nup50 234 6.1 -- -- -- Q6PCB5_C280 RSBN1L Round spermatid basic protein 1- 519 -- -- -- -- like protein P10398_C597 ARAF Serine/threonine-protein kinase A-Raf 505 -- -- -- 5.0 Q9UL40_C68 ZNF346 Zinc finger protein 346 706 -- -- -- -- P46013_C903 MKI67 Antigen KI-67 595 20.0 -- -- -- Q16667_C39 CDKN3 Cyclin-dependent kinase inhibitor 3 482 -- 0.6 -- -- O75150_C890 RNF40 E3 ubiquitin-protein ligase BRE1B 405 -- -- -- -- Q00610_C870 CLTC Clathrin heavy chain 1 113 -- 2.1 -- 2.8 Q9Y5T5_C205 USP16 Ubiquitin carboxyl-terminal hydrolase 474 7.9 20.0 -- -- 16 O95881_C66 TXNDC12 Thioredoxin domain-containing 342 -- -- -- -- protein 12 Q7Z5K2_C160 WAPAL Wings apart-like protein homolog 800 20.0 -- -- -- P42166_C518 TMPO Lamina-associated polypeptide 2, 801 -- -- -- 3.4 isoform alpha Q9Y2S7_C143 POLDIP2 Polymerase delta-interacting 574 -- -- -- 2.3 protein 2 E2QRD5_C183 C15orf38-AP3S2 Protein C15orf38-AP3S2 581 -- -- -- 4.9 O95833_C22 CLIC3 Chloride intracellular channel 531 -- -- -- 20.0 protein 3 O94953_C694 KDM4B Lysine-specific demethylase 4B 395 20.0 -- -- -- O00541_C272 PES1 Pescadillo homolog 511 -- -- -- 5.0 Q9NXJ5_C149 PGPEP1 Pyroglutamyl-peptidase 1 587 -- 20.0 -- -- Q8N5L8_C131 RPP25L Ribonuclease P protein subunit 670 -- -- -- -- p25-like protein Q8IZ73_C246 RPUSD2 RNA pseudouridylate synthase 441 -- -- -- -- domain-containing protein Q99798_C385 ACO2 Aconitate hydratase, mitochondrial 685 20.0 1.0 -- -- Q9GZR2_C382 REXO4 RNA exonuclease 4 621 -- -- -- -- Q13613_C117 MTMR1 Myotubularin-related protein 1 717 -- -- -- -- Q9NUI1_C22 DECR2 Peroxisomal 2,4-dienoyl-CoA 698 -- -- -- -- reductase Q02556_C306 IRF8 Interferon regulatory factor 8 513 -- -- -- -- Q9UPT9_C171 USP22 Ubiquitin carboxyl-terminal 802 -- -- -- -- hydrolase 22 Q8N999_C302 C12orf29 Uncharacterized protein C12orf29 484 -- -- -- -- Q8IU81_C363 IRF2BP1 Interferon regulatory factor 2- 803 -- -- -- -- binding protein 1 Q9C0I1_C152 MTMR12 Myotubularin-related protein 12 671 -- -- -- -- Q9P2X3_C195 IMPACT Protein IMPACT 678 -- 20.0 -- -- Q6QNY0_C168 BLOC1S3 Biogenesis of lysosome-related 411 -- -- -- -- organelles complex Q15796_C81 SMAD2 Mothers against decapentaplegic 561 20.0 -- -- -- homolog 2 Q9NZB2_C531 FAM120A Constitutive coactivator of PPAR- 492 -- -- -- -- gamma-like protein Q9HB90_C377 RRAGC Ras-related GTP-binding protein C 417 3.3 -- -- 4.3 Q9BR61_C267 ACBD6 Acyl-CoA-binding domain- 472 -- -- -- -- containing protein 6 P16455_C145 MGMT Methylated-DNA-protein-cysteine 470 -- -- -- -- methyltransferase Q86UV5_C39 USP48 Ubiquitin carboxyl-terminal hydrolase 381 20.0 -- -- -- 48 A2A288_C367 ZC3H12D Probable ribonuclease ZC3H12D 515 -- -- -- -- Q8NEC7_C140 GSTCD Glutathione S-transferase C-terminal 602 -- -- -- -- domain-containing protein Q6PJG6_C673 BRAT1 BRCA1-associated ATM activator 1 695 -- -- -- -- Q13232_C158 NME3 Nucleoside diphosphate kinase 3 653 -- -- -- 2.7 Q86X76_C165 NIT1 Nitrilase homolog 1 345 -- 0.9 -- -- P42695_C541 NCAPD3 Condensin-2 complex subunit D3 573 -- -- -- -- P41226_C599 UBA7 Ubiquitin-like modifier-activating 702 -- -- -- -- enzyme 7 Q99986_C50 VRK1 Serine/threonine-protein kinase VRK1 497 -- -- -- -- Q8WUM4_C90 PDCD6IP Programmed cell death 6- 527 -- -- -- -- interacting protein P29590_C213 PML Protein PML 477 -- -- -- -- Q9P0K7_C973 RAI14 Ankycorbin 638 -- 8.7 -- -- P53992_C78 SEC24C Protein transport protein Sec24C 498 -- -- 5.4 -- Q13867_C73 BLMH Bleomycin hydrolase 431 -- -- -- 3.9 Q8ND24_C655 RNF214 RING finger protein 214 451 6.5 -- -- 4.0 Q96EK_C48 THAP11 THAP domain-containing protein 11 538 7.9 -- -- -- Q96IV0_C309 NGLY1 Peptide-N(4)-(N-acetyl-beta- 660 -- -- -- -- glucosaminyl)asparagin Q5T1V6_C414 DDX59 Probable ATP-dependent RNA 439 -- -- -- -- helicase DDX59 Q9UHQ1_C99 NARF Nuclear prelamin A recognition factor 740 -- -- -- -- O43396_C34 TXNL1 Thioredoxin-like protein 1 310 -- -- -- -- Q8IV53_C174 DENND1C DENN domain-containing protein 804 -- -- -- -- 1C Q8N9T8_C673 KRI1 Protein KI1 homolog 563 -- -- 5.3 --
TABLE-US-00012 TABLE 2B 9_200 .mu.M.sub.-- 10_200 .mu.M.sub.-- 10_200 .mu.M.sub.-- 11_100 .mu.M.sub.-- 12_200 .mu.M.sub.-- Identifier insitu_ramos insitu_231 insitu_ramos insitu_231 insitu_231 P04406_C152 1.1 1.0 0.9 0.8 0.9 P61978_C132 1.4 5.6 1.6 0.9 7.5 Q13526_C113 1.3 2.0 1.3 0.7 0.6 P24752_C119 4.3 4.5 1.5 1.2 4.1 P24752_C413 20.0 7.8 2.2 1.2 9.9 Q9NUY8_C283 1.5 2.0 -- 1.1 2.9 P13667_C206 1.4 3.8 -- 4.1 16.6 P12268_C140 1.1 0.7 0.9 0.6 1.2 Q15365_C194 1.5 1.6 2.0 0.4 1.3 Q9NVC6_C649 1.3 2.3 2.1 0.8 4.0 P42166_C561 2.1 17.8 -- 0.6 16.1 Q9Y696_C35 1.6 2.7 1.6 1.9 20.0 P10599_C32 2.1 3.8 -- 13.0 20.0 P31943_C267 1.5 4.8 2.5 0.9 5.0 Q86SX6_C67 14.0 1.3 1.1 1.4 1.6 P15121_C299 1.5 20.0 -- 0.7 0.7 P52597_C267 1.6 2.5 2.4 1.0 6.8 Q9ULV4_C420 1.4 3.4 -- 0.9 2.2 P62888_C92 2.4 1.3 4.5 0.8 1.1 Q9NQR4_C153 20.0 20.0 -- 3.7 0.9 P42765_C92 1.2 3.7 1.1 -- 20.0 Q15084_C55 1.4 3.1 -- 4.3 15.4 Q96HE7_C241 1.8 2.0 -- 1.8 16.4 Q99439_C164 1.3 1.2 1.3 0.6 0.9 P25205_C119 1.8 2.3 -- 0.7 1.0 Q9NS86_C187 1.7 6.3 -- 0.8 2.0 Q15233_C145 1.4 -- 2.2 1.2 2.4 Q9BRA2_C43 5.4 20.0 -- 17.7 20.0 P35611_C68 2.3 1.9 -- 0.9 3.6 O75521_C380 1.1 4.8 -- 0.8 1.9 Q9BXW7_C392 -- 20.0 -- 1.1 5.1 P30101_C406 1.6 3.3 1.4 -- 20.0 Q96AB3_C114 3.7 20.0 -- 0.9 1.6 P13667_C555 1.6 3.3 1.4 -- 20.0 Q09161_C44 1.4 12.4 -- 1.1 4.7 P78417_C32 20.0 20.0 20.0 -- 20.0 Q9ULW0_C536 1.6 17.6 -- 1.4 20.0 Q9NRG0_C55 2.5 2.7 -- 1.1 20.0 Q96T76_C848 1.8 2.5 -- -- 20.0 Q8TAQ2_C145 2.4 -- 20.0 7.6 -- Q9BVC5_C10 1.4 3.1 -- 1.1 2.9 Q7Z2W4_C645 1.3 3.8 -- 1.0 3.3 Q9BQ69_C186 -- 2.4 -- 1.0 2.7 Q16831_C162 -- 7.3 -- 0.8 0.9 P30101_C57 1.5 3.3 -- 2.2 20.0 P12268_C331 2.6 -- 2.1 1.0 -- O95571_C170 1.8 9.0 -- 1.2 6.3 O00299_C24 1.4 5.0 -- 2.5 20.0 O14879_C343 -- 4.9 -- 0.4 3.5 Q96CM8_C64 20.0 20.0 -- 1.5 17.9 P51946_C244 2.0 1.7 -- 1.3 1.3 P49588_C773 2.0 2.1 1.7 0.9 0.9 Q96RN5_C618 -- -- -- 1.0 20.0 O15294_C758 -- 2.3 -- 1.0 20.0 P46734_C207 1.8 0.8 -- 0.6 0.7 Q96S55_C272 2.5 1.2 -- -- 2.4 O95229_C54 -- 2.3 -- 1.2 20.0 O60610_C1227 -- 1.5 1.4 0.9 0.8 Q13428_C38 1.7 4.2 -- 1.1 4.7 Q9Y277_C65 0.8 3.3 2.5 1.4 3.9 P57764_C268 4.9 -- 2.2 0.7 -- Q9Y3A3_C134 20.0 1.9 -- 1.8 1.4 Q02252_C317 2.6 1.5 0.7 0.9 3.2 Q9NYL9_C132 -- -- -- 0.5 0.6 P83731_C6 1.3 0.4 2.0 0.5 0.3 O95336_C32 2.6 -- -- 0.9 20.0 Q13155_C291 -- 1.7 -- 0.9 1.1 Q13418_C346 -- 1.1 8.3 0.6 0.6 A6NDU8_C179 3.7 1.3 -- 0.7 -- Q9UKF6_C498 1.7 20.0 2.8 1.3 4.1 Q96F86_C413 4.0 20.0 20.0 20.0 -- P42224_C492 -- 20.0 -- 0.7 1.0 P11216_C326 -- 8.6 -- 0.8 3.9 P21980_C277 -- 0.6 -- 0.6 0.3 Q9HAV7_C124 3.8 1.0 -- 0.9 1.1 P24752_C126 20.0 5.9 -- 1.2 5.8 Q9NQ88_C161 2.0 4.6 2.1 0.9 -- Q13155_C23 1.6 2.3 -- 1.2 1.6 Q9NQW6_C712 -- 11.2 -- 0.9 16.7 P51649_C340 1.5 13.4 20.0 -- 20.0 Q15021_C439 -- 5.2 -- 0.9 6.7 Q5T0N5_C69 -- 2.0 -- 1.3 20.0 P38606_C138 -- 20.0 -- 9.2 20.0 Q9HCC0_C216 3.3 2.3 -- 1.9 20.0 Q9NQC3_C1101 20.0 20.0 -- 1.1 20.0 P35754_C23 5.7 -- 13.0 20.0 20.0 Q99757_C90 -- -- -- 2.7 5.0 Q9Y3D0_C93 2.1 2.1 -- 0.9 -- Q9UMS0_C213 -- 20.0 -- -- 20.0 Q9NXV6_C516 13.8 7.7 -- 1.0 3.3 Q96RS6_C376 1.7 5.7 -- 0.8 1.1 Q14997_C1840 -- 20.0 -- -- 20.0 P50570_C27 1.4 3.1 -- 0.7 0.7 Q86YH6_C71 6.6 20.0 -- 1.0 20.0 Q99497_C106 2.6 1.7 -- 1.3 3.0 Q9UJW0_C258 20.0 20.0 20.0 20.0 20.0 Q9BUH6_C180 -- 3.8 -- -- 1.4 P24752_C196 6.8 5.1 2.3 1.1 4.6 Q13162_C51 -- 2.0 -- 0.9 1.0 Q9BTA9_C553 2.1 20.0 -- 1.7 -- P48643_C253 1.1 -- 0.9 0.6 5.1 O75362_C286 1.8 2.6 -- 1.0 20.0 O60825_C158 1.8 0.7 1.2 -- 0.8 Q8NBS9_C350 1.8 -- 1.4 3.4 9.4 Q9NYL2_C22 -- 1.2 -- 0.7 0.7 P27707_C9 1.5 1.9 1.7 0.5 1.4 Q93009_C223 20.0 2.5 -- 1.0 3.1 O14929_C101 -- 20.0 -- 0.8 20.0 Q9UPQ0_C140 -- 3.9 -- 1.0 1.9 Q96NY7_C487 -- 3.2 -- 3.1 20.0 Q9NQ88_C114 1.4 2.1 -- 0.8 -- Q14790_C360 3.2 -- 5.8 -- -- P04183_C230 2.0 1.3 -- -- -- P68366_C54 1.8 7.9 2.8 0.3 -- Q13428_C1298 2.5 5.9 -- 0.8 20.0 Q5MNZ6_C63 11.2 3.6 -- 0.9 20.0 O14980_C528 1.8 1.3 0.7 0.7 -- Q86W42_C35 2.7 -- -- 1.3 -- Q9Y6G9_C51 -- 4.9 -- -- 6.5 Q9NY27_C22 1.8 -- 2.1 2.3 20.0 Q8NFH5_C255 1.9 3.5 -- 1.3 12.4 Q9Y676_C128 1.3 2.9 -- 0.8 1.4 P35658_C728 4.2 20.0 -- 0.8 -- Q9NTX5_C133 -- 1.6 -- 1.0 1.2 Q15118_C71 -- 18.0 2.7 0.6 -- Q00765_C18 -- 4.2 -- 0.8 20.0 P22307_C71 -- 8.3 -- 2.0 3.7 O75521_C312 -- 5.0 -- -- 4.2 P49189_C288 20.0 20.0 -- -- 1.8 Q5T440_C170 2.4 5.7 -- -- -- Q15084_C190 1.6 3.7 -- -- 20.0 Q96C19_C172 -- 2.4 -- 0.9 0.9 P22061_C102 1.3 1.6 -- 0.8 5.8 Q9NP73_C86 -- -- -- 0.7 -- Q9BRF8_C54 1.9 3.8 -- 1.0 1.2 Q6ICB0_C108 -- -- -- 0.7 -- P29590_C389 -- 7.9 -- 0.5 -- P07858_C211 -- 7.4 -- 1.1 -- Q9NX18_C83 -- 4.4 -- 1.1 20.0 P46109_C249 4.0 20.0 -- -- 20.0 P45984_C177 3.8 20.0 -- 0.9 2.1 P19447_C342 2.7 20.0 -- 0.7 6.7 P42166_C341 1.5 -- -- 0.8 -- Q8N1F7_C522 3.7 -- -- -- 20.0 Q86UY8_C276 -- 5.8 -- 1.4 20.0 Q8WWI1_C228 1.4 17.2 -- 1.3 20.0 Q9NWA0_C139 -- 2.9 -- -- 20.0 P09110_C381 0.9 -- -- 0.8 4.5 Q2NL82_C126 -- 4.9 -- -- 20.0 Q5JPI3_C308 4.0 -- -- 1.0 -- P23919_C163 2.4 -- -- 0.2 -- Q96EB1_C218 3.7 1.1 -- -- -- Q96FX7_C209 16.1 -- -- -- 20.0 O14933_C98 2.8 20.0 3.2 -- -- Q29RF7_C242 -- -- -- 0.9 12.9 Q96T76_C819 4.0 -- 3.0 0.8 -- P23919_C117 1.4 0.4 -- -- 0.2 Q15149_C4574 -- 2.2 -- 1.1 -- Q96RP9_C153 2.8 14.8 -- -- 20.0 P04818_C199 11.7 1.5 -- -- -- P27708_C73 2.2 -- -- -- -- P55265_C1224 -- 10.7 -- -- -- Q9Y3D2_C105 -- 20.0 -- 1.3 20.0 O00244_C12 1.6 2.1 -- 0.7 -- Q8WV74_C207 20.0 -- 20.0 -- -- Q9NRW3_C130 6.2 -- -- -- -- P24468_C326 -- 20.0 -- 0.9 -- P42166_C684 -- 5.2 -- 0.7 -- Q96EY5_C231 2.1 -- -- 0.7 -- P14635_C238 2.1 6.7 -- 0.7 -- Q8NDH3_C81 -- 5.7 -- -- 20.0 Q9P0J1_C149 -- 18.0 -- 1.4 18.5 Q96P48_C900 -- 1.8 -- -- 1.9 Q96HE7_C37 -- -- -- -- -- Q07065_C100 -- -- -- 1.0 20.0 Q9BRJ7_C88 4.0 20.0 -- -- -- O75439_C265 1.7 -- 2.1 1.0 -- O43175_C369 2.0 -- 2.4 -- -- Q9UNI6_C265 -- 1.2 -- -- 1.2 Q06203_C100 2.9 -- 20.0 -- -- A0AVT1_C347 2.6 -- 20.0 1.6 -- Q86X76_C203 20.0 -- -- 0.8 3.9 Q6XZF7_C691 10.0 20.0 20.0 -- -- Q15398_C129 -- -- -- 0.6 -- O75717_C773 -- 2.2 -- 1.2 -- Q01433_C107 1.4 -- -- 0.6 -- Q8WVV9_C464 -- -- -- 0.9 2.0.0 O14733_C131 -- 1.8 -- -- 1.7 Q14137_C404 2.1 20.0 -- -- -- Q96RU2_C171 -- -- -- 2.2 20.0 Q9Y679_C391 -- -- -- 3.0 -- P51610_C1872 1.0 1.5 -- 0.5 -- P22307_C307 -- -- -- 2.9 20.0 Q9BTE3_C325 5.4 -- -- -- -- Q9HA64_C24 1.6 -- -- -- -- Q5TFE4_C119 20.0 -- 20.0 -- -- Q96N67_C2125 -- -- -- -- -- P52948_C1312 -- 20.0 -- -- 20.0 Q5UIP0_C2298 -- 20.0 -- 1.3 -- P51812_C436 1.8 -- -- 0.5 1.4 Q92616_C1692 -- -- -- -- -- Q15345_C297 -- -- -- 1.1 -- Q9NPH0_C267 2.6 13.4 -- -- -- P04183_C66 1.8 -- -- 0.6 -- P42166_C629 -- 2.5 -- 0.6 -- Q15013_C124 -- -- -- 1.0 -- Q9Y5Y2_C72 1.3 1.3 -- 0.5 -- O15446_C86 -- 4.7 -- -- 3.3 Q13630_C116 1.6 20.0 -- -- -- Q8IYQ7_C324 -- 5.3 -- -- -- P05091_C319 20.0 -- -- -- 20.0 Q29RF7_C532 -- 20.0 -- -- -- Q9Y570_C381 -- 4.7 -- -- -- Q14980_C961 -- -- -- -- 20.0 P53384_C235 -- -- -- 0.5 -- Q15003_C418 2.5 -- -- 1.6 -- P53634_C258 -- 5.5 -- -- 13.4 Q8NFF5_C499 12.1 -- -- 1.6 -- Q9ULA0_C144 1.3 5.2 -- -- -- P22307_C94 -- -- -- 3.3 20.0 O15294_C620 -- -- 12.0 -- -- Q9Y5S2_C1517 -- -- -- 1.2 -- Q8TD19_C623 20.0 -- -- -- -- Q8N2W9_C326 2.6 4.5 -- -- -- Q13158_C98 -- -- -- -- -- Q9UKX7_C151 1.8 -- -- 1.0 6.7 Q6PCB5_C280 2.5 -- -- -- 20.0 P10398_C597 1.3 1.4 -- -- -- Q9UL40_C68 20.0 -- -- -- 20.0 P46013_C903 -- -- -- -- -- Q16667_C39 4.5 2.3 -- -- -- O75150_C890 -- 4.2 -- 1.1 -- Q00610_C870 -- -- -- 0.9 1.7 Q9Y5T5_C205 -- -- -- -- -- O95881_C66 20.0 -- -- -- 20.0 Q7Z5K2_C160 -- 20.0 -- 1.0 -- P42166_C518 1.3 4.2 -- 0.8 -- Q9Y2S7_C143 -- 13.9 -- -- -- E2QRD5_C183 -- -- -- 0.9 -- O95833_C22 -- -- -- -- -- O94953_C694 4.2 -- 3.9 -- -- O00541_C272 -- -- -- 1.0 -- Q9NXJ5_C149 20.0 -- 11.0 -- 3.9 Q8N5L8_C131 -- -- -- -- --
Q8IZ73_C246 6.5 -- -- -- -- Q99798_C385 -- 0.7 -- -- -- Q9GZR2_C382 1.0 -- -- -- 20.0 Q13613_C117 4.3 -- -- -- -- Q9NUI1_C22 -- -- -- 2.1 -- Q02556_C306 5.5 -- -- -- -- Q9UPT9_C171 4.4 -- 9.3 0.8 -- Q8N999_C302 20.0 -- -- -- -- Q8IU81_C363 -- 5.9 -- -- -- Q9C0I1_C152 -- -- -- -- 20.0 Q9P2X3_C195 -- -- -- -- -- Q6QNY0_C168 4.6 -- -- -- -- Q15796_C81 5.6 -- -- -- -- Q9NZB2_C531 -- -- -- -- 20.0 Q9HB90_C377 20.0 -- -- -- -- Q9BR61_C267 -- -- -- -- -- P16455_C145 20.0 -- 20.0 -- -- Q86UV5_C39 -- -- -- -- -- A2A288_C367 5.2 -- -- -- -- Q8NEC7_C140 -- -- -- -- -- Q6PJG6_C673 -- -- -- -- -- Q13232_C158 -- -- -- -- -- Q86X76_C165 20.0 -- -- -- -- P42695_C541 -- 20.0 6.5 -- -- P41226_C599 20.0 8.0 -- -- -- Q99986_C50 -- -- 0.7 -- -- Q8WUM4_C90 5.0 -- -- -- -- P29590_C213 4.5 -- -- -- -- Q9P0K7_C973 -- -- -- 0.8 -- P53992_C78 -- -- -- -- -- Q13867_C73 -- 4.2 -- -- -- Q8ND24_C655 -- -- -- -- -- Q96EK4_C48 2.3 -- -- -- -- Q96IV0_C309 6.8 -- -- -- -- Q5T1V6_C414 3.7 11.3 -- -- -- Q9UHQ1_C99 20.0 -- -- -- -- O43396_C34 -- -- -- -- -- Q8IV53_C174 -- -- 5.0 -- -- Q8N9T8_C673 -- -- -- -- -- 13_200 .mu.M.sub.-- 13_200 .mu.M.sub.-- 14_200 .mu.M.sub.-- 14_200 .mu.M.sub.-- 21_200 .mu.M.sub.-- Identifier insitu_231 insitu_ramos insitu_231 insitu_ramos insitu_231 P04406_C152 0.9 0.9 0.9 0.6 1.3 P61978_C132 1.1 1.1 1.3 0.9 1.4 Q13526_C113 0.9 1.0 0.8 1.0 0.7 P24752_C119 2.1 2.8 1.0 -- 1.6 P24752_C413 3.5 20.0 0.9 0.8 1.5 Q9NUY8_C283 1.1 1.1 0.8 0.6 0.8 P13667_C206 1.1 -- 1.0 0.7 0.8 P12268_C140 0.7 -- 10.3 -- 0.6 Q15365_C194 0.9 1.4 1.0 -- 1.1 Q9NVC6_C649 1.1 1.0 1.0 1.4 0.8 P42166_C561 1.2 1.0 2.9 -- 1.4 Q9Y696_C35 2.3 3.2 0.7 -- 1.1 P10599_C32 7.9 4.1 3.1 -- 3.9 P31943_C267 1.1 1.2 1.3 -- 1.5 Q86SX6_C67 10.3 12.4 3.1 -- 1.4 P15121_C299 -- 0.8 0.9 -- 2.7 P52597_C267 1.2 1.3 1.5 -- 1.5 Q9ULV4_C420 1.1 1.1 1.4 -- 1.3 P62888_C92 0.8 2.0 1.0 -- 0.9 Q9NQR4_C153 6.2 12.2 0.8 -- 0.9 P42765_C92 -- 2.2 1.2 0.8 1.3 Q15084_C55 -- 1.5 1.0 -- 0.9 Q96HE7_C241 1.5 1.3 1.1 -- 1.6 Q99439_C164 0.9 1.3 0.9 -- 0.9 P25205_C119 1.1 1.3 1.2 -- 1.1 Q9NS86_C187 1.0 1.1 1.0 3.3 0.9 Q15233_C145 1.0 1.1 1.0 -- -- Q9BRA2_C43 20.0 5.2 1.4 -- 16.0 P35611_C68 1.1 1.0 1.6 -- 0.8 O75521_C380 1.0 -- 1.1 -- 1.8 Q9BXW7_C392 2.0 -- 1.6 1.1 1.6 P30101_C406 -- 2.0 1.1 -- -- Q96AB3_C114 2.4 -- 1.0 -- 4.9 P13667_C555 -- 2.0 1.1 -- -- Q09161_C44 1.0 -- 2.0 -- 1.3 P78417_C32 20.0 20.0 1.7 -- -- Q9ULW0_C536 1.5 -- 1.5 -- 0.9 Q9NRG0_C55 1.2 -- 1.7 -- -- Q96T76_C848 0.6 1.5 5.0 20.0 0.7 Q8TAQ2_C145 1.1 -- 1.3 -- 1.6 Q9BVC5_C10 1.6 -- 1.2 -- 1.5 Q7Z2W4_C645 1.3 0.9 1.4 -- 0.9 Q9BQ69_C186 1.4 -- 1.1 -- 1.4 Q16831_C162 1.0 -- 0.6 -- 1.6 P30101_C57 -- 1.6 1.0 -- 1.0 P12268_C331 -- 1.4 1.7 -- 0.8 O95571_C170 1.2 1.9 1.0 -- 1.7 O00299_C24 -- 1.8 0.8 -- 0.7 O14879_C343 1.0 -- 1.5 -- 0.8 Q96CM8_C64 1.4 -- 1.5 -- 2.0 P51946_C244 1.0 1.2 1.6 -- 1.4 P49588_C773 0.8 1.1 1.0 -- 1.1 Q96RN5_C618 1.0 1.4 1.6 -- 1.0 O15294_C758 1.0 -- 1.6 2.9 1.1 P46734_C207 0.8 -- 13.8 -- 0.9 Q96S55_C272 0.7 -- 1.4 -- 1.3 O95229_C54 0.8 1.4 5.0 20.0 0.8 O60610_C1227 0.7 0.9 -- -- 20.0 Q13428_C38 1.6 -- 3.6 -- 1.5 Q9Y277_C65 1.0 -- 1.7 -- 2.9 P57764_C268 -- 1.5 1.6 -- 0.7 Q9Y3A3_C134 -- 1.9 1.1 -- -- Q02252_C317 1.3 -- 1.1 -- 1.5 Q9NYL9_C132 -- -- 0.5 -- 1.4 P83731_C6 0.7 1.0 -- -- 1.0 O95336_C32 1.6 2.2 3.2 -- 0.9 Q13155_C291 -- -- 1.6 -- 0.8 Q13418_C346 0.7 2.1 0.8 -- 0.5 A6NDU8_C179 0.8 -- 1.9 -- 0.9 Q9UKF6_C498 -- 1.4 1.9 -- 1.4 Q96F86_C413 1.6 2.0 1.0 -- 0.8 P42224_C492 1.0 -- 20.0 -- 0.7 P11216_C326 1.3 -- 0.8 -- 0.9 P21980_C277 0.8 -- 20.0 -- 0.7 Q9HAV7_C124 -- -- 1.3 -- 1.7 P24752_C126 -- -- 0.9 -- 1.4 Q9NQ88_C161 1.1 1.5 20.0 -- 1.0 Q13155_C23 0.9 -- 1.1 -- 0.9 Q9NQW6_C712 1.3 -- 2.1 -- -- P51649_C340 1.1 1.3 0.7 -- 1.2 Q15021_C439 -- -- 5.9 -- 0.7 Q5T0N5_C69 1.0 -- 1.5 -- 0.8 P38606_C138 20.0 -- 1.9 -- 20.0 Q9HCC0_C216 -- 1.8 1.0 -- 2.2 Q9NQC3_C1101 -- -- 20.0 -- 2.1 P35754_C23 -- -- 0.7 -- -- Q99757_C90 -- -- 3.2 -- 4.3 Q9Y3D0_C93 -- 1.4 2.7 -- 0.5 Q9UMS0_C213 1.6 -- 3.5 -- -- Q9NXV6_C516 2.4 1.6 1.4 -- 0.8 Q96RS6_C376 0.7 1.2 0.9 1.8 1.4 Q14997_C1840 0.9 1.6 -- 20.0 1.0 P50570_C27 -- 1.0 1.0 -- 0.8 Q86YH6_C71 1.2 1.8 20.0 -- 2.1 Q99497_C106 -- 3.3 0.8 -- -- Q9UJW0_C258 -- 1.9 3.0 -- -- Q9BUH6_C180 1.1 -- 1.3 -- -- P24752_C196 -- -- 1.0 -- 1.5 Q13162_C51 -- -- 0.9 -- 1.7 Q9BTA9_C553 1.4 -- 1.7 -- 2.8 P48643_C253 -- -- 0.8 -- -- O75362_C286 1.1 20.0 1.4 -- 1.1 O60825_C158 -- 0.9 1.5 14.2 1.4 Q8NBS9_C350 1.3 -- 1.0 -- -- Q9NYL2_C22 1.3 -- 0.9 -- -- P27707_C9 1.0 1.3 -- -- 1.3 Q93009_C223 -- 1.3 1.1 -- 1.8 O14929_C101 0.9 1.2 1.6 1.9 1.2 Q9UPQ0_C140 1.1 -- 4.6 -- 0.8 Q96NY7_C487 1.9 -- 0.8 -- 1.2 Q9NQ88_C114 1.1 1.2 2.2 5.1 0.9 Q14790_C360 1.2 -- -- -- 0.9 P04183_C230 0.7 -- -- -- 0.7 P68366_C54 -- 1.3 -- 5.1 0.8 Q13428_C1298 -- -- 3.0 -- -- Q5MNZ6_C63 1.1 -- 1.2 -- 0.8 O14980_C528 1.1 1.1 20.0 -- 0.8 Q86W42_C35 -- -- 0.9 -- -- Q9Y6G9_C51 -- 1.5 2.1 4.3 0.6 Q9NY27_C22 -- 1.5 1.3 -- -- Q8NFH5_C255 1.5 -- 1.0 -- 1.8 Q9Y676_C128 -- -- 1.2 -- 1.5 P35658_C728 1.0 -- 1.2 -- 1.3 Q9NTX5_C133 1.1 -- 1.0 -- 1.1 Q15118_C71 1.6 -- 4.4 -- 1.7 Q00765_C18 1.2 -- 20.0 -- 0.7 P22307_C71 4.9 -- 1.1 -- 8.3 O75521_C312 0.7 -- 1.4 -- 1.6 P49189_C288 -- 12.0 0.9 -- 1.0 Q5T440_C170 1.2 -- 1.2 -- 1.6 Q15084_C190 -- -- 1.1 -- -- Q96C19_C172 0.7 -- 1.0 -- -- P22061_C102 0.9 -- 1.0 -- -- Q9NP73_C86 0.9 1.4 1.1 -- -- Q9BRF8_C54 1.4 -- 0.9 -- -- Q6ICB0_C108 -- 4.0 -- -- 0.6 P29590_C389 1.3 -- 1.2 -- 20.0 P07858_C211 1.6 -- 1.0 -- 2.6 Q9NX18_C83 -- -- 2.2 -- 1.3 P46109_C249 -- 1.8 1.3 -- 0.9 P45984_C177 -- -- 1.5 -- -- P19447_C342 1.1 -- 2.5 -- 2.0 P42166_C341 1.3 1.4 2.3 -- 1.7 Q8N1F7_C522 -- 2.2 3.2 20.0 -- Q86UY8_C276 1.5 -- 2.8 -- 1.8 Q8WWI1_C228 2.0 1.0 -- -- 1.1 Q9NWA0_C139 1.1 -- -- -- 1.5 P09110_C381 -- -- 1.0 -- -- Q2NL82_C126 -- -- 0.8 -- 1.5 Q5JPI3_C308 1.1 1.7 3.2 -- 0.9 P23919_C163 -- -- 0.9 -- -- Q96EB1_C218 1.2 1.7 -- -- 0.7 Q96FX7_C209 2.1 6.8 2.7 -- 2.0 O14933_C98 -- -- 2.5 -- 0.7 Q29RF7_C242 -- -- 1.8 -- 0.9 Q96T76_C819 -- -- 3.4 -- -- P23919_C117 0.6 -- 1.2 -- 0.9 Q15149_C4574 1.4 -- 1.2 -- 1.0 Q96RP9_C153 -- 0.8 1.2 -- 1.4 P04818_C199 -- -- -- -- -- P27708_C73 -- 5.0 20.0 3.7 0.6 P55265_C1224 1.9 -- 4.2 -- 2.2 Q9Y3D2_C105 -- -- 2.7 -- -- O00244_C12 1.5 -- 0.7 -- 0.7 Q8WV74_C207 13.1 -- 20.0 -- -- Q9NRW3_C130 -- -- -- 1.4 1.3 P24468_C326 -- -- 4.9 -- 1.1 P42166_C684 1.2 -- 3.1 -- -- Q96EY5_C231 -- -- -- -- 0.7 P14635_C238 1.3 1.5 2.6 -- -- Q8NDH3_C81 -- -- 1.4 -- 1.6 Q9P0J1_C149 -- -- 1.3 -- -- Q96P48_C900 1.2 -- -- -- 0.7 Q96HE7_C37 -- 1.4 1.4 -- -- Q07065_C100 1.6 -- 11.2 -- 2.2 Q9BRJ7_C88 -- -- -- -- -- O75439_C265 -- -- 20.0 -- -- O43175_C369 -- 2.0 -- 20.0 1.0 Q9UNI6_C265 -- -- 0.7 -- -- Q06203_C100 -- 2.0 -- -- -- A0AVT1_C347 1.3 -- -- -- -- Q86X76_C203 -- -- 0.7 -- 0.8 Q6XZF7_C691 1.6 -- -- -- 0.8 Q15398_C129 -- -- -- -- -- O75717_C773 1.1 -- 1.7 -- -- Q01433_C107 -- -- -- -- -- Q8WVV9_C464 -- -- 2.2 -- 1.4 O14733_C131 -- 1.6 -- 20.0 0.7 Q14137_C404 1.1 -- -- -- 1.1 Q96RU2_C171 -- -- 1.3 -- -- Q9Y679_C391 -- -- -- -- 1.5 P51610_C1872 1.0 -- 1.1 -- -- P22307_C307 -- -- 0.9 -- 20.0 Q9BTE3_C325 -- -- 5.7 -- -- Q9HA64_C24 -- -- 1.7 -- 0.6 Q5TFE4_C119 -- 1.7 3.8 -- 1.2 Q96N67_C2125 -- -- 1.8 -- -- P52948_C1312 1.2 -- 1.7 -- 1.1 Q5UIP0_C2298 -- -- 20.0 -- 0.9 P51812_C436 1.0 -- 0.6 -- -- Q92616_C1692 -- 0.9 20.0 -- -- Q15345_C297 1.6 -- 5.8 -- 1.1 Q9NPH0_C267 -- -- 1.2 -- -- P04183_C66 1.0 -- -- -- -- P42166_C629 1.0 -- 20.0 -- 1.6 Q15013_C124 -- -- 1.4 -- -- Q9Y5Y2_C72 -- 1.5 -- -- --
O15446_C86 1.3 -- 1.0 -- 1.7 Q13630_C116 -- 1.2 -- -- 0.8 Q8IYQ7_C324 1.3 -- 1.6 -- 2.0 P05091_C319 -- -- 3.3 -- -- Q29RF7_C532 -- 2.0 2.5 -- -- Q9Y570_C381 0.9 -- -- -- -- Q14980_C961 2.2 -- -- -- 3.7 P53384_C235 -- -- -- -- -- Q15003_C418 -- -- -- -- -- P53634_C258 1.6 -- 1.9 -- 2.8 Q8NFF5_C499 -- -- 20.0 -- -- Q9ULA0_C144 -- -- -- -- -- P22307_C94 -- -- 1.0 -- -- O15294_C620 -- -- 1.0 -- -- Q9Y5S2_C1517 -- -- 0.9 -- -- Q8TD19_C623 -- 20.0 -- -- 0.8 Q8N2W9_C326 -- 20.0 -- -- 1.0 Q13158_C98 1.6 -- -- -- -- Q9UKX7_C151 -- -- 1.8 -- -- Q6PCB5_C280 -- 0.8 20.0 -- -- P10398_C597 -- 1.0 -- -- 0.7 Q9UL40_C68 -- 20.0 20.0 -- -- P46013_C903 -- -- -- -- -- Q16667_C39 -- 1.2 -- -- 0.8 O75150_C890 -- -- 1.1 -- -- Q00610_C870 -- -- 20.0 -- -- Q9Y5T5_C205 -- 20.0 -- -- 0.9 O95881_C66 20.0 -- 1.3 -- -- Q7Z5K2_C160 -- -- 2.4 -- -- P42166_C518 -- -- -- -- 1.4 Q9Y2S7_C143 -- -- 1.3 -- -- E2QRD5_C183 -- -- -- -- 0.8 O95833_C22 -- -- 0.8 -- 1.0 O94953_C694 -- 1.5 -- -- -- O00541_C272 1.4 -- 3.8 -- -- Q9NXJ5_C149 -- 20.0 -- -- -- Q8N5L8_C131 1.2 -- 1.3 -- 0.8 Q8IZ73_C246 -- -- -- -- 1.8 Q99798_C385 -- -- -- -- 2.0 Q9GZR2_C382 -- 0.9 1.2 -- -- Q13613_C117 -- -- 1.0 1.7 0.9 Q9NUI1_C22 -- -- 1.1 -- -- Q02556_C306 -- 1.5 -- -- -- Q9UPT9_C171 -- 3.2 -- -- -- Q8N999_C302 -- -- -- -- -- Q8IU81_C363 -- -- -- -- -- Q9C0I1_C152 -- 6.5 -- -- 0.7 Q9P2X3_C195 -- -- 1.6 -- -- Q6QNY0_C168 -- 1.2 -- -- -- Q15796_C81 -- -- -- -- 1.3 Q9NZB2_C531 -- -- -- -- 1.6 Q9HB90_C377 -- -- 1.1 -- -- Q9BR61_C267 1.1 -- -- 6.9 -- P16455_C145 -- -- -- -- -- Q86UV5_C39 -- -- -- -- 1.4 A2A288_C367 -- -- -- -- -- Q8NEC7_C140 1.8 20.0 -- -- -- Q6PJG6_C673 -- -- -- -- -- Q13232_C158 -- -- -- -- -- Q86X76_C165 -- -- 0.8 -- -- P42695_C541 -- -- -- -- 2.3 P41226_C599 -- -- -- -- 1.0 Q99986_C50 -- -- 1.6 -- -- Q8WUM4_C90 -- -- 1.0 -- -- P29590_C213 -- -- -- -- -- Q9P0K7_C973 -- -- -- -- -- P53992_C78 0.7 -- -- -- -- Q13867_C73 -- -- -- -- -- Q8ND24_C655 -- -- -- -- -- Q96EK4_C48 -- -- -- -- -- Q96IV0_C309 -- -- -- -- -- Q5T1V6_C414 -- -- -- -- -- Q9UHQ1_C99 -- 20.0 -- -- -- O43396_C34 -- -- 7.3 -- -- Q8IV53_C174 -- -- -- -- -- Q8N9T8_C673 -- -- -- -- --
TABLE-US-00013 TABLE 2C 27_200 .mu.M.sub.-- 28_200 .mu.M.sub.-- 29_200 .mu.M.sub.-- 31_200 .mu.M.sub.-- 31_200 .mu.M.sub.-- 33_200 .mu.M.sub.-- Identifier insitu_231 insitu_231 insitu_ramos insitu_231 insitu_ramos insitu_231 P04406_C152 0.5 0.8 1.2 0.8 1.0 1.1 P61978_C132 0.9 1.0 1.2 0.9 1.1 1.3 Q13526_C113 0.5 0.7 1.0 0.6 1.1 1.2 P24752_C119 1.6 1.0 1.2 0.6 0.7 2.3 P24752_C413 1.9 1.0 1.2 1.0 -- 20.0 Q9NUY8_C283 0.6 0.7 1.3 0.6 -- 1.6 P13667_C206 1.0 1.3 1.4 0.8 1.1 2.7 P12268_C140 0.5 0.6 1.6 0.6 1.0 1.4 Q15365_C194 0.5 -- 1.0 0.7 1.6 2.2 Q9NVC6_C649 1.3 1.1 1.6 0.8 -- 1.2 P42166_C561 0.8 1.1 0.9 1.3 2.5 2.9 Q9Y696_C35 1.0 2.7 1.0 0.6 -- 8.9 P10599_C32 2.5 20.0 1.9 1.2 1.7 20.0 P31943_C267 1.1 -- 1.4 0.7 0.9 1.3 Q86SX6_C67 1.5 -- 1.2 -- 0.7 1.0 P15121_C299 0.5 0.8 1.1 0.8 1.3 0.7 P52597_C267 1.0 -- 1.2 1.2 1.3 1.6 Q9ULV4_C420 0.8 0.7 -- 0.8 1.1 1.6 P62888_C92 0.6 0.9 2.0 -- -- 1.1 Q9NQR4_C153 1.3 -- -- 0.6 1.4 1.0 P42765_C92 1.2 -- 0.9 0.8 1.2 1.3 Q15084_C55 1.2 1.4 1.0 0.7 -- 3.7 Q96HE7_C241 0.9 1.1 1.3 0.7 -- 2.3 Q99439_C164 -- 0.9 1.0 0.7 1.3 1.5 P25205_C119 1.0 -- 1.3 0.5 -- 1.2 Q9NS86_C187 0.4 -- 1.7 0.5 -- 1.5 Q15233_C145 1.2 1.1 -- 0.7 0.8 1.3 Q9BRA2_C43 20.0 -- 2.3 0.6 -- 20.0 P35611_C68 0.9 1.7 -- -- -- 1.6 O75521_C380 1.6 0.8 1.2 -- -- 0.7 Q9BXW7_C392 1.3 1.0 1.5 -- -- 1.1 P30101_C406 1.2 -- 1.5 0.8 1.1 -- Q96AB3_C114 1.0 0.8 1.1 0.7 -- 0.9 P13667_C555 1.2 -- 1.5 0.8 1.1 -- Q09161_C44 0.8 -- 1.1 0.6 -- 1.1 P78417_C32 20.0 20.0 20.0 0.8 2.6 20.0 Q9ULW0_C536 1.1 1.0 1.8 -- -- 2.2 Q9NRG0_C55 0.9 -- 1.5 -- 1.6 1.1 Q96T76_C848 0.5 0.8 1.0 20.0 -- -- Q8TAQ2_C145 2.1 0.9 0.8 1.1 1.3 Q9BVC5_C10 1.1 0.9 1.8 -- -- 1.3 Q7Z2W4_C645 0.8 -- 1.6 -- -- 2.1 Q9BQ69_C186 2.4 -- 1.5 0.5 -- 0.9 Q16831_C162 0.5 0.7 -- -- -- 1.3 P30101_C57 1.1 1.6 1.2 0.9 -- -- P12268_C331 0.7 0.8 1.3 1.0 1.1 1.5 O95571_C170 1.1 -- 1.5 -- -- 1.2 O00299_C24 0.7 -- 1.1 0.6 -- 8.4 O14879_C343 0.4 0.7 -- 0.9 -- 2.4 Q96CM8_C64 1.8 -- 2.3 0.8 -- 2.3 P51946_C244 1.1 -- 1.1 -- -- 0.9 P49588_C773 0.6 -- 1.1 -- 1.2 -- Q96RN5_C618 0.8 0.7 1.3 0.9 -- 2.0 O15294_C758 1.0 1.0 -- -- -- 1.2 P46734_C207 0.8 -- -- -- -- 1.2 Q96S55_C272 0.9 0.5 -- -- -- 1.2 O95229_C54 0.5 0.7 1.2 -- -- -- O60610_C1227 0.5 -- 1.6 0.6 1.2 -- Q13428_C38 1.3 1.0 1.5 -- -- -- Q9Y277_C65 1.6 1.2 -- -- 1.0 -- P57764_C268 0.6 -- 1.1 -- 3.1 -- Q9Y3A3_C134 -- -- 1.2 1.0 1.8 1.2 Q02252_C317 2.0 1.1 1.2 -- -- 2.3 Q9NYL9_C132 0.6 0.7 0.9 0.7 0.7 -- P83731_C6 0.4 -- 1.6 -- -- 1.6 O95336_C32 0.5 -- 3.0 -- 1.7 2.3 O13155_C291 0.7 0.9 -- 0.8 -- -- Q13418_C346 0.6 -- -- 0.6 -- 1.4 A6NDU8_C179 0.7 0.8 1.0 -- -- 7.4 Q9UKF6_C498 1.5 -- 2.3 -- -- 1.6 Q96F86_C413 0.7 -- 1.9 -- 1.3 3.0 P42224_C492 -- 0.8 3.8 0.8 1.9 1.3 P11216_C326 0.7 -- -- 0.5 -- 1.6 P21980_C277 0.6 0.4 -- -- -- -- O9HAV7_C124 0.6 -- 1.0 -- -- -- P24752_C126 -- 1.2 1.2 0.7 -- 20.0 Q9NQ88_C161 0.6 -- 1.3 1.0 2.6 -- Q13155_C23 0.7 -- 1.6 -- -- 1.4 Q9NQW6_C712 1.0 0.9 -- 0.7 -- 1.5 P51649_C340 0.9 -- 1.0 -- 1.0 0.9 Q15021_C439 0.6 0.9 1.4 2.0.0 -- -- Q5T0N5_C69 0.6 -- -- -- -- 2.6 P38606_C138 4.6 -- -- -- 1.7 20.0 Q9HCC0_C216 1.0 -- -- -- -- 1.0 Q9NQC3_C1101 1.4 -- -- 12.8 -- 2.1 P35754_C23 0.7 1.4 -- 0.6 1.1 20.0 Q99757_C90 6.3 -- 1.6 1.4 -- -- Q9Y3D0_C93 0.7 0.8 1.0 -- -- -- Q9UMS0_C213 1.3 -- 2.0 1.5 -- 1.9 Q9NXV6_C516 1.4 -- 1.1 -- -- 1.4 Q96RS6_C376 -- -- 1.3 0.8 -- -- Q14997_C1840 20.0 0.9 -- -- 1.2 -- P50570_C27 0.5 -- 1.0 -- -- -- Q86YH6_C71 1.3 -- 1.1 -- -- -- Q99497_C106 0.9 1.0 -- 0.8 1.0 -- Q9UJW0_C258 0.8 -- 20.0 -- -- -- Q9BUH6_C180 -- 1.1 1.5 0.8 -- 1.4 P24752_C196 -- -- 1.1 -- -- 3.4 Q13162_C51 0.9 -- -- -- 1.3 1.7 Q9BTA9_C553 -- -- 1.1 -- -- -- P48643_C253 -- 0.8 -- 0.5 1.0 -- O75362_C286 1.0 0.9 1.0 -- -- -- O60825_C158 -- -- 0.9 -- 1.3 -- Q8NBS9_C350 1.1 1.1 -- 0.8 -- 2.6 Q9NYL2_C22 0.7 -- 1.2 -- -- 1.5 P97707_C9 -- -- 1.2 -- 1.2 1.9 Q93009_C223 1.1 1.0 1.0 -- -- -- O14929_C101 -- -- 1.0 -- -- 1.3 Q9UPQ0_C140 0.7 -- -- -- -- 2.5 Q96NY7_C487 -- -- -- 0.5 -- 14.9 Q9NQ88_C114 0.4 -- 0.9 -- -- -- Q14790_C360 0.4 -- 1.6 0.5 -- 3.2 P04183_C230 -- -- 1.2 -- 2.1 1.6 P68366_C54 0.3 -- 1.0 -- 1.1 -- Q13428_C1298 1.0 0.9 -- -- 2.2 -- Q5MNZ6_C63 -- 1.7 -- -- -- 1.6 O14980_C528 -- -- 1.1 -- -- -- Q86W42_C35 1.3 1.3 -- 0.5 -- 1.6 Q9Y6G9_C51 0.6 0.7 -- -- 1.6 1.8 Q9NY27_C22 -- -- 1.7 -- -- -- Q8NFN5_C255 -- -- 1.6 -- -- -- Q9Y676_C128 1.0 -- -- -- -- -- P35658_C728 -- -- 1.7 0.7 -- 6.4 Q9NTX5_C133 1.5 -- -- -- -- 1.0 Q15118_C71 1.5 -- 1.8 -- -- -- Q00765_C18 1.1 -- 1.4 -- -- 1.1 P22307_C71 20.0 -- -- -- -- -- O75521_C312 1.1 -- -- -- -- -- P49189_C288 -- -- -- -- -- 20.0 Q5T440_C170 -- -- 1.3 -- -- 2.5 Q15084_C190 1.0 -- 1.0 1.0 -- -- Q96C19_C172 -- -- 1.2 1.6 -- -- P22061_C102 2.2 -- 1.2 -- -- -- Q9NP73_C86 0.4 -- 2.0 -- -- -- Q9BRF8_C54 -- -- 1.5 -- -- 1.8 Q6ICB0_C108 1.9 -- 1.0 1.2 1.3 -- P29590_C389 0.5 -- -- 1.1 -- -- P07858_C211 0.8 -- -- -- -- 2.2 Q9NX18_C83 1.5 -- 1.9 -- -- -- P46109_C249 -- -- 4.2 -- -- -- P45984_C177 0.5 -- 1.7 -- -- 2.0 P19447_C342 -- -- -- -- -- -- P42166_C341 0.6 -- -- -- 2.0 -- Q8N1F7_C522 -- -- 1.7 -- 3.6 1.7 Q86UY8_C276 1.7 -- -- -- -- 0.8 Q8WWI1_C228 0.8 -- 1.0 -- -- -- Q9NWA0_C139 1.3 -- 0.7 -- -- 1.6 P09110_C381 -- 0.8 -- 0.7 -- -- Q2NL82_C126 0.4 -- -- -- -- -- Q5JPI3_C308 0.7 -- 1.3 -- -- -- P23919_C163 0.2 0.4 -- -- 1.0 -- Q96EB1_C218 0.6 -- 1.1 -- -- -- Q96FX7_C209 -- -- -- -- -- -- O14933_C98 -- -- 2.4 -- 1.9 -- Q29RF7_C242 0.9 1.0 -- -- -- -- Q96T76_C819 0.5 -- -- 20.0 20.0 -- P23919_C117 -- -- 1.6 -- -- 2.5 Q15149_C4574 1.3 -- -- -- -- 1.4 Q96RP9_C153 1.6 -- 1.2 -- -- -- P04818_C199 0.5 -- 2.5 -- -- -- P27708_C73 0.5 -- 1.8 -- -- -- P55265_C1224 1.4 -- 1.8 -- -- 1.6 Q9Y3D2_C105 1.5 -- -- -- -- -- O00244_C12 0.4 -- 1.4 -- -- 1.8 Q8WV74_C207 2.2 -- -- -- -- -- Q9NRW3_C130 1.3 -- 13.3 -- -- -- P24468_C326 1.2 -- -- -- -- -- P42166_C684 -- -- -- -- 3.3 -- Q96EY5_C231 0.6 -- 1.1 -- -- 1.2 P14635_C238 -- -- -- -- -- 1.2 Q8NDH3_C81 -- -- -- 1.0 -- -- Q9P0J1_C149 -- -- -- 1.0 -- -- Q96P48_C900 -- -- 1.1 1.1 -- 1.7 Q96HE7_C37 -- -- 2.1 0.6 -- -- Q07065_C100 -- -- -- -- -- 1.7 Q9BRJ7_C88 -- -- 1.7 -- 2.9 -- O75439_C265 1.6 0.6 -- 1.0 1.4 -- O43175_C369 -- -- 1.1 20.0 20.0 -- Q9UNI6_C265 0.6 0.8 -- -- -- -- Q06203_C100 0.8 -- 1.6 0.6 1.2 -- A0AVT1_C347 0.8 -- -- -- 1.9 -- Q86X76_C203 -- -- -- -- -- -- Q6XZF7_C691 -- -- -- 2.3 -- 2.5 Q15398_C129 0.5 -- 1.2 -- 2.4 1.3 O75717_C773 -- -- -- -- -- -- Q01433_C107 0.4 -- -- 0.7 -- -- Q8WV9_C464 -- -- -- -- -- 2.2 O14733_C131 -- -- 1.0 -- -- -- Q14137_C404 0.6 -- 1.5 -- -- -- Q96RU2_C171 1.1 1.1 -- -- -- -- Q9Y679_C391 1.2 -- -- -- -- -- P51610_C1872 -- -- -- -- -- -- P22307_C307 -- -- -- -- -- -- Q9BTE3_C325 -- 1.0 -- 1.9 3.5 -- Q9HA64_C24 -- -- -- -- -- -- Q5TFE4_C119 -- -- -- 3.1 -- -- Q96N67_C2125 0.9 -- -- 0.8 -- -- P52948_C1312 1.3 -- -- -- -- 1.8 Q5UIP0_C2298 -- -- -- -- -- 3.4 P51812_C436 -- -- 1.3 -- -- -- Q92616_C1692 0.5 -- -- 0.6 2.0 1.2 Q15345_C297 -- -- -- 0.9 -- 1.9 Q9NPH0_C267 1.3 -- 2.1 -- -- -- P04183_C66 -- -- 1.6 0.7 -- -- P42166_C629 -- -- 1.6 -- -- -- Q15013_C124 1.0 1.0 -- -- -- -- Q9Y5Y2_C72 -- -- 1.1 -- -- -- O15446_C86 -- -- -- -- -- 1.6 Q13630_C116 -- -- -- -- -- -- Q8IYQ7_C324 -- -- -- -- -- 1.4 P05091_C319 -- -- 10.5 -- -- -- Q29RF7_C532 -- -- -- 5.1 8.1 -- Q9Y570_C381 -- -- 1.2 -- -- -- Q14980_C961 2.4 -- -- 1.1 -- 4.8 P53384_C235 -- -- -- 0.7 -- -- Q15003_C418 -- -- 1.4 -- -- -- P53634_C258 -- -- -- -- -- -- Q8NFF5_C499 -- -- 3.4 -- -- -- Q9ULA0_C144 -- -- 1.3 -- -- -- P22307_C94 -- 20.0 -- -- -- -- O15294_C620 -- -- 2.6 1.1 -- -- Q9Y5S2_C1517 -- -- -- -- -- -- Q8TD19_C623 -- -- -- 0.7 1.4 -- Q8N2W9_C326 -- -- -- -- -- -- Q13158_C98 0.6 -- 1.3 0.9 -- -- Q9UKX7_C151 -- -- -- -- -- -- Q6PCB5_C280 1.0 -- 1.4 -- -- -- P10398_C597 -- -- 1.2 -- -- -- Q9UL40_C68 1.9 -- 2.4 -- -- -- P46013_C903 -- -- 1.6 -- -- -- Q16667_C39 -- -- 1.7 -- -- -- O75150_C890 1.2 -- -- 0.7 -- -- Q00610_C870 -- -- -- -- -- -- Q9Y5T5_C205 1.2 -- -- -- -- -- O95881_C66 -- -- -- -- -- -- Q7Z5K2_C160 -- -- 0.7 -- -- -- P42166_C518 -- -- -- -- -- -- Q9Y2S7_C143 -- -- -- -- -- -- E2QRD5_C183 -- -- -- 0.8 -- -- O95833_C22 -- -- -- 0.5 -- -- O94953_C694 -- -- 1.1 -- -- -- O00541_C272 -- -- -- -- -- -- Q9NXJ5_C149 -- -- -- -- -- --
Q8N5L8_C131 -- -- -- -- 1.7 Q8IZ73_C246 -- -- -- 0.4 1.0 -- Q99798_C385 -- -- -- -- -- -- Q9GZR2_C382 -- -- -- -- -- -- Q13613_C117 -- -- 1.5 -- -- -- Q9NUI1_C22 -- -- -- 0.7 -- -- Q02556_C306 -- -- 1.4 -- 2.0 -- Q9UPT9_C171 -- -- -- -- -- -- Q8N999_C302 -- 0.6 -- -- -- -- Q8IU81_C363 -- -- -- 0.7 -- -- Q9C0I1_C152 -- -- 4.6 -- -- -- Q9P2X3_C195 -- -- -- 0.5 -- -- Q6QNY0_C168 -- -- 1.6 -- -- -- Q15796_C81 -- -- 1.3 -- -- -- Q9NZB2_C531 -- -- -- -- -- 2.4 Q9HB90_C377 -- -- -- -- -- -- Q9BR61_C267 -- -- 2.5 -- -- -- Pl6455_C145 -- -- -- -- 1.2 -- Q86UV5_C39 -- -- -- -- 1.3 -- A2A288_C367 -- -- 1.3 -- 1.9 -- Q8NEC7_C140 -- -- -- -- -- -- Q6PJG6_C673 -- -- 4.6 -- 20.0 -- Q13232_C158 -- -- 1.5 -- -- -- Q86X76_C165 -- -- -- -- -- -- P42695_C541 -- -- -- -- -- -- P41226_C599 -- -- -- -- -- -- Q99986_C50 -- -- -- -- -- -- Q8WUM4_C90 0.6 -- -- -- -- -- P29590_C213 0.8 -- -- -- -- -- Q9P0K7_C973 -- -- -- -- -- -- P53992_C78 -- -- -- -- -- -- Q13867_C73 -- -- -- -- -- -- Q8ND24_C655 -- -- -- -- -- -- Q96EK4_C48 -- -- -- -- -- -- Q96IV0_C309 -- -- 3.0 -- -- -- Q5T1V6_C414 -- -- -- -- -- -- Q9UHQ1_C99 -- -- -- -- -- -- O43396_C34 -- -- -- -- -- -- Q8IV53_C174 -- -- -- -- -- -- Q8N9T8_C673 -- -- -- -- -- -- 38_200 .mu.M.sub.-- 41_200 .mu.M.sub.-- 45_200 .mu.M.sub.-- 51_200 .mu.M.sub.-- 56_200 .mu.M.sub.-- Identifier insitu_231 insitu_231 insitu_231 insitu_231 insitu_231 P04406_C152 0.9 0.6 20.0 1.0 0.9 P61978_C132 1.0 0.9 9.3 1.2 0.8 Q13526_C113 1.0 0.6 10.0 1.4 -- P24752_C119 1.2 1.9 12.4 0.7 0.8 P24752_C413 1.1 0.9 20.0 1.1 0.7 Q9NUY8_C283 1.0 0.7 2.7 2.1 0.9 P13667_C206 0.9 1.0 1.9 5.3 0.8 P12268_C140 1.2 0.7 1.9 1.0 1.1 Q15365_C194 2.5 0.3 1.5 0.6 1.2 Q9NVC6_C649 1.2 0.8 20.0 -- 0.8 P42166_C561 20.0 0.7 20.0 1.3 1.1 Q9Y696_C35 1.0 0.6 1.4 2.6 0.9 P10599_C32 2.1 0.4 5.7 13.9 1.5 P31943_C267 1.2 1.0 3.6 1.3 0.8 Q86SX6_C67 1.0 1.0 9.1 1.0 0.8 P15121_C299 1.0 0.9 1.0 0.9 0.9 P52597_C267 1.8 0.8 4.1 1.3 0.9 Q9ULV4_C420 1.4 0.8 3.2 1.1 1.0 P62888_C92 3.2 0.8 2.2 0.9 0.9 Q9NQR4_C153 1.1 0.7 2.0 0.6 0.8 P42765_C92 0.9 1.1 15.1 1.1 -- Q15084_C55 0.8 0.9 2.0 5.0 0.8 Q96HE7_C241 1.6 0.7 2.1 1.7 0.9 Q99439_C164 1.5 0.4 1.8 -- 1.2 P25205_C119 0.9 0.9 6.2 2.6 1.0 Q9NS86_C187 2.0 0.6 3.1 2.0 1.2 Q15233_C145 0.9 1.1 3.7 1.6 0.8 Q9BRA2_C43 1.1 0.6 8.0 -- 3.9 P35611_C68 1.4 0.9 20.0 1.0 0.8 O75521_C380 0.9 1.1 1.2 1.0 1.1 Q9BXW7_C392 0.8 1.0 4.9 1.1 0.8 P30101_C406 0.8 1.0 2.6 6.0 0.7 Q96AB3_C114 1.2 0.7 3.1 0.8 -- P13667_C555 0.8 1.0 2.6 6.0 0.7 Q09161_C44 1.2 0.8 2.5 1.2 1.0 P78417_C32 1.0 0.3 1.9 -- 1.3 Q9ULW0_C536 1.2 1.2 20.0 1.4 -- Q9NRG0_C55 1.0 0.9 20.0 1.1 1.0 Q96T76_C848 -- 0.7 20.0 -- 1.4 Q8TAQ2_C145 1.3 1.2 2.9 1.2 0.8 Q9BVC5_C10 0.9 1.0 3.4 1.0 0.8 Q7Z2W4_C645 0.9 0.9 4.6 1.6 0.9 Q9BQ69_C186 3.6 1.2 3.3 1.2 0.8 Q16831_C162 1.4 0.8 1.3 1.1 1.1 P30101_C57 0.9 1.3 2.6 -- 0.7 P12268_C331 1.3 0.8 4.1 -- -- O95571_C170 -- 1.1 3.1 -- 0.8 O00299_C24 1.0 0.6 -- -- 0.9 O14879_C343 -- 0.3 4.0 1.3 1.2 Q96CM8_C64 1.1 0.8 20.0 -- 0.8 P51946_C244 1.0 1.2 2.4 0.7 1.0 P49588_C773 0.7 0.7 10.1 -- -- Q96RN5_C618 1.4 -- 20.0 2.1 0.8 O15294_C758 -- 0.9 2.9 1.1 1.0 P46734_C207 1.5 0.7 1.5 0.7 1.3 Q96S55_C272 1.3 0.8 2.5 0.9 0.9 O95229_C54 -- 0.6 -- 1.0 1.0 O60610_C1227 0.3 0.6 3.0 -- 1.2 Q13428_C38 1.1 1.1 2.8 1.1 0.8 Q9Y277_C65 20.0 1.4 -- -- 0.9 P57764_C268 1.8 0.6 20.0 1.8 0.8 Q9Y3A3_C134 1.0 0.9 2.3 -- 0.9 Q02252_C317 2.4 0.8 2.6 -- -- Q9NYL9_C132 2.6 0.3 0.9 0.8 -- P83731_C6 2.1 0.3 1.2 -- -- O95336_C32 2.8 0.6 -- -- 0.9 O13155_C291 1.7 0.7 7.3 1.1 1.1 Q13418_C346 1.0 0.6 -- 0.8 -- A6NDU8_C179 1.4 0.6 20.0 -- 1.2 Q9UKF6_C498 -- 1.0 20.0 -- 1.0 Q96F86_C413 -- -- 20.0 20.0 1.2 P42224_C492 -- 0.6 20.0 2.4 1.2 P11216_C326 1.2 0.7 5.3 0.8 -- P21980_C277 1.4 0.6 1.1 1.0 -- O9HAV7_C124 1.0 0.6 2.1 -- 0.7 P24752_C126 -- 0.8 -- -- 0.8 Q9NQ88_C161 -- -- 20.0 -- 1.4 Q13155_C23 1.4 -- 4.4 1.2 0.9 Q9NQW6_C712 1.5 0.7 5.9 -- 1.0 P51649_C340 -- -- 2.4 -- -- Q15021_C439 1.1 0.8 -- 1.1 1.2 Q5T0N5_C69 1.5 0.6 5.4 1.7 1.0 P38606_C138 2.1 0.8 -- 10.6 1.1 Q9HCC0_C216 -- 1.0 7.0 0.9 0.7 Q9NQC3_C1101 20.0 1.0 -- 1.2 0.8 P35754_C23 0.9 0.5 16.3 -- 0.9 Q99757_C90 3.6 0.5 6.7 5.0 -- Q9Y3D0_C93 2.4 0.6 20.0 20.0 -- Q9UMS0_C213 3.5 0.9 7.1 1.8 0.7 Q9NXV6_C516 1.8 0.9 -- -- -- Q96RS6_C376 1.3 0.7 -- -- -- Q14997_C1840 20.0 0.9 -- 20.0 -- P50570_C271 0.9 0.5 15.4 1.1 -- Q86YH6_C71 -- 0.9 20.0 -- 1.0 Q99497_C106 1.1 0.6 20.0 1.4 -- Q9UJW0_C258 -- 0.7 2.7 1.1 1.3 Q9BUH6_C180 1.4 0.6 5.5 -- 1.3 P24752_C196 1.0 0.9 -- -- 0.8 Q13162_C51 2.4 -- 2.3 -- 1.0 Q9BTA9_C553 1.1 1.0 19.1 1.2 0.9 P48643_C253 1.1 0.8 1.2 0.8 0.9 O75362_C286 -- 1.0 20.0 -- 0.9 O60825_C158 -- 0.7 1.3 0.9 1.2 Q8NBS9_C350 -- 0.9 2.1 -- 0.8 Q9NYL2_C22 -- 0.6 2.3 -- 1.0 P277Q07_C9 1.8 0.4 -- -- -- Q93009_C223 0.8 -- 20.0 1.0 -- O14929_C101 1.1 0.6 20.0 -- -- Q9UPQ0_C140 2.5 0.5 20.0 -- 1.1 Q96NY7_C487 -- 0.4 1.8 -- 1.3 Q9NQ88_C114 -- 0.6 4.6 -- -- Q14790_C360 1.0 0.5 20.0 -- 1.2 P04183_C230 2.8 0.6 2.0 0.7 -- P68366_C54 2.0 -- -- -- -- Q13428_C1298 2.0 1.0 4.7 -- -- Q5MNZ6_C63 -- 0.8 2.6 -- 0.7 O14980_C528 2.1 0.9 -- 1.1 -- Q86W42_C35 0.8 1.1 20.0 1.4 0.7 Q9Y6G9_C51 -- 0.7 -- -- -- Q9NY27_C22 1.0 0.8 20.0 2.2 -- Q8NFH5_C255 1.1 0.8 5 5 -- -- Q9Y676_C128 1.0 -- 2.0 1.0 -- P35658_C728 -- 1.1 3.1 -- -- Q9NTX5_C133 -- -- 4.2 -- 0.9 Q15118_C71 1.3 -- 20.0 -- -- Q00765_C18 -- -- 20.0 -- 0.9 P22307_C71 -- 1.2 16.8 5.6 -- O75521_C312 1.1 -- 0.7 -- 0.9 P49189_C288 0.7 0.8 20.0 -- 0.9 Q5T440_C170 -- 0.7 3.5 -- 1.0 Q15084_C190 1.0 0.9 2.8 -- -- Q96C19_C172 0.7 0.5 -- 1.2 -- P22061_C102 -- 0.7 -- -- -- Q9NP73_C86 1.3 0.5 20.0 1.3 -- Q9BRF8_C54 -- 0.7 20.0 -- -- Q6ICB0_C108 1.1 -- 3.1 1.1 -- P29590_C389 2.2 0.4 3.4 0.9 -- P07858_C211 1.8 0.4 2.4 -- -- Q9NX18_C83 -- -- 3.7 1.0 0.7 P46109_C249 1.6 -- 20.0 -- 0.8 P45984_C177 -- 0.5 -- -- 1.1 P19447_C342 1.0 0.7 5.9 -- 0.7 P42166_C341 -- 0.5 -- 1.2 -- Q8N1F7_C522 1.7 0.8 20.0 -- -- Q86UY8_C276 -- 1.7 2.1 1.2 -- Q8WWI1_C228 -- 0.8 20.0 -- -- Q9NWA0_C139 -- 0.8 -- -- 1.1 P09110_C381 0.8 0.9 1.2 -- 0.8 Q2NL82_C126 0.9 0.8 20.0 1.9 -- Q5JPI3_C308 -- 0.7 -- -- 0.7 P23919_C163 5.7 0.2 -- -- 0.9 Q96EB1_C218 1.3 -- 20.0 -- -- Q96FX7_C209 1.0 -- 20.0 -- 0.9 O14933_C98 1.6 0.5 20.0 -- -- Q29RF7_C242 1.0 -- -- 1.8 1.0 Q96T76_C819 4.6 0.6 20.0 -- 0.9 P23919_C117 4.4 -- -- -- -- Q15149_C4574 -- 0.9 -- -- 1.0 Q96RP9_C153 -- 1.1 -- -- -- P04818_C199 20.0 0.7 20.0 20.0 -- P27708_C73 -- 0.7 -- -- -- P55265_C1224 -- -- 20.0 1.3 -- Q9Y3D2_C105 1.6 1.2 20.0 -- -- O00244_C12 -- -- -- -- -- Q8WV74_C207 -- 0.9 20.0 -- -- Q9NRW3_C130 -- 0.9 20.0 -- 0.9 P24468_C326 -- 0.8 20.0 -- -- P42166_C684 3.0 -- 6.8 1.2 1.1 Q96EY5_C231 1.8 -- -- -- -- P14635_C238 1.8 0.5 -- -- -- Q8NDH3_C81 2.0 -- -- -- 1.0 Q9P0J1_C149 -- 0.9 20.0 -- 0.8 Q96P48_C900 -- 0.5 -- -- -- Q96HE7_C37 3.4 0.4 20.0 -- -- Q07065_C100 -- 1.0 20.0 -- -- Q9BRJ7_C88 -- 0.4 -- 1.6 1.0 O75439_C265 -- 1.3 -- -- -- O43175_C369 -- -- -- -- -- Q9UNI6_C265 1.0 1.0 7.5 -- -- Q06203_C100 1.2 -- -- -- -- A0AVT1_C347 -- 0.6 -- -- -- Q86X76_C203 -- 1.3 20.0 -- 0.8 Q6XZF7_C691 -- -- -- -- -- Q15398_C129 1.4 -- -- 7.4 1.3 O75717_C773 2.3 1.0 3.6 -- 0.9 Q01433_C107 -- 0.5 -- -- -- Q8WVV9_C464 1.5 -- 20.0 3.2 -- O14733_C131 20.0 0.8 -- -- -- Q14137_C404 -- -- -- -- -- Q96RU2_C171 0.8 -- -- -- -- Q9Y679_C391 -- -- 20.0 20.0 0.7 P51610_C1872 1.3 0.5 -- -- -- P22307_C307 -- 1.1 -- -- -- Q9BTE3_C325 2.2 0.6 20.0 -- -- Q9HA64_C24 1.7 -- -- -- 1.0 Q5TFE4_C119 -- 0.5 -- -- -- Q96N67_C2125 1.5 0.7 -- -- 0.9 P52948_C1312 1.3 -- -- -- -- Q5UIP0_C2298 -- 1.2 20.0 -- -- P51812_C436 -- 4.5 -- -- -- Q92616_C1692 -- 0.7 -- -- -- Q15345_C297 1.4 0.8 -- -- -- Q9NPH0_C267 -- -- 20.0 -- -- P04183_C66 -- 0.4 -- -- -- P42166_C629 -- -- -- -- -- Q15013_C124 -- -- 20.0 1.2 --
Q9Y5Y2_C72 1.7 -- -- -- -- O15446_C86 -- -- 4.6 -- -- Q13630_C116 -- -- 20.0 -- -- Q8IYQ7_C324 -- -- -- -- -- P05091_C319 -- 0.9 20.0 -- -- Q29RF7_C532 -- 0.8 -- -- -- Q9Y570_C381 -- 0.2 -- -- -- Q14980_C961 2.6 -- -- -- -- P53384_C235 2.0 0.2 -- -- -- Q15003_C418 -- -- 20.0 -- 1.3 P53634_C258 -- -- -- -- -- Q8NFF5_C499 -- -- -- -- 1.3 Q9ULA0_C144 -- 0.8 -- -- 0.9 P22307_C94 -- -- 20.0 -- -- O15294_C620 -- 1.0 -- -- -- Q9Y5S2_C1517 -- 0.4 20.0 -- -- Q8TD19_C623 -- 0.6 -- -- -- Q8N2W9_C326 0.9 0.8 -- -- -- Q13158_C98 1.5 -- -- -- -- Q9UKX7_C151 -- 0.8 -- -- -- Q6PCB5_C280 -- -- -- -- -- P10398_C597 -- -- -- -- -- Q9UL40_C68 -- -- -- -- -- P46013_C903 1.1 1.0 20.0 1.1 -- Q16667_C39 -- -- -- -- -- O75150_C890 -- 1.3 -- -- -- Q00610_C870 20.0 -- -- -- -- Q9Y5T5_C205 1.5 -- -- -- -- O95881_C66 1.0 1.0 -- -- -- Q7Z5K2_C160 -- 1.0 -- -- -- P42166_C518 2.2 -- -- -- -- Q9Y2S7_C143 -- 1.2 -- -- 0.8 E2QRD5_C183 -- 0.5 -- -- -- O95833_C22 -- -- 13.8 -- -- O94953_C694 -- -- -- -- -- O00541_C272 -- 0.9 -- -- -- Q9NXJ5_C149 -- -- -- -- -- Q8N5L8_C131 -- -- 7.3 -- -- Q8IZ73_C246 1.7 -- -- -- -- Q99798_C385 -- 0.1 -- -- -- Q9GZR2_C382 -- -- 20.0 -- -- Q13613_C117 -- -- -- -- -- Q9NUI1_C22 -- -- 17.0 -- -- Q02556_C306 -- -- -- -- Q9UPT9_C171 -- -- -- -- -- Q8N999_C302 -- 0.3 20.0 -- -- Q8IU81_C363 1.3 -- 1.7 -- -- Q9C0I1_C152 -- -- -- -- -- Q9P2X3_C195 -- -- 20.0 -- -- Q6QNY0_C168 1.0 -- -- -- -- Q15796_C81 -- -- -- -- -- Q9NZB2_C531 -- -- -- -- 0.6 Q9HB90_C377 -- -- -- -- -- Q9BR61_C267 -- 0.5 -- -- -- Pl6455_C145 -- -- -- -- -- Q86UV5_C39 -- -- -- -- -- A2A288_C367 -- -- -- -- -- Q8NEC7_C140 -- -- 20.0 -- -- Q6PJG6_C673 -- -- -- -- 0.9 Q13232_C158 -- -- 8.8 -- -- Q86X76_C165 -- -- -- -- -- P42695_C541 -- -- -- -- -- P41226_C599 -- -- -- -- -- Q99986_C50 -- -- 4.1 -- -- Q8WUM4_C90 -- -- -- -- -- P29590_C213 -- -- -- -- -- Q9P0K7_C973 -- -- -- -- -- P53992_C78 -- -- -- -- -- Q13867_C73 -- -- -- -- -- Q8ND24_C655 -- -- -- -- -- Q96EK4_C48 -- -- -- -- -- Q96IV0_C309 -- -- -- -- -- Q5T1V6_C414 -- -- -- -- -- Q9UHQ1_C99 -- -- -- -- -- O43396_C34 -- -- -- -- -- Q8IV53_C174 -- -- -- -- -- Q8N9T8_C673 -- -- -- -- --
[0507] Table 3 illustrates a list of cysteine containing proteins and potential cysteine site of conjugation.
TABLE-US-00014 Cysteine Identifier Protein Name Location Protein Class O00170 AIP AH receptor-interacting protein C122 Uncategorized O00541 PES1 Pescadillo homolog C272; C361 Uncategorized O00622 CYR61 Protein CYR61 C39; C70; C134 Uncategorized O14920 IKBKB Inhibitor of nuclear factor kappa-B C464 Enzyme kinase subunit O14933 UBE2L6 Ubiquitin/ISG15-conjugating enzyme C98 Enzyme E2 L6 PCTK O14980 XPO1 Exportin-1 C34; C528; Uncategorized C1070 O75362 ZNF217 Zinc finger protein 217 C286 Transcription factors and regulators O94953 KDM4B Lysine-specific demethylase 4B C694 Enzyme P00813 ADA Adenosine deaminase C75 Enzyme P04150 NR3C1 Glucocorticoid receptor C302; C622 Transcription factors and regulators P09086 POU2F2 POU domain, class 2, transcription C346 Transcription factor 2 factors and regulators P09211 GSTP1 Glutathione S-transferase P C48 Enzyme P14598 NCF1 Neutrophil cytosol factor 1 C378 Adapter, scaffolding, modulator proteins P15374 UCHL3 Ubiquitin carboxyl-terminal hydrolase C95 Enzyme isozyme L3 P16455 MGMT Methylated-DNA--protein-cysteine C145; C150 Enzyme methyltransferase P17812 CTP synthase 1 C491 Enzyme P19447 ERCC3 TFIIH basal transcription factor C342 Enzyme complex helicase P21580 TNFAIP3 Tumor necrosis factor alpha-induced C54 Enzyme protein 3 P24752 ACAT1 Acetyl-CoA acetyltransferase, C119; C126; Enzyme mitochondrial C196; C413 P40261 Nicotinamide N-methyltransferase C165 Enzyme P40763 STAT3 Signal transducer and activator of C259 Transcription transcription 3 factors and regulators P41226 UBA7 Ubiquitin-like modifier-activating C599 Enzyme enzyme 7 P42575 CASP2 Caspase-2 C370 Enzyme P43403 ZAP70 Tyrosine-protein kinase ZAP-70 C117 Enzyme P48200 IREB2 Iron-responsive element-binding C137 Transcription protein 2 factors and regulators P48735 IDH2 Isocitrate dehydrogenase C308 Enzyme P50851 LRBA Lipopolysaccharide-responsive and C1704; C2675 Uncategorized beige-like anchor protein P51617 IRAK1 Interleukin-1 receptor-associated C608 Enzyme kinase 1 P61081 NEDD8-conjugating enzyme Ubc12 C47 Enzyme P61088 Ubiquitin-conjugating enzyme E2 N C87 Enzyme P63244 GNB2L1 Guanine nucleotide-binding protein C182 Channels, subunit beta-2-like 1 Transporters, Receptors P68036 UBE2L3 Ubiquitin-conjugating enzyme E2 L3 C86 Enzyme Q00535 CDK5 Cyclin-dependent kinase 5 C157 Enzyme Q01201 RELB Transcription factor RelB C109 Transcription factors and regulators Q02556 IRF8 Interferon regulatory factor 8 C306 Transcription factors and regulators Q04759 PRKCQ Protein kinase C theta type C14; C17 Enzyme Q06124 Tyrosine-protein phosphatase non-receptor C573 Enzyme type 11 Q09472 EP300 Histone acetyltransferase p300 C1738 Enzyme Q14790 CASP8 Caspase-8 C360 Enzyme Q15084 PDIA6 Protein disulfide-isomerase A6 C55; C58; C190; Enzyme C193 Q15306 IRF4 Interferon regulatory factor 4 C194 Transcription factors and regulators Q15910 EZH2 Histone-lysine N-methyltransferase C503 Enzyme EZH2 Q16186 Proteasomal ubiquitin receptor ADRM1 C88 Channels, Transporters, Receptors Q16763 UBE2S Ubiquitin-conjugating enzyme E2 S C118 Enzyme Q16822 PCK2 Phosphoenolpyruvate carboxykinase C306 Enzyme Q16875 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase C155 Enzyme 3 Q16877 PFKFB4 6-phosphofructo-2-kinase/fructose- C159 Enzyme 2,6-bisphosphata Q6L8Q7 PDE12 2,5-phosphodiesterase 12 C108 Enzyme Q70CQ2 USP34 Ubiquitin carboxyl-terminal hydrolase C741; C1090 Enzyme 34 Q7Z2W4 ZC3HAV1 Zinc finger CCCH-type antiviral C645 Transcription protein 1 factors and regulators Q86UV5 USP48 Ubiquitin carboxyl-terminal hydrolase C39 Enzyme 48 Q8TAQ2 SMARCC2 SWI/SNF complex subunit C145 Transcription SMARCC2 factors and regulators Q92851 Caspase-10 C401 Enzyme Q93009 USP7 Ubiquitin carboxyl-terminal hydrolase 7 C223; C315 Enzyme Q96FA3 PELI1 E3 ubiquitin-protein ligase pellino C282 Enzyme homolog 1 Q96GG9 DCUN1D1 DCN1-like protein 1 C115 Uncategorized Q96JH7 VCPIP1 Deubiquitinating protein VCIP135 C219 Enzyme Q96RU2 USP28 Ubiquitin carboxyl-terminal hydrolase C171; C733 Enzyme 28 Q99873 PRMT1 Protein arginine N-methyltransferase C109 Enzyme 1 Q9C0C9 UBE2O Ubiquitin-conjugating enzyme E2 O C375 Enzyme Q9HB90 RRAGC Ras-related GTP-binding protein C C358; C377 Channels, transporters, and receptors Q9NRW4 Dual specificity protein phosphatase 22 C124 Enzyme Q9NWZ3 IRAK4 Interleukin-1 receptor-associated C13 Enzyme kinase 4 Q9NYL2 MLTK Mitogen-activated protein kinase C22 Enzyme kinase kinase MLT Q9UPT9 USP22 Ubiquitin carboxyl-terminal hydrolase C44; C171 Enzyme 22 Q9Y3Z3 SAMHD1 SAM domain and HD domain-containing C522 Enzyme protein 1 Q9Y4C1 KDM3A Lysine-specific demethylase 3A C251 Enzyme Q9Y5T5 USP16 Ubiquitin carboxyl-terminal hydrolase C205 Enzyme 16
[0508] Table 4 shows representative cysteines with known covalent ligands targeted by fragment electrophiles in isoTOP-ABPP experiments.
TABLE-US-00015 Other cysteines Previous Liganded quantified by covalent Cysteine Protein cysteine Fragment(s) isoTOP-ABPP inhibitor(s) location BTK C481 2, 3, 14, 31 C145, C337 Ibrutinib Active site TGM2 C277 12, 14, 32 C10, C27, C230, 18d Active Site C269, C290, C336, C370, C524, C545, C620 Map2k7 C131 2, 3, 11, 14, 20, C260, C280 Ibrutinib Active Site 21, 38 XPO1 C528 2, 3, 5, 14, 24, C34, C119, C164, KPT-330 Non-active 31, 43, 56 C199, C327, C498, site C723, C1070 Casp5 C315 3, 50 -- Z-WEHD-CHO/FMK Active Site ("WEHD" disclosed as SEQ ID NO: 863) Casp8 C360 2, 4, 11 C236, C409 Z-VAD-FMK, Active Site CV8/9-AOMK ERCC3 C342 2, 3, 5, 8, 14, 21 -- Triptolide Active Site Park 7 C106 2, 9, 8, 11, 13, , C46, C53 WRR-086 Active Site (Toxoplasma 43, 45, 50, 52 DJ-1) GSTO1 C32 2-13, 16, 18-22, C90, C192, C237 KT53 Active Site 33, 27-30, 32-34, 36, 39, 43, 49, 50, 52, 54, 55 ALDH2 C319 3, 8-10, 12, 27, C66, C179, C386, Disulfiram Active Site 28, 32, 39, 40, C472 43, 49, 50 CTSZ C92 4, 11, 20, 28, 32 C89, C126, C132, Cy5DCG04 Active Site C154, C170, C173, C179, C214
[0509] Table 5 shows Reactive docking results for liganded cysteines.
TABLE-US-00016 Most ligandable Most ligandable cysteine by Cysteine cysteine by Protein PDB ID: docking location isoTOP-ABPP Match Aldh2 1O05 C319 Active site C319 Yes BTK 1K2P C481 Active site C481 Yes CASP8 1QTN C360 Active Site C360 Yes CCNB1 2JGZ C238 Non-active site C238 Yes CDKN3 1FQ1 C39 Non-active site C39 Yes CLIC4 2AEH C35 Non-active site C35 Yes DTYMK 1E2G C163 Non-active site C163 Yes IDH1 3MAP C269 Non-active site C269 Yes IMPDH2 1NF7 C331 Active site C331, C140 Yes GLRX5 2WUL C67 Active site C67 Yes GSTO1 1EEM C32 Active site C32 Yes NME3 1ZS6 C158 Non-active site C158 Yes PKM 4JPG C423 Non-active site C423 Yes SRC 2SRC C277 Active Site C277 Yes TIGAR 3DCY C114 Non-active site C114, C161 Yes TXNDC 1WOU C43 Active site C43 Yes UGDH 3ITK C276 Active site C276 Yes UPP1 3EUF C162 Non-active site C162 Yes XPO1 3GB8 C528 Non-active site C528 Yes CDK5 1UNG C157 Non-active site C269 Second EDC3 3D3K C311 Non-active site C137, C413, C499 Second NR2F2 3CJW C213 Non-active site C326, C213 (in situ) Second PDCD6IP 2R02 C231 Non-active site C90 Second PRMT1 1ORI C285 Active site C109 Second UBE2S 1ZDN C118 Non-active site C95 Second FNBP1 2EFL C145 Non-active site C70 No HAT1 2P0W C120 Non-active site C101 No MAPK9 3NPC C163 Active site C177 No STAT1 1YVL C543 Non-active site C492, C255 No
[0510] Table 6 shows site of fragment labeling for recombinant proteins. The underlines portion indicates the fragment-modified cysteines.
TABLE-US-00017 M + H M + H SEQ calculated observed Protein Cysteine Fragment # Peptide ID NO: (m/z) (m/z) Charge IMPDH2 C140 14 R.HGFCGIPITDTGR.M 45 715.86 715.86 2 TIGAR C114 5 R.EECPVFTPPGGETLDQVK.M 143 1123.97 1123.97 2 CASP8 C360 7 K.VFFIQACQGDNYQK.G 335 660.98 660.98 3 IDH1 C269 20 K.SEGGFIWACK.N 260 702.84 702.84 2
[0511] Table 7 illustrates a list of DMF-sensitive Cys residues in human T cells, defined as Cys residues that showed R values (DMSO/DMF)>4 in isoTOP-ABPP experiments comparing DMSO-versus DMF-treated T cells.
TABLE-US-00018 Conserved Role in Name Full name Protein function Residue in mice immunology ADA Adenosine Adenosine C75 yes Positive regulator deaminase deaminase of T cell co-activation AGFG2 Arf-GAP domain GTPase activator C39 yes Unknown and FG repeat- containing protein 2 AIP AH receptor- Transcription factor C122 yes Unknown interacting protein binding CRKL Crk-like protein Poly(A) RNA C249 yes Unknown binding FLII Protein flightless-1 Actin binding C46 yes Unknown homolog GAK Cyclin-G-associated Serine/threonine C87 yes Unknown kinase protein kinase HUWE1 E3 ubiquitin-protein E3 ubiquitin- C3372 yes Unknown ligase HUWE1 protein ligase IKBKB Inhibitor of nuclear Serine kinase C464 yes Phosphorylates factor kappa-B IkB-.alpha. in NF-.kappa.B kinase subunit pathway 1L16 Pro-interleukin-16 Cytokine C1004 yes Influences migration of CD4+ lymphocytes IRF4 Interferon DNA binding C194 yes Regulates regulatory factor 4 dendritic cell and B cell development, as well as T/B cell differentiation IRF8 Interferon DNA binding C306 yes Plays a negative regulatory factor 8 regulatory role in immune cells. Binds to upstream regulatory region of MHC class I genes. Regulates the development and differentiation of myeloid cells. KIAA0528 Uncharacterized Calcium-dependent C993 yes Unknown protein phospholipid binding LAS1L Ribosomal Poly(A) RNA C456 yes Unknown biogenesis protein binding MARS2 Methionine--tRNA Methionine-tRNA C425 yes Unknown ligase, ligase mitochondrial MAT2A S-adenosylmethionine Methionine C56 yes Unknown synthase isoform adenosyltransferase type-2 MAT2A S-adenosylmethionine Methionine C104 yes Unknown synthase isoform adenosyltransferase type-2 MTCH2 Mitochondrial Induces C296 yes Unknown carrier homolog 2 mitochondrial depolarization PGP Phosphoglycolate Phosphatase C297 yes Unknown phosphatase PML Protein RNA/DNA binding C479 yes Modulates TGF- Promyelocytic beta signaling, leukemia induced by interferon to promote antiviral responses PRKCQ Protein kinase C Serine/threonine C14 yes Promotes TCR theta type protein kinase signaling through activation of NF- .kappa.B and other transcription factors PYGB Glycogen Phosphorylase C326 yes Unknown phosphorylase, brain form RARS Arginine--tRNA tRNA binding C32 yes Unknown ligase, cytoplasmic SON Protein SON RNA/DNA binding C92 yes Unknown SYNE2 Nesprin-2 Actin binding C553 yes Unknown TDRKH Tudor and KH RNA binding C109 yes Unknown domain-containing protein THNSL1 Threonine synthase- Threonine synthase C324 yes Unknown like 1 THOC1 THO complex RNA/DNA binding C49 yes Unknown subunit 1 TNFAIP3 Tumor necrosis Ubiquitin-specific C54 yes Inhibits NF-.kappa.B factor alpha-induced protease signaling upon protein 3 TCR-mediated T cell activation UBR4 E3 ubiquitin-protein Ubiquitin ligase C2554 yes Unknown ligase USP7 Ubiquitin carboxyl- Ubiquitin-specific C315 yes Deubiquitinates terminal hydrolase 7 protease FOXP3, increasing Treg suppressive capacity VDAC3 Voltage-dependent Mitochondrial C65 yes Unknown anion-selective outer membrane channel protein channel VDAC3 Voltage-dependent Voltage-gated C36 yes Unknown anion-selective anion channel channel protein ZC3HAV1 Zinc finger CCCH- Poly(A) RNA C645 yes Inhibits viral type antiviral binding replication protein 1 ZNF346 Zinc finger protein RNA binding C68 yes Unknown 346 AARS Alanine--tRNA Alanine-tRNA C773 no Unknown ligase, cytoplasmic ligase APOBEC3C Probable DNA dC- Cytidine deaminase C130 no Inhibits retrovirus dU- editing enzyme replication BCL2A1 Bcl-2-related Scaffolding protein C55 no Expression protein A1 induced by inflammatory cytokines BCL2A1 Bcl-2-related Scaffolding protein C19 no Unknown protein A1 CHRAC1 Chromatin Chromatin C55 no Unknown accessibility remodeling complex protein 1 DCXR L-xylulose Xylulose reductase C244 no Unknown reductase GHDC GH3 domain- Uncharacterized C502 no Unknown containing protein IRAK4 Interleukin-1 Serine/threonine C13 no Helps initiate receptor-associated protein kinase innate immune kinase 4 response by promoting ubiquitination of IRAK1 upon TLR activation. Also implicated in T cell activation NADSYN1 Glutamine- NAD(+) synthase C428 no Unknown dependent NAD(+) synthetase PGLS 6-phospho- Hydrolysis of 6- C32 no Unknown gluconolactonase phosphogluconolactone PRKDC DNA-dependent Serine/threonine C4045 no Regulates DNA protein kinase protein kinase damage response, catalytic subunit involved in V(D)J recombination PUSL1 tRNA pseudouridine Pseudouridine C292 no Unknown synthase-like 1 synthase RIN3 Ras and Rab GTPase activator C942 no Unknown interactor 3 SCLY Selenocysteine lyase Selenocysteine C22 no Unknown lyase SPCS2 Signal peptidase Peptidase C17 no Unknown complex subunit 2 TRNT1 CCA tRNA tRNA binding C373 no Mutations lead to nucleotidyltransferase B-cell 1, mitochondrial immunodeficiency as well as progressive reductions in T and NK cells (OMIM number 616084) TUBGCP3 Gamma-tubulin Gamma-tubulin C194 no Unknown complex component binding 3 UBE2L6 Ubiquitin/ISG 15- Ubiquitin- C98 no Acts as an E2 conjugating enzyme conjugating enzyme for an E2 L6 enzyme IFN-induced ubiquitin-like protein
[0512] Table 8 illustrates an exemplary list of DMF sensitive cysteine-containing proteins in human T cell targets. Table 8 further shows the accession number (or the protein identifier) of the protein.
TABLE-US-00019 SEQ ID DMF_50 DMF_50 DMF_50 DMF_25 DMF_10 MMF_50 Identifier Protein Name NO: .mu.M_4 h .mu.M_2 h .mu.M_1 h .mu.M_4 h .mu.M_4h .mu.M_4 h Q9NRW3_C130 APOBEC3C Probable DNA dC- 805 20 20 8.6 -- 1.92 1.48 dU-editing enzyme APOBEC-3C Q9NWZ3_C13 IRAK4 Interleukin-1 receptor- 806 20 -- 8.3 -- -- 1.48 associated kinase 4 Q9Y2W6_C109 TDRKH Tudor and KH domain- 807 20 20 4 -- 2.34 1.36 containing protein Q6IA69_C428 NADSYN1 Glutamine-dependent 808 20 2.31 1.81 -- 1.43 1.33 NAD(+) synthetase O14920_C464 IKBKB Inhibitor of nuclear factor 809 20 10.12 3.96 -- 2.59 -- kappa-B kinase subunit P00513_C75 ADA Adenosine deaminase 810 20 5.08 2.51 -- 2.29 -- Q9Y277_C65 VDAC3 Voltage-dependent anion- 811 15.94 7.53 3.35 5.64 1.73 1.39 selective channel protein P49588_C773 AARS Alanine-t-RNA ligase, 812 12.75 10.16 9.34 -- 2.84 1.24 cytoplasmic O14933_C98 UBE2L6 Ubiquitin/ISG15- 813 12.55 2.92 2.44 -- 1.49 1.7 conjugating enzyme E2 L6 O95336_C32 PGLS 6-phosphogluconolactonase 814 11.51 9.49 3.42 5.32 1.9 1.26 A6NDG6_C297 PGP Phosphoglycolate phosphatase 815 10.77 4.21 3.06 -- -- 1.52 Q7Z6Z7_C3372 HUWE1 E3 ubiquitin-protein ligase 816 10.48 4.43 2.28 -- 1.58 1.2 HUWE1 Q16548_C55 BCL2A1 Bcl-2-related protein A1 817 7.18 -- -- -- -- 0.97 P11216_C326 PYGB Glycogen phosphorylase, 818 6.76 3.73 2.47 3.53 1.65 1.29 brain form O95081_C39 AGFG2 Arf-GAP domain and FG 819 6.39 3.85 -- -- 1.42 1.24 repeat-containing protein 2 Q7Z2W4_C645 ZC3HAV1 Zinc finger CCCH-type 820 6.28 3.13 2.36 2.52 1.46 1.3 antiviral protein 1 O00170_C122 AIP AH receptor-interacting protein 821 6.14 3.05 -- -- -- 1.24 Q96Q11_C373 TRNT1 CCA tRNA 822 5.83 2.66 1.97 -- -- 1.29 nucleotidyltransferase 1, mitochondrial Q8TB24_C942 RIN3 Ras and Rab interactor 3 823 5.7 3 -- -- -- 1.23 Q9Y4W2_C456 LAS1L Ribosomal biogenesis 824 5.61 3.42 1.8 -- 1.29 1.14 protein LAS1L Q02556_C306 IRF8 Interferon regulatory factor 8 825 5.32 -- 1.66 -- 1.9 -- Q96GW9_C425 MARS2 Methionine--tRNA ligase, 826 5.3 4.16 2.23 2.86 1.84 1.3 mitochondrial A Q15306_C194 IRF4 Interferon regulatory factor 4 827 5.25 3.13 1.32 1.78 1.69 1.33 Q15005_C17 SPCS2 Signal peptidase complex 828 5.09 3.86 2.25 2.41 1.42 1.32 subunit 2 P54136_C32 RARS Arginine--tRNA ligase, cytoplasmic 829 5.02 3.58 2.58 4.03 0.62 1.78 Q96CW5_C194 TUBGCP3 Gamma-tubulin complex 830 4.94 2.44 -- -- -- -- component 3 P46109_C249 CRKL Crk-like protein 831 4.86 3.21 2.21 -- 1.38 1.27 Q8N0Z8_C292 PUSL1 tRNA pseudouridine 832 4.68 -- -- -- -- 1.36 synthase-like 1 Q5T4S7_C2554 UBR4 E3 ubiquitin-protein ligase 833 4.63 2.1 1.6 -- 1.52 1.2 UBR4 Q9UL40_C68 ZNF346 Zinc finger protein 346 834 4.6 3.91 2.5 -- 1.98 1.26 Q13045_C46 FLII Protein flightless-1 homolog 835 4.5 3.57 2.05 -- 1.55 1.27 Q86YS7_C993 KIAA0528 Uncharacterized protein 836 4.38 -- -- -- 1.4 1.4 KIAA0528 Q9Y6C9_C296 MITCH2 Mitochondrial carrier 837 4.3 2.44 1.81 -- 1.68 1.35 homolog 2 Q7Z4W1_C244 DCXR L-xylulose reductase 838 4.24 2.76 1.29 -- 2.3 -- Q04759_C14 PRKCQ Protein kinase C theta type 839 4.21 2.92 -- 3.29 1.62 1.14 P18583_C92 SON Protein SON 840 4.17 6.31 2.5 -- -- 1.31 P31153_C56 MAT2A S-adenosylmethionine 841 4.17 -- -- -- -- -- synthase isoform type-2 Q16548_C19 BCL2A1 Bcl-2-related protein A1 842 4.16 2.09 -- 2.19 1.15 1.28 Q14005_C1004 IL16 Pro-interleukin-16 843 4.13 3.32 1.95 -- 1.37 1.31 P31153_C104 MAT2A S-adenosylmethionine 844 4.11 -- -- -- 1.5 1.31 synthase isoform type-2 Q9Y277_C36 VDAC3 Voltage-dependent anion- 845 4.11 3.98 3.21 -- -- 1.18 selective channel protein Q8WXH0_C553 SYNE2 Nesprin-2 846 4.05 3.29 -- -- 1.61 -- Q96I15_C22 SCLY Selenocysteine lyase 847 4.04 2.16 1.9 2.16 1.31 1.27 P29590_C479 PML Protein PML 848 -- 4.57 -- 2.17 2.1 1.27 Q8IYQ7_C324 THNSL1 Threonine synthase-like 1 849 -- 19.36 15.93 -- -- 1.4 Q93009_C315 USP7 Ubiquitin carboxvy-terminal 850 -- 14.06 5.33 -- 1.9 1.4 hydrolase 7 P21580_C54 TNFAIP3 Tumor necrosis factor 851 -- 5.34 -- -- 1.58 -- alpha-induced protein 3 O14976_C87 GAK Cyclin-G-associated kinase 852 -- 4.79 -- -- 1.36 -- Q96FV9_C49 THOC1 THO complex subunit 1 853 -- 5.7 3.93 -- -- 0.97 ki 854 -- 10.53 4.14 -- -- 1.23 Q9NRG0_C55 CHRAC1 Chromatin accessibility 855 -- 11.72 12.59 -- 5.07 1.27 complex protein 1 Q8N2G8_C502 GHDC GH3 domain-containing 856 -- 20 4.23 -- -- -- protein
[0513] Table 9 illustrates the full protein sequence of exemplary cysteine-containing proteins described herein. The cysteine residue of interest is denoted with (*).
TABLE-US-00020 Protein Cysteine Identifier Residue SEQ (Accession No.) Protein Name Number Sequence ID NO: O75874 Isocitrate C269 MSKKISGGSV VEMQGDEMTR IIWELIKEKL 1 dehydrogenase IFPYVELDLH SYDLGIENRD ATNDQVTKDA 1 (IDH1) AEAIKKHNVG VKCATITPDE KRVEEFKLKQ MWKSPNGTIR NILGGTVFRE AIICKNIPRL VSGWVKPIII GRHAYGDQVR ATDFVVPGPG KVEITYTPSD GTQKVTYLVH NFEEGGGVAM GMYNQDKSIE DFAHSSFQMA LSKGWPLYLS TKNTILKKYD GRFKDIFQEI YDKQYKSQFE AQKIWYEHRL IDDMVAQAMK SEGGFIWAC*K NYDGDVQSDS VAQGYGSLGM MTSVLVCPDG KTVEAEAAHG TVTRHYRMYQ KGQETSTNPI ASIFAWTRGL AHRAKLDNNK ELAFFANALE EVSIETIEAG FMTKDLAACI KGLPNVQRSD YLNTFEFMDK LGENLKIKLA QAKL P48735 Isocitrate C308 MAGYLRVVRS LCRASGSRPA WAPAALTAPT 2 dehydrogenase SQEQPRRHYA DKRIKVAKPV VEMDGDEMTR 2 (IDH2) IIWQFIKEKL ILPHVDIQLK YFDLGLPNRD QTDDQVTIDS ALATQKYSVA VKCATITPDE ARVEEFKLKK MWKSPNGTIR NILGGTVFRE PIICKNIPRL VPGWTKPITI GRHAHGDQYK ATDFVADRAG TFKMVFTPKD GSGVKEWEVY NFPAGGVGMG MYNTDESISG FAHSCFQYAI QKKWPLYMST KNTILKAYDG RFKDIFQEIF DKHYKTDFDK NKIWYEHRLI DDMVAQVLKS SGGFVWAC*KN YDGDVQSDIL AQGFGSLGLM TSVLVCPDGK TIEAEAAHGT VTRHYREHQK GRPTSTNPIA SIFAWTRGLE HRGKLDGNQD LIRFAQMLEK VCVETVESGA MTKDLAGCIH GLSNVKLNEH FLNTTDFLDT IKSNLDRALG RQ Q14790 CASP8 C360 MDFSRNLYDI GEQLDSEDLA SLKFLSLDYI 3 PQRKQEPIKD ALMLFQRLQE KRMLEESNLS FLKELLFRIN RLDLLITYLN TRKEEMEREL QTPGRAQISA YRVMLYQISE EVSRSELRSF KFLLQEEISK CKLDDDMNLL DIFIEMEKRV ILGEGKLDIL KRVCAQINKS LLKIINDYEE FSKERSSSLE GSPDEFSNGE ELCGVMTISD SPREQDSESQ TLDKVYQMKS KPRGYCLIIN NHNFAKAREK VPKLHSIRDR NGTHLDAGAL TTTFEELHFE IKPHDDCTVE QIYEILKIYQ LMDHSNMDCF ICCILSHGDK GIIYGTDGQE APIYELTSQF TGLKCPSLAG KPKVFFIQAC* QGDNYQKGIP VETDSEEQPY LEMDLSSPQT RYIPDEADFL LGMATVNNCV SYRNPAEGTW YIQSLCQSLR ERCPRGDDIL TILTEVNYEV SNKDDKKNMG KQMPQPTFTL RKKLVFPSD Q92851 CASP10 C401 MKSQGQHWYS SSDKNCKVSF REKLLIIDSN 4 LGVQDVENLK FLCIGLVPNK KLEKSSSASD VFEHLLAEDL LSEEDPFFLA ELLYIIRQKK LLQHLNCTKE EVERLLPTRQ RVSLFRNLLY ELSEGIDSEN LKDMIFLLKD SLPKTEMTSL SFLAFLEKQG KIDEDNLTCL EDLCKTVVPK LLRNIEKYKR EKAIQIVTPP VDKEAESYQG EEELVSQTDV KTFLEALPQE SWQNKHAGSN GNRATNGAPS LVSRGMQGAS ANTLNSETST KRAAVYRMNR NHRGLCVIVN NHSFTSLKDR QGTHKDAEIL SHVFQWLGFT VHIHNNVTKV EMEMVLQKQK CNPAHADGDC FVFCILTHGR FGAVYSSDEA LIPIREIMSH FTALQCPRLA EKPKLFFIQA C* QGEEIQPSV SIEADALNPE QAPTSLQDSI PAEADFLLGL ATVPGYVSFR HVEEGSWYIQ SLCNHLKKLV PRMLKFLEKT MEIRGRKRTV WGAKQISATS LPTAISAQTP RPPMRRWSSV S Q99873 PRMT1 C109 MENFVATLAN GMSLQPPLEE VSCGQAESSE 5 KPNAEDMTSK DYYFDSYAHF GIHEEMLKDE VRTLTYRNSM FHNRHLFKDK VVLDVGSGTG ILCMFAAKAG ARKVIGIEC* S SISDYAVKIV KANKLDHVVT IIKGKVEEVE LPVEKVDIII SEWMGYCLFY ESMLNTVLYA RDKWLAPDGL IFPDRATLYV TAIEDRQYKD YKIHWWENVY GFDMSCIKDV AIKEPLVDVV DPKQLVTNAC LIKEVDIYTV KVEDLTFTSP FCLQVKRNDY VHALVAYFNI EFTRCHKRTG FSTSPESPYT HWKQTVFYME DYLTVKTGEE IFGTIGMRPN AKNNRDLDFT IDLDFKGQLC ELSCSTDYRM R Q9NYL2 MAP3 kinase C22 MSSLGASFVQ IKFDDLQFFE NC*GGGSFGSV 6 MLTK (or ZAK) YRAKWISQDK EVAVKKLLKI EKEAEILSVL SHRNIIQFYG VILEPPNYGI VTEYASLGSL YDYINSNRSE EMDMDHIMTW ATDVAKGMHY LHMEAPVKVI HRDLKSRNVV IAADGVLKIC DFGASRFHNH TTHMSLVGTF PWMAPEVIQS LPVSETCDTY SYGVVLWEML TREVPFKGLE GLQVAWLVVE KNERLTIPSS CPRSFAELLH QCWEADAKKR PSFKQIISIL ESMSNDTSLP DKCNSFLHNK AEWRCEIEAT LERLKKLERD LSFKEQELKE RERRLKMWEQ KLTEQSNTPL LPSFEIGAWT EDDVYCWVQQ LVRKGDSSAE MSVYASLFKE NNITGKRLLL LEEEDLKDMG IVSKGHIIHF KSAIEKLTHD YINLFHFPPL IKDSGGEPEE NEEKIVNLEL VFGFHLKPGT GPQDCKWKMY MEMDGDEIAI TYIKDVTFNT NLPDAEILKM TKPPFVMEKW IVGIAKSQTV ECTVTYESDV RTPKSTKHVH SIQWSRTKPQ DEVKAVQLAI QTLFTNSDGN PGSRSDSSAD CQWLDTLRMR QIASNTSLQR SQSNPILGSP FFSHFDGQDS YAAAVRRPQV PIKYQQITPV NQSRSSSPTQ YGLTKNFSSL HLNSRDSGFS SGNTDTSSER GRYSDRSRNK YGRGSISLNS SPRGRYSGKS QHSTPSRGRY PGKFYRVSQS ALNPHQSPDF KRSPRDLHQP NTIPGMPLHP ETDSRASEED SKVSEGGWTK VENRKKPHRP SPAKTNKERA RGDHRGWRNF P12268 IMPDH2 C140, C331 MADYLISGGT SYVPDDGLTA QQLFNCGDGL 7 TYNDFLILPG YIDFTADQVD LTSALTKKIT LKTPLVSSPM DTVTEAGMAI AMALTGGIGF IHHNCTPEFQ ANEVRKVKKY EQGFITDPVV LSPKDRVRDV FEAKARHGFC* GIPITDTGRM GSRLVGIISS RDIDFLKEEE HDCFLEEIMT KREDLVVAPA GITLKEANEI LQRSKKGKLP IVNEDDELVA IIARTDLKKN RDYPLASKDA KKQLLCGAAI GTHEDDKYRL DLLAQAGVDV VVLDSSQGNS IFQINMIKYI KDKYPNLQVI GGNVVTAAQA KNLIDAGVDA LRVGMGSGSI C*ITQEVLACG RPQATAVYKV SEYARRFGVP VIADGGIQNV GHIAKALALG ASTVMMGSLL AATTEAPGEY FFSDGIRLKK YRGMGSLDAM DKHLSSQNRY FSEADKIKVA QGVSGAVQDK GSIHKFVPYL IAGIQHSCQD IGAKSLTQVR AMMYSGELKF EKRTSSAQVE GGVHSLHSYE KRLF Q9NQ88 TIGAR C114, C161 MARFALTVVR HGETRFNKEK IIQGQGVDEP 8 LSETGFKQAA AAGIFLNNVK FTHAFSSDLM RTKQTMHGIL ERSKFCKDMT VKYDSRLRER KYGVVEGKAL SELRAMAKAA REEC*PVFTPP GGETLDQVKM RGIDFFEFLC QLILKEADQK EQFSQGSPSN C*LETSLAEIF PLGKNHSSKV NSDSGIPGLA ASVLVVSHGA YMRSLFDYFL TDLKCSLPAT LSRSELMSVT PNTGMSLFII NFEEGREVKP TVQCICMNLQ DHLNGLTETR Q04759 PKC.theta. C14, C17 MSPFLRIGLS NFDC*GSC*QSC QGEAVNPYCA 9 VLNKEYVESE NGQMYIQKKP TMYPPWDSTF DAHINKGRVM QIIVKGKNVD LISETTVELY SLAERCRKNN GKTEIWLELK PQGRMLMNAR YFLEMSDTKD MNEFETEGFF ALHQRRGAIK QAKVHHVKCH EFTATFFPQP TFCSVCHEFV WGLNKQGYQC RQCNAAIHKK CIDKVIAKCT GSAINSRETM FHKERFKIDM PHRFKVYNYK SPTFCEHCGT LLWGLARQGL KCDACGMNVH HRCQTKVANL CGINQKLMAE ALAMIESTQQ ARCLRDTEQI FREGPVEIGL PCSIKNEARP PCLPTPGKRE PQGISWESPL DEVDKMCHLP EPELNKERPS LQIKLKIEDF ILHKMLGKGS FGKVFLAEFK KTNQFFAIKA LKKDVVLMDD DVECTMVEKR VLSLAWEHPF LTHMFCTFQT KENLFFVMEY LNGGDLMYHI QSCHKFDLSR ATFYAAEIIL GLQFLHSKGI VYRDLKLDNI LLDKDGHIKI ADFGMCKENM LGDAKTNTFC GTPDYIAPEI LLGQKYNHSV DWWSFGVLLY EMLIGQSPFH GQDEEELFHS IRMDNPFYPR WLEKEAKDLL VKLFVREPEK RLGVRGDIRQ HPLFREINWE ELERKEIDPP FRPKVKSPFD CSNFDKEFLN EKPRLSFADR ALINSMDQNM FRNFSFMNPG MERLIS
[0514] Table 10A-Table 10E illustrate a list of cysteine containing proteins and potential cysteine site of conjugation separated by protein class. Table 10 .ANG. illustrates cysteine containing enzymes and potential cysteine conjugation site. Table 10B shows a list of cysteine containing transcription factors and regulators. Table 10C shows an exemplary list of cysteine containing channels, transporters and receptors. Table 10D illustrates an exemplary cysteine containing adapter, scaffolding, and modulator protein. Table 10E provides an exemplary list of uncategorized cysteine containing proteins.
TABLE-US-00021 TABLE 10A Cysteine Protein Identifier Protein Name Location Class O14920 IKBKB Inhibitor of nuclear factor kappa-B kinase subunit C464 Enzyme O14933 UBE2L6 Ubiquitin/ISG15-conjugating enzyme E2 L6 PCTK C98 Enzyme O94953 KDM4B Lysine-specific demethylase 4B C694 Enzyme P00813 ADA Adenosine deaminase C75 Enzyme P09211 GSTP1 Glutathione S-transferase P C48 Enzyme P15374 UCHL3 Ubiquitin carboxyl-terminal hydrolase isozyme L3 C95 Enzyme P16455 MGMT Methylated-DNA--protein-cysteine methyltransferase C145; Enzyme C150 P17812 CTP synthase 1 C491 Enzyme P19447 ERCC3 TFIIH basal transcription factor complex helicase C342 Enzyme P21580 TNFAIP3 Tumor necrosis factor alpha-induced protein 3 C54 Enzyme P24752 ACAT1 Acetyl-CoA acetyltransferase, mitochondrial C119; Enzyme C126; C196; C413 P40261 Nicotinamide N-methyltransferase C165 Enzyme P41226 UBA7 Ubiquitin-like modifier-activating enzyme 7 C599 Enzyme P42575 CASP2 Caspase-2 C370 Enzyme P43403 ZAP70 Tyrosine-protein kinase ZAP-70 C117 Enzyme P48735 IDH2 Isocitrate dehydrogenase C308 Enzyme P51617 IRAKI Interleukin-1 receptor-associated kinase 1 C608 Enzyme P61081 NEDD8-conjugating enzyme Ubc12 C47 Enzyme P61088 Ubiquitin-conjugating enzyme E2 N C87 Enzyme P68036 UBE2L3 Ubiquitin-conjugating enzyme E2 L3 C86 Enzyme Q00535 CDK5 Cyclin-dependent kinase 5 C157 Enzyme Q04759 PRKCQ Protein kinase C theta type C14; C17 Enzyme Q06124 Tyrosine-protein phosphatase non-receptor type 11 C573 Enzyme Q09472 EP300 Histone acetyltransferase p300 C1738 Enzyme Q14790 CASP8 Caspase-8 C360 Enzyme Q15084 PDIA6 Protein disulfide-isomerase A6 C55; C58; Enzyme C190; C193 Q15910 EZH2 Histone-lysine N-methyltransferase EZH2 C503 Enzyme Q16763 UBE2S Ubiquitin-conjugating enzyme E2 S C118 Enzyme Q16822 PCK2 Phosphoenolpyruvate carboxykinase C306 Enzyme Q16875 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 C155 Enzyme Q16877 PFKFB4 6-phosphofructo-2-kinase/fructose-2,6-bisphosphata C159 Enzyme Q6L8Q7 PDE12 2,5-phosphodiesterase 12 C108 Enzyme Q70CQ2 USP34 Ubiquitin carboxyl-terminal hydrolase 34 C741; Enzyme C1090 Q86UV5 USP48 Ubiquitin carboxyl-terminal hydrolase 48 C39 Enzyme Q92851 Caspase-10 C401 Enzyme Q93009 USP7 Ubiquitin carboxyl-terminal hydrolase 7 C223; Enzyme C315 Q96FA3 PELI1 E3 ubiquitin-protein ligase pellino homolog 1 C282 Enzyme Q96JH7 VCPIP1 Deubiquitinating protein VCIP135 C219 Enzyme Q96RU2 USP28 Ubiquitin carboxyl-terminal hydrolase 28 C171; Enzyme C733 Q99873 PRMT1 Protein arginine N-methyltransferase 1 C109 Enzyme Q9C0C9 UBE2O Ubiquitin-conjugating enzyme E2 O C375 Enzyme Q9NRW4 Dual specificity protein phosphatase 22 C124 Enzyme Q9NWZ3 IRAK4 Interleukin-1 receptor-associated kinase 4 C13 Enzyme Q9NYL2 MLTK Mitogen-activated protein kinase kinase kinase MLT C22 Enzyme Q9UPT9 USP22 Ubiquitin carboxyl-terminal hydrolase 22 C44; Enzyme C171 Q9Y3Z3 SAMHD1 SAM domain and HD domain-containing protein 1 C522 Enzyme Q9Y4C1 KDM3A Lysine-specific demethylase 3A C251 Enzyme Q9Y5T5 USP16 Ubiquitin carboxyl-terminal hydrolase 16 C205 Enzyme
TABLE-US-00022 TABLE 10B Cysteine Identifier Protein Name Location Protein Class O75362 ZNF217 Zinc finger protein 217 C286 Transcription factors and regulators P04150 NR3C1 Glucocorticoid receptor C302; C622 Transcription factors and regulators P09086 POU2F2 POU domain, class 2, transcription factor 2 C346 Transcription factors and regulators P40763 STAT3 Signal transducer and activator of transcription 3 C259 Transcription factors and regulators P48200 IREB2 Iron-responsive element-binding protein 2 C137 Transcription factors and regulators Q01201 RELB Transcription factor RelB C109 Transcription factors and regulators Q02556 IRF8 Interferon regulatory factor 8 C306 Transcription factors and regulators Q15306 IRF4 Interferon regulatory factor 4 C194 Transcription factors and regulators Q7Z2W4 ZC3HAV1 Zinc finger CCCH-type antiviral protein 1 C645 Transcription factors and regulators Q8TAQ2 SMARCC2 SWI/SNF complex subunit SMARCC2 C145 Transcription factors and regulators
TABLE-US-00023 TABLE 10C Cysteine Identifier Protein Name Location Protein Class P63244 GNB2L1 Guanine nucleotide-binding protein subunit C182 Channels, beta-2-like 1 Transporters, Receptors Q16186 Proteasomal ubiquitin receptor ADRM1 C88 Channels, Transporters, Receptors Q9HB90 RRAGC Ras-related GTP-binding protein C C358; C377 Channels, transporters, and receptors
TABLE-US-00024 TABLE 10D Cysteine Identifier Protein Name Location Protein Class P14598 NCF1 Neutrophil cytosol factor 1 C378 Adapter, scaffolding, modulator proteins
TABLE-US-00025 TABLE 10E Cysteine Identifier Protein Name Location Protein Class O00170 AIP AH receptor-interacting protein C122 Uncategorized O00541 PES1 Pescadillo homolog C272; C361 Uncategorized O00622 CYR61 Protein CYR61 C39; C70; C134 Uncategorized O14980 XPO1 Exportin-1 C34; C528; Uncategorized C1070 P50851 LRBA Lipopolysaccharide-responsive and beige- C1704; C2675 Uncategorized like anchor protein Q96GG9 DCUN1D1 DCN1-like protein 1 C115 Uncategorized
[0515] The examples and embodiments described herein are for illustrative purposes only and various modifications or changes suggested to persons skilled in the art are to be included within the spirit and purview of this application and scope of the appended claims.
Sequence CWU
1
1
8681414PRTHomo sapiensIsocitrate dehydrogenase 1 (IDH1); O75874
C269MOD_RES(269)..(269)Site of chemical conjugation 1Met Ser Lys Lys Ile
Ser Gly Gly Ser Val Val Glu Met Gln Gly Asp1 5
10 15Glu Met Thr Arg Ile Ile Trp Glu Leu Ile Lys
Glu Lys Leu Ile Phe 20 25
30Pro Tyr Val Glu Leu Asp Leu His Ser Tyr Asp Leu Gly Ile Glu Asn
35 40 45Arg Asp Ala Thr Asn Asp Gln Val
Thr Lys Asp Ala Ala Glu Ala Ile 50 55
60Lys Lys His Asn Val Gly Val Lys Cys Ala Thr Ile Thr Pro Asp Glu65
70 75 80Lys Arg Val Glu Glu
Phe Lys Leu Lys Gln Met Trp Lys Ser Pro Asn 85
90 95Gly Thr Ile Arg Asn Ile Leu Gly Gly Thr Val
Phe Arg Glu Ala Ile 100 105
110Ile Cys Lys Asn Ile Pro Arg Leu Val Ser Gly Trp Val Lys Pro Ile
115 120 125Ile Ile Gly Arg His Ala Tyr
Gly Asp Gln Tyr Arg Ala Thr Asp Phe 130 135
140Val Val Pro Gly Pro Gly Lys Val Glu Ile Thr Tyr Thr Pro Ser
Asp145 150 155 160Gly Thr
Gln Lys Val Thr Tyr Leu Val His Asn Phe Glu Glu Gly Gly
165 170 175Gly Val Ala Met Gly Met Tyr
Asn Gln Asp Lys Ser Ile Glu Asp Phe 180 185
190Ala His Ser Ser Phe Gln Met Ala Leu Ser Lys Gly Trp Pro
Leu Tyr 195 200 205Leu Ser Thr Lys
Asn Thr Ile Leu Lys Lys Tyr Asp Gly Arg Phe Lys 210
215 220Asp Ile Phe Gln Glu Ile Tyr Asp Lys Gln Tyr Lys
Ser Gln Phe Glu225 230 235
240Ala Gln Lys Ile Trp Tyr Glu His Arg Leu Ile Asp Asp Met Val Ala
245 250 255Gln Ala Met Lys Ser
Glu Gly Gly Phe Ile Trp Ala Cys Lys Asn Tyr 260
265 270Asp Gly Asp Val Gln Ser Asp Ser Val Ala Gln Gly
Tyr Gly Ser Leu 275 280 285Gly Met
Met Thr Ser Val Leu Val Cys Pro Asp Gly Lys Thr Val Glu 290
295 300Ala Glu Ala Ala His Gly Thr Val Thr Arg His
Tyr Arg Met Tyr Gln305 310 315
320Lys Gly Gln Glu Thr Ser Thr Asn Pro Ile Ala Ser Ile Phe Ala Trp
325 330 335Thr Arg Gly Leu
Ala His Arg Ala Lys Leu Asp Asn Asn Lys Glu Leu 340
345 350Ala Phe Phe Ala Asn Ala Leu Glu Glu Val Ser
Ile Glu Thr Ile Glu 355 360 365Ala
Gly Phe Met Thr Lys Asp Leu Ala Ala Cys Ile Lys Gly Leu Pro 370
375 380Asn Val Gln Arg Ser Asp Tyr Leu Asn Thr
Phe Glu Phe Met Asp Lys385 390 395
400Leu Gly Glu Asn Leu Lys Ile Lys Leu Ala Gln Ala Lys Leu
405 4102452PRTHomo sapiensIsocitrate
dehydrogenase 2 (IDH2); P48735 C308MOD_RES(308)..(308)Site of
chemical conjugation 2Met Ala Gly Tyr Leu Arg Val Val Arg Ser Leu Cys Arg
Ala Ser Gly1 5 10 15Ser
Arg Pro Ala Trp Ala Pro Ala Ala Leu Thr Ala Pro Thr Ser Gln 20
25 30Glu Gln Pro Arg Arg His Tyr Ala
Asp Lys Arg Ile Lys Val Ala Lys 35 40
45Pro Val Val Glu Met Asp Gly Asp Glu Met Thr Arg Ile Ile Trp Gln
50 55 60Phe Ile Lys Glu Lys Leu Ile Leu
Pro His Val Asp Ile Gln Leu Lys65 70 75
80Tyr Phe Asp Leu Gly Leu Pro Asn Arg Asp Gln Thr Asp
Asp Gln Val 85 90 95Thr
Ile Asp Ser Ala Leu Ala Thr Gln Lys Tyr Ser Val Ala Val Lys
100 105 110Cys Ala Thr Ile Thr Pro Asp
Glu Ala Arg Val Glu Glu Phe Lys Leu 115 120
125Lys Lys Met Trp Lys Ser Pro Asn Gly Thr Ile Arg Asn Ile Leu
Gly 130 135 140Gly Thr Val Phe Arg Glu
Pro Ile Ile Cys Lys Asn Ile Pro Arg Leu145 150
155 160Val Pro Gly Trp Thr Lys Pro Ile Thr Ile Gly
Arg His Ala His Gly 165 170
175Asp Gln Tyr Lys Ala Thr Asp Phe Val Ala Asp Arg Ala Gly Thr Phe
180 185 190Lys Met Val Phe Thr Pro
Lys Asp Gly Ser Gly Val Lys Glu Trp Glu 195 200
205Val Tyr Asn Phe Pro Ala Gly Gly Val Gly Met Gly Met Tyr
Asn Thr 210 215 220Asp Glu Ser Ile Ser
Gly Phe Ala His Ser Cys Phe Gln Tyr Ala Ile225 230
235 240Gln Lys Lys Trp Pro Leu Tyr Met Ser Thr
Lys Asn Thr Ile Leu Lys 245 250
255Ala Tyr Asp Gly Arg Phe Lys Asp Ile Phe Gln Glu Ile Phe Asp Lys
260 265 270His Tyr Lys Thr Asp
Phe Asp Lys Asn Lys Ile Trp Tyr Glu His Arg 275
280 285Leu Ile Asp Asp Met Val Ala Gln Val Leu Lys Ser
Ser Gly Gly Phe 290 295 300Val Trp Ala
Cys Lys Asn Tyr Asp Gly Asp Val Gln Ser Asp Ile Leu305
310 315 320Ala Gln Gly Phe Gly Ser Leu
Gly Leu Met Thr Ser Val Leu Val Cys 325
330 335Pro Asp Gly Lys Thr Ile Glu Ala Glu Ala Ala His
Gly Thr Val Thr 340 345 350Arg
His Tyr Arg Glu His Gln Lys Gly Arg Pro Thr Ser Thr Asn Pro 355
360 365Ile Ala Ser Ile Phe Ala Trp Thr Arg
Gly Leu Glu His Arg Gly Lys 370 375
380Leu Asp Gly Asn Gln Asp Leu Ile Arg Phe Ala Gln Met Leu Glu Lys385
390 395 400Val Cys Val Glu
Thr Val Glu Ser Gly Ala Met Thr Lys Asp Leu Ala 405
410 415Gly Cys Ile His Gly Leu Ser Asn Val Lys
Leu Asn Glu His Phe Leu 420 425
430Asn Thr Thr Asp Phe Leu Asp Thr Ile Lys Ser Asn Leu Asp Arg Ala
435 440 445Leu Gly Arg Gln
4503479PRTHomo sapiensCASP8; Q14790 C360MOD_RES(360)..(360)Site of
chemical conjugation 3Met Asp Phe Ser Arg Asn Leu Tyr Asp Ile Gly Glu Gln
Leu Asp Ser1 5 10 15Glu
Asp Leu Ala Ser Leu Lys Phe Leu Ser Leu Asp Tyr Ile Pro Gln 20
25 30Arg Lys Gln Glu Pro Ile Lys Asp
Ala Leu Met Leu Phe Gln Arg Leu 35 40
45Gln Glu Lys Arg Met Leu Glu Glu Ser Asn Leu Ser Phe Leu Lys Glu
50 55 60Leu Leu Phe Arg Ile Asn Arg Leu
Asp Leu Leu Ile Thr Tyr Leu Asn65 70 75
80Thr Arg Lys Glu Glu Met Glu Arg Glu Leu Gln Thr Pro
Gly Arg Ala 85 90 95Gln
Ile Ser Ala Tyr Arg Val Met Leu Tyr Gln Ile Ser Glu Glu Val
100 105 110Ser Arg Ser Glu Leu Arg Ser
Phe Lys Phe Leu Leu Gln Glu Glu Ile 115 120
125Ser Lys Cys Lys Leu Asp Asp Asp Met Asn Leu Leu Asp Ile Phe
Ile 130 135 140Glu Met Glu Lys Arg Val
Ile Leu Gly Glu Gly Lys Leu Asp Ile Leu145 150
155 160Lys Arg Val Cys Ala Gln Ile Asn Lys Ser Leu
Leu Lys Ile Ile Asn 165 170
175Asp Tyr Glu Glu Phe Ser Lys Glu Arg Ser Ser Ser Leu Glu Gly Ser
180 185 190Pro Asp Glu Phe Ser Asn
Gly Glu Glu Leu Cys Gly Val Met Thr Ile 195 200
205Ser Asp Ser Pro Arg Glu Gln Asp Ser Glu Ser Gln Thr Leu
Asp Lys 210 215 220Val Tyr Gln Met Lys
Ser Lys Pro Arg Gly Tyr Cys Leu Ile Ile Asn225 230
235 240Asn His Asn Phe Ala Lys Ala Arg Glu Lys
Val Pro Lys Leu His Ser 245 250
255Ile Arg Asp Arg Asn Gly Thr His Leu Asp Ala Gly Ala Leu Thr Thr
260 265 270Thr Phe Glu Glu Leu
His Phe Glu Ile Lys Pro His Asp Asp Cys Thr 275
280 285Val Glu Gln Ile Tyr Glu Ile Leu Lys Ile Tyr Gln
Leu Met Asp His 290 295 300Ser Asn Met
Asp Cys Phe Ile Cys Cys Ile Leu Ser His Gly Asp Lys305
310 315 320Gly Ile Ile Tyr Gly Thr Asp
Gly Gln Glu Ala Pro Ile Tyr Glu Leu 325
330 335Thr Ser Gln Phe Thr Gly Leu Lys Cys Pro Ser Leu
Ala Gly Lys Pro 340 345 350Lys
Val Phe Phe Ile Gln Ala Cys Gln Gly Asp Asn Tyr Gln Lys Gly 355
360 365Ile Pro Val Glu Thr Asp Ser Glu Glu
Gln Pro Tyr Leu Glu Met Asp 370 375
380Leu Ser Ser Pro Gln Thr Arg Tyr Ile Pro Asp Glu Ala Asp Phe Leu385
390 395 400Leu Gly Met Ala
Thr Val Asn Asn Cys Val Ser Tyr Arg Asn Pro Ala 405
410 415Glu Gly Thr Trp Tyr Ile Gln Ser Leu Cys
Gln Ser Leu Arg Glu Arg 420 425
430Cys Pro Arg Gly Asp Asp Ile Leu Thr Ile Leu Thr Glu Val Asn Tyr
435 440 445Glu Val Ser Asn Lys Asp Asp
Lys Lys Asn Met Gly Lys Gln Met Pro 450 455
460Gln Pro Thr Phe Thr Leu Arg Lys Lys Leu Val Phe Pro Ser Asp465
470 4754521PRTHomo sapiensCASP10;
Q92851 C401MOD_RES(401)..(401)Site of chemical conjugation 4Met Lys Ser
Gln Gly Gln His Trp Tyr Ser Ser Ser Asp Lys Asn Cys1 5
10 15Lys Val Ser Phe Arg Glu Lys Leu Leu
Ile Ile Asp Ser Asn Leu Gly 20 25
30Val Gln Asp Val Glu Asn Leu Lys Phe Leu Cys Ile Gly Leu Val Pro
35 40 45Asn Lys Lys Leu Glu Lys Ser
Ser Ser Ala Ser Asp Val Phe Glu His 50 55
60Leu Leu Ala Glu Asp Leu Leu Ser Glu Glu Asp Pro Phe Phe Leu Ala65
70 75 80Glu Leu Leu Tyr
Ile Ile Arg Gln Lys Lys Leu Leu Gln His Leu Asn 85
90 95Cys Thr Lys Glu Glu Val Glu Arg Leu Leu
Pro Thr Arg Gln Arg Val 100 105
110Ser Leu Phe Arg Asn Leu Leu Tyr Glu Leu Ser Glu Gly Ile Asp Ser
115 120 125Glu Asn Leu Lys Asp Met Ile
Phe Leu Leu Lys Asp Ser Leu Pro Lys 130 135
140Thr Glu Met Thr Ser Leu Ser Phe Leu Ala Phe Leu Glu Lys Gln
Gly145 150 155 160Lys Ile
Asp Glu Asp Asn Leu Thr Cys Leu Glu Asp Leu Cys Lys Thr
165 170 175Val Val Pro Lys Leu Leu Arg
Asn Ile Glu Lys Tyr Lys Arg Glu Lys 180 185
190Ala Ile Gln Ile Val Thr Pro Pro Val Asp Lys Glu Ala Glu
Ser Tyr 195 200 205Gln Gly Glu Glu
Glu Leu Val Ser Gln Thr Asp Val Lys Thr Phe Leu 210
215 220Glu Ala Leu Pro Gln Glu Ser Trp Gln Asn Lys His
Ala Gly Ser Asn225 230 235
240Gly Asn Arg Ala Thr Asn Gly Ala Pro Ser Leu Val Ser Arg Gly Met
245 250 255Gln Gly Ala Ser Ala
Asn Thr Leu Asn Ser Glu Thr Ser Thr Lys Arg 260
265 270Ala Ala Val Tyr Arg Met Asn Arg Asn His Arg Gly
Leu Cys Val Ile 275 280 285Val Asn
Asn His Ser Phe Thr Ser Leu Lys Asp Arg Gln Gly Thr His 290
295 300Lys Asp Ala Glu Ile Leu Ser His Val Phe Gln
Trp Leu Gly Phe Thr305 310 315
320Val His Ile His Asn Asn Val Thr Lys Val Glu Met Glu Met Val Leu
325 330 335Gln Lys Gln Lys
Cys Asn Pro Ala His Ala Asp Gly Asp Cys Phe Val 340
345 350Phe Cys Ile Leu Thr His Gly Arg Phe Gly Ala
Val Tyr Ser Ser Asp 355 360 365Glu
Ala Leu Ile Pro Ile Arg Glu Ile Met Ser His Phe Thr Ala Leu 370
375 380Gln Cys Pro Arg Leu Ala Glu Lys Pro Lys
Leu Phe Phe Ile Gln Ala385 390 395
400Cys Gln Gly Glu Glu Ile Gln Pro Ser Val Ser Ile Glu Ala Asp
Ala 405 410 415Leu Asn Pro
Glu Gln Ala Pro Thr Ser Leu Gln Asp Ser Ile Pro Ala 420
425 430Glu Ala Asp Phe Leu Leu Gly Leu Ala Thr
Val Pro Gly Tyr Val Ser 435 440
445Phe Arg His Val Glu Glu Gly Ser Trp Tyr Ile Gln Ser Leu Cys Asn 450
455 460His Leu Lys Lys Leu Val Pro Arg
Met Leu Lys Phe Leu Glu Lys Thr465 470
475 480Met Glu Ile Arg Gly Arg Lys Arg Thr Val Trp Gly
Ala Lys Gln Ile 485 490
495Ser Ala Thr Ser Leu Pro Thr Ala Ile Ser Ala Gln Thr Pro Arg Pro
500 505 510Pro Met Arg Arg Trp Ser
Ser Val Ser 515 5205361PRTHomo sapiensPRMT1;
Q99873 C109MOD_RES(109)..(109)Site of chemical conjugation 5Met Glu Asn
Phe Val Ala Thr Leu Ala Asn Gly Met Ser Leu Gln Pro1 5
10 15Pro Leu Glu Glu Val Ser Cys Gly Gln
Ala Glu Ser Ser Glu Lys Pro 20 25
30Asn Ala Glu Asp Met Thr Ser Lys Asp Tyr Tyr Phe Asp Ser Tyr Ala
35 40 45His Phe Gly Ile His Glu Glu
Met Leu Lys Asp Glu Val Arg Thr Leu 50 55
60Thr Tyr Arg Asn Ser Met Phe His Asn Arg His Leu Phe Lys Asp Lys65
70 75 80Val Val Leu Asp
Val Gly Ser Gly Thr Gly Ile Leu Cys Met Phe Ala 85
90 95Ala Lys Ala Gly Ala Arg Lys Val Ile Gly
Ile Glu Cys Ser Ser Ile 100 105
110Ser Asp Tyr Ala Val Lys Ile Val Lys Ala Asn Lys Leu Asp His Val
115 120 125Val Thr Ile Ile Lys Gly Lys
Val Glu Glu Val Glu Leu Pro Val Glu 130 135
140Lys Val Asp Ile Ile Ile Ser Glu Trp Met Gly Tyr Cys Leu Phe
Tyr145 150 155 160Glu Ser
Met Leu Asn Thr Val Leu Tyr Ala Arg Asp Lys Trp Leu Ala
165 170 175Pro Asp Gly Leu Ile Phe Pro
Asp Arg Ala Thr Leu Tyr Val Thr Ala 180 185
190Ile Glu Asp Arg Gln Tyr Lys Asp Tyr Lys Ile His Trp Trp
Glu Asn 195 200 205Val Tyr Gly Phe
Asp Met Ser Cys Ile Lys Asp Val Ala Ile Lys Glu 210
215 220Pro Leu Val Asp Val Val Asp Pro Lys Gln Leu Val
Thr Asn Ala Cys225 230 235
240Leu Ile Lys Glu Val Asp Ile Tyr Thr Val Lys Val Glu Asp Leu Thr
245 250 255Phe Thr Ser Pro Phe
Cys Leu Gln Val Lys Arg Asn Asp Tyr Val His 260
265 270Ala Leu Val Ala Tyr Phe Asn Ile Glu Phe Thr Arg
Cys His Lys Arg 275 280 285Thr Gly
Phe Ser Thr Ser Pro Glu Ser Pro Tyr Thr His Trp Lys Gln 290
295 300Thr Val Phe Tyr Met Glu Asp Tyr Leu Thr Val
Lys Thr Gly Glu Glu305 310 315
320Ile Phe Gly Thr Ile Gly Met Arg Pro Asn Ala Lys Asn Asn Arg Asp
325 330 335Leu Asp Phe Thr
Ile Asp Leu Asp Phe Lys Gly Gln Leu Cys Glu Leu 340
345 350Ser Cys Ser Thr Asp Tyr Arg Met Arg
355 3606800PRTHomo sapiensMAP3 kinase MLTK (or ZAK);
Q9NYL2 C22MOD_RES(22)..(22)Site of chemical conjugation 6Met Ser Ser Leu
Gly Ala Ser Phe Val Gln Ile Lys Phe Asp Asp Leu1 5
10 15Gln Phe Phe Glu Asn Cys Gly Gly Gly Ser
Phe Gly Ser Val Tyr Arg 20 25
30Ala Lys Trp Ile Ser Gln Asp Lys Glu Val Ala Val Lys Lys Leu Leu
35 40 45Lys Ile Glu Lys Glu Ala Glu Ile
Leu Ser Val Leu Ser His Arg Asn 50 55
60Ile Ile Gln Phe Tyr Gly Val Ile Leu Glu Pro Pro Asn Tyr Gly Ile65
70 75 80Val Thr Glu Tyr Ala
Ser Leu Gly Ser Leu Tyr Asp Tyr Ile Asn Ser 85
90 95Asn Arg Ser Glu Glu Met Asp Met Asp His Ile
Met Thr Trp Ala Thr 100 105
110Asp Val Ala Lys Gly Met His Tyr Leu His Met Glu Ala Pro Val Lys
115 120 125Val Ile His Arg Asp Leu Lys
Ser Arg Asn Val Val Ile Ala Ala Asp 130 135
140Gly Val Leu Lys Ile Cys Asp Phe Gly Ala Ser Arg Phe His Asn
His145 150 155 160Thr Thr
His Met Ser Leu Val Gly Thr Phe Pro Trp Met Ala Pro Glu
165 170 175Val Ile Gln Ser Leu Pro Val
Ser Glu Thr Cys Asp Thr Tyr Ser Tyr 180 185
190Gly Val Val Leu Trp Glu Met Leu Thr Arg Glu Val Pro Phe
Lys Gly 195 200 205Leu Glu Gly Leu
Gln Val Ala Trp Leu Val Val Glu Lys Asn Glu Arg 210
215 220Leu Thr Ile Pro Ser Ser Cys Pro Arg Ser Phe Ala
Glu Leu Leu His225 230 235
240Gln Cys Trp Glu Ala Asp Ala Lys Lys Arg Pro Ser Phe Lys Gln Ile
245 250 255Ile Ser Ile Leu Glu
Ser Met Ser Asn Asp Thr Ser Leu Pro Asp Lys 260
265 270Cys Asn Ser Phe Leu His Asn Lys Ala Glu Trp Arg
Cys Glu Ile Glu 275 280 285Ala Thr
Leu Glu Arg Leu Lys Lys Leu Glu Arg Asp Leu Ser Phe Lys 290
295 300Glu Gln Glu Leu Lys Glu Arg Glu Arg Arg Leu
Lys Met Trp Glu Gln305 310 315
320Lys Leu Thr Glu Gln Ser Asn Thr Pro Leu Leu Pro Ser Phe Glu Ile
325 330 335Gly Ala Trp Thr
Glu Asp Asp Val Tyr Cys Trp Val Gln Gln Leu Val 340
345 350Arg Lys Gly Asp Ser Ser Ala Glu Met Ser Val
Tyr Ala Ser Leu Phe 355 360 365Lys
Glu Asn Asn Ile Thr Gly Lys Arg Leu Leu Leu Leu Glu Glu Glu 370
375 380Asp Leu Lys Asp Met Gly Ile Val Ser Lys
Gly His Ile Ile His Phe385 390 395
400Lys Ser Ala Ile Glu Lys Leu Thr His Asp Tyr Ile Asn Leu Phe
His 405 410 415Phe Pro Pro
Leu Ile Lys Asp Ser Gly Gly Glu Pro Glu Glu Asn Glu 420
425 430Glu Lys Ile Val Asn Leu Glu Leu Val Phe
Gly Phe His Leu Lys Pro 435 440
445Gly Thr Gly Pro Gln Asp Cys Lys Trp Lys Met Tyr Met Glu Met Asp 450
455 460Gly Asp Glu Ile Ala Ile Thr Tyr
Ile Lys Asp Val Thr Phe Asn Thr465 470
475 480Asn Leu Pro Asp Ala Glu Ile Leu Lys Met Thr Lys
Pro Pro Phe Val 485 490
495Met Glu Lys Trp Ile Val Gly Ile Ala Lys Ser Gln Thr Val Glu Cys
500 505 510Thr Val Thr Tyr Glu Ser
Asp Val Arg Thr Pro Lys Ser Thr Lys His 515 520
525Val His Ser Ile Gln Trp Ser Arg Thr Lys Pro Gln Asp Glu
Val Lys 530 535 540Ala Val Gln Leu Ala
Ile Gln Thr Leu Phe Thr Asn Ser Asp Gly Asn545 550
555 560Pro Gly Ser Arg Ser Asp Ser Ser Ala Asp
Cys Gln Trp Leu Asp Thr 565 570
575Leu Arg Met Arg Gln Ile Ala Ser Asn Thr Ser Leu Gln Arg Ser Gln
580 585 590Ser Asn Pro Ile Leu
Gly Ser Pro Phe Phe Ser His Phe Asp Gly Gln 595
600 605Asp Ser Tyr Ala Ala Ala Val Arg Arg Pro Gln Val
Pro Ile Lys Tyr 610 615 620Gln Gln Ile
Thr Pro Val Asn Gln Ser Arg Ser Ser Ser Pro Thr Gln625
630 635 640Tyr Gly Leu Thr Lys Asn Phe
Ser Ser Leu His Leu Asn Ser Arg Asp 645
650 655Ser Gly Phe Ser Ser Gly Asn Thr Asp Thr Ser Ser
Glu Arg Gly Arg 660 665 670Tyr
Ser Asp Arg Ser Arg Asn Lys Tyr Gly Arg Gly Ser Ile Ser Leu 675
680 685Asn Ser Ser Pro Arg Gly Arg Tyr Ser
Gly Lys Ser Gln His Ser Thr 690 695
700Pro Ser Arg Gly Arg Tyr Pro Gly Lys Phe Tyr Arg Val Ser Gln Ser705
710 715 720Ala Leu Asn Pro
His Gln Ser Pro Asp Phe Lys Arg Ser Pro Arg Asp 725
730 735Leu His Gln Pro Asn Thr Ile Pro Gly Met
Pro Leu His Pro Glu Thr 740 745
750Asp Ser Arg Ala Ser Glu Glu Asp Ser Lys Val Ser Glu Gly Gly Trp
755 760 765Thr Lys Val Glu Tyr Arg Lys
Lys Pro His Arg Pro Ser Pro Ala Lys 770 775
780Thr Asn Lys Glu Arg Ala Arg Gly Asp His Arg Gly Trp Arg Asn
Phe785 790 795
8007514PRTHomo sapiensIMPDH2; P12268 C140,
C331MOD_RES(140)..(140)Site of chemical
conjugationMOD_RES(331)..(331)Site of chemical conjugation 7Met Ala Asp
Tyr Leu Ile Ser Gly Gly Thr Ser Tyr Val Pro Asp Asp1 5
10 15Gly Leu Thr Ala Gln Gln Leu Phe Asn
Cys Gly Asp Gly Leu Thr Tyr 20 25
30Asn Asp Phe Leu Ile Leu Pro Gly Tyr Ile Asp Phe Thr Ala Asp Gln
35 40 45Val Asp Leu Thr Ser Ala Leu
Thr Lys Lys Ile Thr Leu Lys Thr Pro 50 55
60Leu Val Ser Ser Pro Met Asp Thr Val Thr Glu Ala Gly Met Ala Ile65
70 75 80Ala Met Ala Leu
Thr Gly Gly Ile Gly Phe Ile His His Asn Cys Thr 85
90 95Pro Glu Phe Gln Ala Asn Glu Val Arg Lys
Val Lys Lys Tyr Glu Gln 100 105
110Gly Phe Ile Thr Asp Pro Val Val Leu Ser Pro Lys Asp Arg Val Arg
115 120 125Asp Val Phe Glu Ala Lys Ala
Arg His Gly Phe Cys Gly Ile Pro Ile 130 135
140Thr Asp Thr Gly Arg Met Gly Ser Arg Leu Val Gly Ile Ile Ser
Ser145 150 155 160Arg Asp
Ile Asp Phe Leu Lys Glu Glu Glu His Asp Cys Phe Leu Glu
165 170 175Glu Ile Met Thr Lys Arg Glu
Asp Leu Val Val Ala Pro Ala Gly Ile 180 185
190Thr Leu Lys Glu Ala Asn Glu Ile Leu Gln Arg Ser Lys Lys
Gly Lys 195 200 205Leu Pro Ile Val
Asn Glu Asp Asp Glu Leu Val Ala Ile Ile Ala Arg 210
215 220Thr Asp Leu Lys Lys Asn Arg Asp Tyr Pro Leu Ala
Ser Lys Asp Ala225 230 235
240Lys Lys Gln Leu Leu Cys Gly Ala Ala Ile Gly Thr His Glu Asp Asp
245 250 255Lys Tyr Arg Leu Asp
Leu Leu Ala Gln Ala Gly Val Asp Val Val Val 260
265 270Leu Asp Ser Ser Gln Gly Asn Ser Ile Phe Gln Ile
Asn Met Ile Lys 275 280 285Tyr Ile
Lys Asp Lys Tyr Pro Asn Leu Gln Val Ile Gly Gly Asn Val 290
295 300Val Thr Ala Ala Gln Ala Lys Asn Leu Ile Asp
Ala Gly Val Asp Ala305 310 315
320Leu Arg Val Gly Met Gly Ser Gly Ser Ile Cys Ile Thr Gln Glu Val
325 330 335Leu Ala Cys Gly
Arg Pro Gln Ala Thr Ala Val Tyr Lys Val Ser Glu 340
345 350Tyr Ala Arg Arg Phe Gly Val Pro Val Ile Ala
Asp Gly Gly Ile Gln 355 360 365Asn
Val Gly His Ile Ala Lys Ala Leu Ala Leu Gly Ala Ser Thr Val 370
375 380Met Met Gly Ser Leu Leu Ala Ala Thr Thr
Glu Ala Pro Gly Glu Tyr385 390 395
400Phe Phe Ser Asp Gly Ile Arg Leu Lys Lys Tyr Arg Gly Met Gly
Ser 405 410 415Leu Asp Ala
Met Asp Lys His Leu Ser Ser Gln Asn Arg Tyr Phe Ser 420
425 430Glu Ala Asp Lys Ile Lys Val Ala Gln Gly
Val Ser Gly Ala Val Gln 435 440
445Asp Lys Gly Ser Ile His Lys Phe Val Pro Tyr Leu Ile Ala Gly Ile 450
455 460Gln His Ser Cys Gln Asp Ile Gly
Ala Lys Ser Leu Thr Gln Val Arg465 470
475 480Ala Met Met Tyr Ser Gly Glu Leu Lys Phe Glu Lys
Arg Thr Ser Ser 485 490
495Ala Gln Val Glu Gly Gly Val His Ser Leu His Ser Tyr Glu Lys Arg
500 505 510Leu Phe8270PRTHomo
sapiensTIGAR; Q9NQ88 C114, C161MOD_RES(114)..(114)Site of chemical
conjugationMOD_RES(161)..(161)Site of chemical conjugation 8Met Ala Arg
Phe Ala Leu Thr Val Val Arg His Gly Glu Thr Arg Phe1 5
10 15Asn Lys Glu Lys Ile Ile Gln Gly Gln
Gly Val Asp Glu Pro Leu Ser 20 25
30Glu Thr Gly Phe Lys Gln Ala Ala Ala Ala Gly Ile Phe Leu Asn Asn
35 40 45Val Lys Phe Thr His Ala Phe
Ser Ser Asp Leu Met Arg Thr Lys Gln 50 55
60Thr Met His Gly Ile Leu Glu Arg Ser Lys Phe Cys Lys Asp Met Thr65
70 75 80Val Lys Tyr Asp
Ser Arg Leu Arg Glu Arg Lys Tyr Gly Val Val Glu 85
90 95Gly Lys Ala Leu Ser Glu Leu Arg Ala Met
Ala Lys Ala Ala Arg Glu 100 105
110Glu Cys Pro Val Phe Thr Pro Pro Gly Gly Glu Thr Leu Asp Gln Val
115 120 125Lys Met Arg Gly Ile Asp Phe
Phe Glu Phe Leu Cys Gln Leu Ile Leu 130 135
140Lys Glu Ala Asp Gln Lys Glu Gln Phe Ser Gln Gly Ser Pro Ser
Asn145 150 155 160Cys Leu
Glu Thr Ser Leu Ala Glu Ile Phe Pro Leu Gly Lys Asn His
165 170 175Ser Ser Lys Val Asn Ser Asp
Ser Gly Ile Pro Gly Leu Ala Ala Ser 180 185
190Val Leu Val Val Ser His Gly Ala Tyr Met Arg Ser Leu Phe
Asp Tyr 195 200 205Phe Leu Thr Asp
Leu Lys Cys Ser Leu Pro Ala Thr Leu Ser Arg Ser 210
215 220Glu Leu Met Ser Val Thr Pro Asn Thr Gly Met Ser
Leu Phe Ile Ile225 230 235
240Asn Phe Glu Glu Gly Arg Glu Val Lys Pro Thr Val Gln Cys Ile Cys
245 250 255Met Asn Leu Gln Asp
His Leu Asn Gly Leu Thr Glu Thr Arg 260 265
2709706PRTHomo sapiensPKCtheta; Q04759 C14,
C17MOD_RES(14)..(14)Site of chemical conjugationMOD_RES(17)..(17)Site of
chemical conjugation 9Met Ser Pro Phe Leu Arg Ile Gly Leu Ser Asn Phe Asp
Cys Gly Ser1 5 10 15Cys
Gln Ser Cys Gln Gly Glu Ala Val Asn Pro Tyr Cys Ala Val Leu 20
25 30Val Lys Glu Tyr Val Glu Ser Glu
Asn Gly Gln Met Tyr Ile Gln Lys 35 40
45Lys Pro Thr Met Tyr Pro Pro Trp Asp Ser Thr Phe Asp Ala His Ile
50 55 60Asn Lys Gly Arg Val Met Gln Ile
Ile Val Lys Gly Lys Asn Val Asp65 70 75
80Leu Ile Ser Glu Thr Thr Val Glu Leu Tyr Ser Leu Ala
Glu Arg Cys 85 90 95Arg
Lys Asn Asn Gly Lys Thr Glu Ile Trp Leu Glu Leu Lys Pro Gln
100 105 110Gly Arg Met Leu Met Asn Ala
Arg Tyr Phe Leu Glu Met Ser Asp Thr 115 120
125Lys Asp Met Asn Glu Phe Glu Thr Glu Gly Phe Phe Ala Leu His
Gln 130 135 140Arg Arg Gly Ala Ile Lys
Gln Ala Lys Val His His Val Lys Cys His145 150
155 160Glu Phe Thr Ala Thr Phe Phe Pro Gln Pro Thr
Phe Cys Ser Val Cys 165 170
175His Glu Phe Val Trp Gly Leu Asn Lys Gln Gly Tyr Gln Cys Arg Gln
180 185 190Cys Asn Ala Ala Ile His
Lys Lys Cys Ile Asp Lys Val Ile Ala Lys 195 200
205Cys Thr Gly Ser Ala Ile Asn Ser Arg Glu Thr Met Phe His
Lys Glu 210 215 220Arg Phe Lys Ile Asp
Met Pro His Arg Phe Lys Val Tyr Asn Tyr Lys225 230
235 240Ser Pro Thr Phe Cys Glu His Cys Gly Thr
Leu Leu Trp Gly Leu Ala 245 250
255Arg Gln Gly Leu Lys Cys Asp Ala Cys Gly Met Asn Val His His Arg
260 265 270Cys Gln Thr Lys Val
Ala Asn Leu Cys Gly Ile Asn Gln Lys Leu Met 275
280 285Ala Glu Ala Leu Ala Met Ile Glu Ser Thr Gln Gln
Ala Arg Cys Leu 290 295 300Arg Asp Thr
Glu Gln Ile Phe Arg Glu Gly Pro Val Glu Ile Gly Leu305
310 315 320Pro Cys Ser Ile Lys Asn Glu
Ala Arg Pro Pro Cys Leu Pro Thr Pro 325
330 335Gly Lys Arg Glu Pro Gln Gly Ile Ser Trp Glu Ser
Pro Leu Asp Glu 340 345 350Val
Asp Lys Met Cys His Leu Pro Glu Pro Glu Leu Asn Lys Glu Arg 355
360 365Pro Ser Leu Gln Ile Lys Leu Lys Ile
Glu Asp Phe Ile Leu His Lys 370 375
380Met Leu Gly Lys Gly Ser Phe Gly Lys Val Phe Leu Ala Glu Phe Lys385
390 395 400Lys Thr Asn Gln
Phe Phe Ala Ile Lys Ala Leu Lys Lys Asp Val Val 405
410 415Leu Met Asp Asp Asp Val Glu Cys Thr Met
Val Glu Lys Arg Val Leu 420 425
430Ser Leu Ala Trp Glu His Pro Phe Leu Thr His Met Phe Cys Thr Phe
435 440 445Gln Thr Lys Glu Asn Leu Phe
Phe Val Met Glu Tyr Leu Asn Gly Gly 450 455
460Asp Leu Met Tyr His Ile Gln Ser Cys His Lys Phe Asp Leu Ser
Arg465 470 475 480Ala Thr
Phe Tyr Ala Ala Glu Ile Ile Leu Gly Leu Gln Phe Leu His
485 490 495Ser Lys Gly Ile Val Tyr Arg
Asp Leu Lys Leu Asp Asn Ile Leu Leu 500 505
510Asp Lys Asp Gly His Ile Lys Ile Ala Asp Phe Gly Met Cys
Lys Glu 515 520 525Asn Met Leu Gly
Asp Ala Lys Thr Asn Thr Phe Cys Gly Thr Pro Asp 530
535 540Tyr Ile Ala Pro Glu Ile Leu Leu Gly Gln Lys Tyr
Asn His Ser Val545 550 555
560Asp Trp Trp Ser Phe Gly Val Leu Leu Tyr Glu Met Leu Ile Gly Gln
565 570 575Ser Pro Phe His Gly
Gln Asp Glu Glu Glu Leu Phe His Ser Ile Arg 580
585 590Met Asp Asn Pro Phe Tyr Pro Arg Trp Leu Glu Lys
Glu Ala Lys Asp 595 600 605Leu Leu
Val Lys Leu Phe Val Arg Glu Pro Glu Lys Arg Leu Gly Val 610
615 620Arg Gly Asp Ile Arg Gln His Pro Leu Phe Arg
Glu Ile Asn Trp Glu625 630 635
640Glu Leu Glu Arg Lys Glu Ile Asp Pro Pro Phe Arg Pro Lys Val Lys
645 650 655Ser Pro Phe Asp
Cys Ser Asn Phe Asp Lys Glu Phe Leu Asn Glu Lys 660
665 670Pro Arg Leu Ser Phe Ala Asp Arg Ala Leu Ile
Asn Ser Met Asp Gln 675 680 685Asn
Met Phe Arg Asn Phe Ser Phe Met Asn Pro Gly Met Glu Arg Leu 690
695 700Ile Ser7051014PRTHomo sapiensRPS3 40S
ribosomal protein S3; P23396_C97MOD_RES(4)..(4)Site of chemical
conjugation 10Arg Gly Leu Cys Ala Ile Ala Gln Ala Glu Ser Leu Arg Tyr1
5 101126PRTHomo sapiensTUBA4A Tubulin
alpha-4A chain; P68366_C295MOD_RES(16)..(16)Site of chemical
conjugation 11Lys Ala Tyr His Glu Gln Leu Ser Val Ala Glu Ile Thr Asn Ala
Cys1 5 10 15Phe Glu Pro
Ala Asn Gln Met Val Lys Cys 20 251216PRTHomo
sapiensTUBA4A Tubulin alpha-4A chain;
P68366_C347MOD_RES(10)..(10)Site of chemical conjugation 12Lys Arg Ser
Ile Gln Phe Val Asp Trp Cys Pro Thr Gly Phe Lys Val1 5
10 151316PRTHomo sapiensTUBA1A Tubulin
alpha-1A chain; Q71U36_C347MOD_RES(10)..(10)Site of chemical
conjugation 13Lys Arg Thr Ile Gln Phe Val Asp Trp Cys Pro Thr Gly Phe Lys
Val1 5 10 151419PRTHomo
sapiensTUBA1A Tubulin alpha-1A chain; Q71U36_C376MOD_RES(4)..(4)Site
of chemical conjugation 14Arg Ala Val Cys Met Leu Ser Asn Thr Thr Ala Ile
Ala Glu Ala Trp1 5 10
15Ala Arg Leu1530PRTHomo sapiensBZW1 Basic leucine zipper and W2 domain-
containing prot; Q7L1Q6_C35MOD_RES(12)..(12)Site of chemical
conjugation 15Lys Glu Arg Phe Asp Pro Thr Gln Phe Gln Asp Cys Ile Ile Gln
Gly1 5 10 15Leu Thr Glu
Thr Gly Thr Asp Leu Glu Ala Val Ala Lys Phe 20
25 301619PRTHomo sapiensGAPDH
Glyceraldehyde-3-phosphate dehydrogenase;
P04406_C152MOD_RES(8)..(8)Site of chemical conjugation 16Lys Ile Ile Ser
Asn Ala Ser Cys Thr Thr Asn Cys Leu Ala Pro Leu1 5
10 15Ala Lys Val1717PRTHomo sapiensPRMT1
Protein arginine N-methyltransferase 1;
Q99873_C109MOD_RES(7)..(7)Site of chemical conjugation 17Lys Val Ile Gly
Ile Glu Cys Ser Ser Ile Ser Asp Tyr Ala Val Lys1 5
10 15Ile1816PRTHomo sapiensPCBP1
Poly(rC)-binding protein 1; Q15365_C109MOD_RES(9)..(9)Site of
chemical conjugation 18Arg Leu Val Val Pro Ala Thr Gln Cys Gly Ser Leu
Ile Gly Lys Gly1 5 10
151924PRTHomo sapiensPIN1 Peptidyl-prolyl cis-trans isomerase NIMA-
interacti; Q13526_C113MOD_RES(19)..(19)Site of chemical conjugation 19Lys
Ile Lys Ser Gly Glu Glu Asp Phe Glu Ser Leu Ala Ser Gln Phe1
5 10 15Ser Asp Cys Ser Ser Ala Lys
Ala 202021PRTHomo sapiensNUDC Nuclear migration protein nudC;
Q9Y266_C188MOD_RES(16)..(16)Site of chemical conjugation 20Arg Trp
Thr Gln Thr Leu Ser Glu Leu Asp Leu Ala Val Pro Phe Cys1 5
10 15Val Asn Phe Arg Leu
202118PRTHomo sapiensCLIC4 Chloride intracellular channel protein 4;
Q9Y696_C35MOD_RES(12)..(12)Site of chemical conjugation 21Lys Ala Gly Ser
Asp Gly Glu Ser Ile Gly Asn Cys Pro Phe Ser Gln1 5
10 15Arg Leu2221PRTHomo sapiensACAT1 Acetyl-CoA
acetyltransferase, mitochondrial; P24752_C119MOD_RES(15)..(15)Site
of chemical conjugation 22Arg Gln Ala Val Leu Gly Ala Gly Leu Pro Ile Ser
Thr Pro Cys Thr1 5 10
15Thr Ile Asn Lys Val 202319PRTHomo sapiensHSPD1 60 kDa heat
shock protein, mitochondrial; P10809_C442MOD_RES(14)..(14)Site of
chemical conjugation 23Arg Ala Ala Val Glu Glu Gly Ile Val Leu Gly Gly
Gly Cys Ala Leu1 5 10
15Leu Arg Cys2411PRTHomo sapiensTUBA4A Tubulin alpha-4A chain;
P68366_C315MOD_RES(5)..(5)Site of chemical conjugation 24Lys Tyr Met Ala
Cys Cys Leu Leu Tyr Arg Gly1 5
102512PRTHomo sapiensGSTP1 Glutathione S-transferase P;
P09211_C48MOD_RES(4)..(4)Site of chemical conjugation 25Lys Ala Ser Cys
Leu Tyr Gly Gln Leu Pro Lys Phe1 5
102621PRTHomo sapiensGLRX5 Glutaredoxin-related protein 5,
mitochondrial; Q86SX6_C67MOD_RES(9)..(9)Site of chemical conjugation
26Lys Gly Thr Pro Glu Gln Pro Gln Cys Gly Phe Ser Asn Ala Val Val1
5 10 15Gln Ile Leu Arg Leu
202712PRTHomo sapiensEEF2 Elongation factor 2;
P13639_C41MOD_RES(10)..(10)Site of chemical conjugation 27Lys Ser Thr Leu
Thr Asp Ser Leu Val Cys Lys Ala1 5
102824PRTHomo sapiensXPO1 Exportin-1;
O14980_C34MOD_RES(13)..(13)Site of chemical conjugation 28Lys Leu Asp Ile
Asn Leu Leu Asp Asn Val Val Asn Cys Leu Tyr His1 5
10 15Gly Glu Gly Ala Gln Gln Arg Met
202925PRTHomo sapiensPCBP1 Poly(rC)-binding protein 1;
Q15365_C194MOD_RES(18)..(18)Site of chemical conjugation 29Arg Val Met
Thr Ile Pro Tyr Gln Pro Met Pro Ala Ser Ser Pro Val1 5
10 15Ile Cys Ala Gly Gly Gln Asp Arg Cys
20 253029PRTHomo sapiensCCT7 T-complex protein 1
subunit eta; Q99832_C29MOD_RES(20)..(20)Site of chemical conjugation
30Lys Glu Gly Thr Asp Ser Ser Gln Gly Ile Pro Gln Leu Val Ser Asn1
5 10 15Ile Ser Ala Cys Gln Val
Ile Ala Glu Ala Val Arg Thr 20 253112PRTHomo
sapiensRPS11 40S ribosomal protein S11;
P62280_C60MOD_RES(2)..(2)Site of chemical conjugation 31Lys Cys Pro Phe
Thr Gly Asn Val Ser Ile Arg Gly1 5
10329PRTHomo sapiensGSTO1 Glutathione S-transferase omega-1;
P78417_C32MOD_RES(3)..(3)Site of chemical conjugation 32Arg Phe Cys Pro
Phe Ala Glu Arg Thr1 53314PRTHomo sapiensACAT1 Acetyl-CoA
acetyltransferase, mitochondrial; P24752_C196MOD_RES(7)..(7)Site of
chemical conjugation 33Lys Ile His Met Gly Ser Cys Ala Glu Asn Thr Ala
Lys Lys1 5 103417PRTHomo sapiensTXN
Thioredoxin; P10599_C32MOD_RES(12)..(12)Site of chemical conjugation
34Lys Leu Val Val Val Asp Phe Ser Ala Thr Trp Cys Gly Pro Cys Lys1
5 10 15Met3518PRTHomo
sapiensCCT8 T-complex protein 1 subunit theta;
P50990_C244MOD_RES(7)..(7)Site of chemical conjugation 35Lys Ile Ala Val
Tyr Ser Cys Pro Phe Asp Gly Met Ile Thr Glu Thr1 5
10 15Lys Gly3628PRTHomo sapiensPDIA4 Protein
disulfide-isomerase A4; P13667_C206MOD_RES(23)..(23)Site of chemical
conjugation 36Lys Glu Asn Phe Asp Glu Val Val Asn Asp Ala Asp Ile Ile Leu
Val1 5 10 15Glu Phe Tyr
Ala Pro Trp Cys Gly His Cys Lys Lys 20
253713PRTHomo sapiensSEC13 Protein SEC13 homolog;
P55735_C187MOD_RES(7)..(7)Site of chemical conjugation 37Arg Phe Ala Ser
Gly Gly Cys Asp Asn Leu Ile Lys Leu1 5
103819PRTHomo sapiensPTGES3 Prostaglandin E synthase 3;
Q15185_C58MOD_RES(11)..(11)Site of chemical conjugation 38Lys His Leu Asn
Glu Ile Asp Leu Phe His Cys Ile Asp Pro Asn Asp1 5
10 15Ser Lys His3913PRTHomo sapiensADK
Adenosine kinase; P55263_C353MOD_RES(4)..(4)Site of chemical
conjugation 39Arg Thr Gly Cys Thr Phe Pro Glu Lys Pro Asp Phe His1
5 104010PRTHomo sapiensRPS5 40S ribosomal
protein S5; P46782_C66MOD_RES(4)..(4)Site of chemical conjugation
40Lys Ala Gln Cys Pro Ile Val Glu Arg Leu1 5
104116PRTHomo sapiensCNN2 Calponin-2;
Q99439_C164MOD_RES(5)..(5)Site of chemical conjugation 41Lys Ala Gly Gln
Cys Val Ile Gly Leu Gln Met Gly Thr Asn Lys Cys1 5
10 154226PRTHomo sapiensEEF2 Elongation factor
2; P13639_C136MOD_RES(17)..(17)Site of chemical conjugation 42Arg
Val Thr Asp Gly Ala Leu Val Val Val Asp Cys Val Ser Gly Val1
5 10 15Cys Val Gln Thr Glu Thr Val
Leu Arg Gln 20 254326PRTHomo sapiensIAH1
Isoamyl acetate-hydrolyzing esterase 1 homolog;
Q2TAA2_C137MOD_RES(11)..(11)Site of chemical conjugation 43Arg Val Ile
Leu Ile Thr Pro Thr Pro Leu Cys Glu Thr Ala Trp Glu1 5
10 15Glu Gln Cys Ile Ile Gln Gly Cys Lys
Leu 20 254424PRTHomo sapiensPRDX5
Peroxiredoxin-5, mitochondrial; P30044_C204MOD_RES(14)..(14)Site of
chemical conjugation 44Lys Ala Leu Asn Val Glu Pro Asp Gly Thr Gly Leu
Thr Cys Ser Leu1 5 10
15Ala Pro Asn Ile Ile Ser Gln Leu 204515PRTHomo sapiensIMPDH2
Inosine-5-monophosphate dehydrogenase 2;
P12268_C140MOD_RES(5)..(5)Site of chemical conjugation 45Arg His Gly Phe
Cys Gly Ile Pro Ile Thr Asp Thr Gly Arg Met1 5
10 154616PRTHomo sapiensHSD17B10 3-hydroxyacyl-CoA
dehydrogenase type-2; Q99714_C214MOD_RES(3)..(3)Site of chemical
conjugation 46Lys Val Cys Asn Phe Leu Ala Ser Gln Val Pro Phe Pro Ser Arg
Leu1 5 10 154712PRTHomo
sapiensNIT2 Omega-amidase NIT2; Q9NQR4_C153MOD_RES(7)..(7)Site of
chemical conjugation 47Arg Val Gly Leu Gly Ile Cys Tyr Asp Met Arg Phe1
5 104813PRTHomo sapiensAKR1B1 Aldose
reductase; P15121_C299MOD_RES(3)..(3)Site of chemical conjugation
48Arg Val Cys Ala Leu Leu Ser Cys Thr Ser His Lys Asp1 5
104920PRTHomo sapiensMTHFD1L Monofunctional
C1-tetrahydrofolate synthase, mitochondrial;
Q6UB35_C906MOD_RES(16)..(16)Site of chemical conjugation 49Lys Ile Asp
Arg Tyr Thr Gln Gln Gly Phe Gly Asn Leu Pro Ile Cys1 5
10 15Met Ala Lys Thr
205027PRTHomo sapiensTUBB Tubulin beta chain;
P07437_C239MOD_RES(24)..(24)Site of chemical conjugation 50Lys Leu Thr
Thr Pro Thr Tyr Gly Asp Leu Asn His Leu Val Ser Ala1 5
10 15Thr Met Ser Gly Val Thr Thr Cys Leu
Arg Phe 20 255125PRTHomo sapiensPDIA6 Protein
disulfide-isomerase A6; Q15084_C55MOD_RES(19)..(19)Site of chemical
conjugation 51Arg Glu Val Ile Gln Ser Asp Ser Leu Trp Leu Val Glu Phe Tyr
Ala1 5 10 15Pro Trp Cys
Gly His Cys Gln Arg Leu 20 255213PRTHomo
sapiensPKM Pyruvate kinase isozymes M1/M2;
P14618_C423MOD_RES(2)..(2)Site of chemical conjugation 52Lys Cys Cys Ser
Gly Ala Ile Ile Val Leu Thr Lys Ser1 5
105329PRTHomo sapiensRPS23 40S ribosomal protein S23;
P62266_C90MOD_RES(11)..(11)Site of chemical conjugation 53Lys Ile Thr Ala
Phe Val Pro Asn Asp Gly Cys Leu Asn Phe Ile Glu1 5
10 15Glu Asn Asp Glu Val Leu Val Ala Gly Phe
Gly Arg Lys 20 255441PRTHomo sapiensTUBA1A
Tubulin alpha-1A chain; Q71U36_C20MOD_RES(20)..(20)Site of chemical
conjugation 54Met Arg Glu Cys Ile Ser Ile His Val Gly Gln Ala Gly Val Gln
Ile1 5 10 15Gly Asn Ala
Cys Trp Glu Leu Tyr Cys Leu Glu His Gly Ile Gln Pro 20
25 30Asp Gly Gln Met Pro Ser Asp Lys Thr
35 405511PRTHomo sapiensPGLS 6-phosphogluconolactonase;
O95336_C32MOD_RES(4)..(4)Site of chemical conjugation 55Arg Ala Ala
Cys Cys Leu Ala Gly Ala Arg Ala1 5
105625PRTHomo sapiensACAT1 Acetyl-CoA acetyltransferase,
mitochondrial; P24752_C413MOD_RES(11)..(11)Site of chemical conjugation
56Lys Gln Gly Glu Tyr Gly Leu Ala Ser Ile Cys Asn Gly Gly Gly Gly1
5 10 15Ala Ser Ala Met Leu Ile
Gln Lys Leu 20 255714PRTHomo sapiensRPS4X 40S
ribosomal protein S4, X isoform; P62701_C41MOD_RES(5)..(5)Site of
chemical conjugation 57Lys Leu Arg Glu Cys Leu Pro Leu Ile Ile Phe Leu
Arg Asn1 5 105822PRTHomo sapiensRPL23 60S
ribosomal protein L23; P62829_C28MOD_RES(14)..(14)Site of chemical
conjugation 58Arg Ile Ser Leu Gly Leu Pro Val Gly Ala Val Ile Asn Cys Ala
Asp1 5 10 15Asn Thr Gly
Ala Lys Asn 205940PRTHomo sapiensTUBA1A Tubulin alpha-1A
chain; Q71U36_C25MOD_RES(24)..(24)Site of chemical conjugation 59Arg
Glu Cys Ile Ser Ile His Val Gly Gln Ala Gly Val Gln Ile Gly1
5 10 15Asn Ala Cys Trp Glu Leu Tyr
Cys Leu Glu His Gly Ile Gln Pro Asp 20 25
30Gly Gln Met Pro Ser Asp Lys Thr 35
406032PRTHomo sapiensERO1L ERO1-like protein alpha;
Q96HE7_C166MOD_RES(10)..(10)Site of chemical conjugation 60Lys His Asp
Asp Ser Ser Asp Asn Phe Cys Glu Ala Asp Asp Ile Gln1 5
10 15Ser Pro Glu Ala Glu Tyr Val Asp Leu
Leu Leu Asn Pro Glu Arg Tyr 20 25
306131PRTHomo sapiensERO1L ERO1-like protein alpha;
Q96HE7_C241MOD_RES(27)..(27)Site of chemical conjugation 61Lys Arg Pro
Leu Asn Pro Leu Ala Ser Gly Gln Gly Thr Ser Glu Glu1 5
10 15Asn Thr Phe Tyr Ser Trp Leu Glu Gly
Leu Cys Val Glu Lys Arg 20 25
306216PRTHomo sapiensTXNDC17 Thioredoxin domain-containing protein
17; Q9BRA2_C43MOD_RES(4)..(4)Site of chemical conjugation 62Lys Ser Trp
Cys Pro Asp Cys Val Gln Ala Glu Pro Val Val Arg Glu1 5
10 156317PRTHomo sapiensPDIA4 Protein
disulfide-isomerase A4; P13667_C555MOD_RES(12)..(12)Site of chemical
conjugation 63Lys Asp Val Leu Ile Glu Phe Tyr Ala Pro Trp Cys Gly His Cys
Lys1 5 10
15Gln6417PRTHomo sapiensPDIA3 Protein disulfide-isomerase A3;
P30101_C406MOD_RES(12)..(12)Site of chemical conjugation 64Lys Asp Val
Leu Ile Glu Phe Tyr Ala Pro Trp Cys Gly His Cys Lys1 5
10 15Asn6511PRTHomo sapiensTUBB Tubulin
beta chain; P07437_C354MOD_RES(5)..(5)Site of chemical conjugation
65Lys Thr Ala Val Cys Asp Ile Pro Pro Arg Gly1 5
106610PRTHomo sapiensRPL24 60S ribosomal protein L24;
P83731_C36MOD_RES(2)..(2)Site of chemical conjugation 66Lys Cys Glu Ser
Ala Phe Leu Ser Lys Arg1 5 106710PRTHomo
sapiensCORO1C Coronin-1C; Q9ULV4_C420MOD_RES(2)..(2)Site of chemical
conjugation 67Lys Cys Asp Leu Ile Ser Ile Pro Lys Lys1 5
106810PRTHomo sapiensRPS27 40S ribosomal protein S27;
P42677_C77MOD_RES(6)..(6)Site of chemical conjugation 68Arg Leu Thr Glu
Gly Cys Ser Phe Arg Arg1 5 106911PRTHomo
sapiensCLIC1 Chloride intracellular channel protein 1;
O00299_C24MOD_RES(5)..(5)Site of chemical conjugation 69Lys Ile Gly Asn
Cys Pro Phe Ser Gln Arg Leu1 5
107013PRTHomo sapiensSRP9 Signal recognition particle 9 kDa protein;
P49458_C48MOD_RES(8)..(8)Site of chemical conjugation 70Lys Val Thr Asp
Asp Leu Val Cys Leu Val Tyr Lys Thr1 5
107121PRTHomo sapiensACAA2 3-ketoacyl-CoA thiolase, mitochondrial;
P42765_C92MOD_RES(3)..(3)Site of chemical conjugation 71Arg Leu Cys Gly
Ser Gly Phe Gln Ser Ile Val Asn Gly Cys Gln Glu1 5
10 15Ile Cys Val Lys Glu
207213PRTHomo sapiensNONO Non-POU domain-containing octamer-binding
protein; Q15233_C145MOD_RES(4)..(4)Site of chemical conjugation 72Arg Phe
Ala Cys His Ser Ala Ser Leu Thr Val Arg Asn1 5
107331PRTHomo sapiensDNM2 Dynamin-2;
P50570_C27MOD_RES(13)..(13)Site of chemical conjugation 73Lys Leu Gln Asp
Ala Phe Ser Ser Ile Gly Gln Ser Cys His Leu Asp1 5
10 15Leu Pro Gln Ile Ala Val Val Gly Gly Gln
Ser Ala Gly Lys Ser 20 25
307421PRTHomo sapiensMCM3 DNA replication licensing factor MCM3;
P25205_C119MOD_RES(6)..(6)Site of chemical conjugation 74Arg Thr Leu Thr
Ser Cys Phe Leu Ser Cys Val Val Cys Val Glu Gly1 5
10 15Ile Val Thr Lys Cys
207513PRTHomo sapiensNT5DC1 5-nucleotidase domain-containing protein
1; Q5TFE4_C119MOD_RES(11)..(11)Site of chemical conjugation 75Lys His Phe
Leu Ser Asp Thr Gly Met Ala Cys Arg Ser1 5
107615PRTHomo sapiensCNN2 Calponin-2;
Q99439_C215MOD_RES(2)..(2)Site of chemical conjugation 76Lys Cys Ala Ser
Gln Val Gly Met Thr Ala Pro Gly Thr Arg Arg1 5
10 157713PRTHomo sapiensPSME1 Proteasome activator
complex subunit 1; Q06323_C22MOD_RES(10)..(10)Site of chemical
conjugation 77Lys Val Asp Val Phe Arg Glu Asp Leu Cys Thr Lys Thr1
5 107815PRTHomo sapiensSARS Serine--tRNA ligase,
cytoplasmic; P49591_C438MOD_RES(4)..(4)Site of chemical conjugation
78Arg Thr Ile Cys Ala Ile Leu Glu Asn Tyr Gln Thr Glu Lys Gly1
5 10 157913PRTHomo sapiensNUDCD1
NudC domain-containing protein 1; Q96RS6_C376MOD_RES(6)..(6)Site of
chemical conjugation 79Arg Asp Ser Ala Gln Cys Ala Ala Ile Ala Glu Arg
Leu1 5 108034PRTHomo sapiensTUBA1A
Tubulin alpha-1A chain; Q71U36_C129MOD_RES(6)..(6)Site of chemical
conjugation 80Lys Leu Ala Asp Gln Cys Thr Gly Leu Gln Gly Phe Leu Val Phe
His1 5 10 15Ser Phe Gly
Gly Gly Thr Gly Ser Gly Phe Thr Ser Leu Leu Met Glu 20
25 30Arg Leu8112PRTHomo sapiensNAA15
N-alpha-acetyltransferase 15, NatA auxiliary subunit;
Q9BXJ9_C721MOD_RES(8)..(8)Site of chemical conjugation 81Arg Leu Phe Asn
Thr Ala Val Cys Glu Ser Lys Asp1 5
108213PRTHomo sapiensCSTF2 Cleavage stimulation factor subunit 2;
P33240_C150MOD_RES(3)..(3)Site of chemical conjugation 82Lys Leu Cys Val
Gln Asn Ser Pro Gln Glu Ala Arg Asn1 5
108338PRTHomo sapiensAIMP2 Aminoacyl tRNA synthase complex-
interacting multifunctional protein 2; Q13155_C291MOD_RES(27)..(27)Site
of chemical conjugation 83Lys Ser Pro Trp Leu Ala Gly Asn Glu Leu Thr Val
Ala Asp Val Val1 5 10
15Leu Trp Ser Val Leu Gln Gln Ile Gly Gly Cys Ser Val Thr Val Pro
20 25 30Ala Asn Val Gln Arg Trp
358413PRTHomo sapiensTIPRL TIP41-like protein;
O75663_C87MOD_RES(4)..(4)Site of chemical conjugation 84Lys Val Ala Cys
Ala Glu Glu Trp Gln Glu Ser Arg Thr1 5
108510PRTHomo sapiensGNB2L1 Guanine nucleotide-binding protein
subunit beta-2-like 1; P63244_C182MOD_RES(8)..(8)Site of chemical
conjugation 85Lys Val Trp Asn Leu Ala Asn Cys Lys Leu1 5
108621PRTHomo sapiensEIF3M Eukaryotic translation
initiation factor 3 subunit; Q7L2H7_C134MOD_RES(6)..(6)Site of
chemical conjugation 86Lys Val Ala Ala Ser Cys Gly Ala Ile Gln Tyr Ile
Pro Thr Glu Leu1 5 10
15Asp Gln Val Arg Lys 208730PRTHomo sapiensFASN Fatty acid
synthase; P49327_C2359MOD_RES(6)..(6)Site of chemical conjugation
87Lys Leu Thr Pro Gly Cys Glu Ala Glu Ala Glu Thr Glu Ala Ile Cys1
5 10 15Phe Phe Val Gln Gln Phe
Thr Asp Met Glu His Asn Arg Val 20 25
308838PRTHomo sapiensTMPO Lamina-associated polypeptide 2,
isoform alpha; P42166_C561MOD_RES(27)..(27)Site of chemical
conjugation 88Lys Val Asp Asp Glu Ile Leu Gly Phe Ile Ser Glu Ala Thr Pro
Leu1 5 10 15Gly Gly Ile
Gln Ala Ala Ser Thr Glu Ser Cys Asn Gln Gln Leu Asp 20
25 30Leu Ala Leu Cys Arg Ala
35899PRTHomo sapiensACAT1 Acetyl-CoA acetyltransferase,
mitochondrial; P24752_C126MOD_RES(3)..(3)Site of chemical conjugation
89Lys Val Cys Ala Ser Gly Met Lys Ala1 59013PRTHomo
sapiensRPL4 60S ribosomal protein L4;
P36578_C96MOD_RES(11)..(11)Site of chemical conjugation 90Arg Ser Gly Gln
Gly Ala Phe Gly Asn Met Cys Arg Gly1 5
109110PRTHomo sapiensTHUMPD1 THUMP domain-containing protein 1;
Q9NXG2_C31MOD_RES(3)..(3)Site of chemical conjugation 91Arg Arg Cys Asp
Ala Gly Gly Pro Arg Gln1 5 109218PRTHomo
sapiensP4HB Protein disulfide-isomerase;
P07237_C397MOD_RES(13)..(13)Site of chemical conjugation 92Lys Lys Asn
Val Phe Val Glu Phe Tyr Ala Pro Trp Cys Gly His Cys1 5
10 15Lys Gln9315PRTHomo sapiensDCK
Deoxycytidine kinase; P27707_C9MOD_RES(3)..(3)Site of chemical
conjugation 93Arg Ser Cys Pro Ser Phe Ser Ala Ser Ser Glu Gly Thr Arg
Ile1 5 10 15949PRTHomo
sapiensEIF3F Eukaryotic translation initiation factor 3 subunit;
O00303_C256MOD_RES(3)..(3)Site of chemical conjugation 94Lys Thr Cys Phe
Ser Pro Asn Arg Val1 59532PRTHomo sapiensSNX6 Sorting
nexin-6; Q9UNH7_C264MOD_RES(26)..(26)Site of chemical conjugation
95Lys Ser Ala Ala Asp Asp Tyr Asn Arg Ile Gly Ser Ser Leu Tyr Ala1
5 10 15Leu Gly Thr Gln Asp Ser
Thr Asp Ile Cys Lys Phe Phe Leu Lys Val 20 25
309636PRTHomo sapiensPDIA6 Protein disulfide-isomerase
A6; Q15084_C190MOD_RES(31)..(31)Site of chemical conjugation 96Lys
Asp Val Ile Glu Leu Thr Asp Asp Ser Phe Asp Lys Asn Val Leu1
5 10 15Asp Ser Glu Asp Val Trp Met
Val Glu Phe Tyr Ala Pro Trp Cys Gly 20 25
30His Cys Lys Asn 359711PRTHomo sapiensHNRNPR
Heterogeneous nuclear ribonucleoprotein R;
O43390_C99MOD_RES(6)..(6)Site of chemical conjugation 97Lys Ser Ala Phe
Leu Cys Gly Val Met Lys Thr1 5
109845PRTHomo sapiensCOG3 Conserved oligomeric Golgi complex subunit
3; Q96JB2_C65MOD_RES(19)..(19)Site of chemical conjugation 98Lys Ala Ala
Ala Glu Asn Leu Pro Val Pro Ala Glu Leu Pro Ile Glu1 5
10 15Asp Leu Cys Ser Leu Thr Ser Gln Ser
Leu Pro Ile Glu Leu Thr Ser 20 25
30Val Val Pro Glu Ser Thr Glu Asp Ile Leu Leu Lys Gly 35
40 459910PRTHomo sapiensCRKL Crk-like
protein; P46109_C249MOD_RES(5)..(5)Site of chemical conjugation
99Lys Arg Val Pro Cys Ala Tyr Asp Lys Thr1 5
1010018PRTHomo sapiensRPL30 60S ribosomal protein L30;
P62888_C92MOD_RES(3)..(3)Site of chemical conjugation 100Arg Val Cys Thr
Leu Ala Ile Ile Asp Pro Gly Asp Ser Asp Ile Ile1 5
10 15Arg Ser10129PRTHomo sapiensTBC1D23 TBC1
domain family member 23; Q9NUY8_C283MOD_RES(26)..(26)Site of
chemical conjugation 101Lys Phe Leu Glu Asn Thr Pro Ser Ser Leu Asn Ile
Glu Asp Ile Glu1 5 10
15Asp Leu Phe Ser Leu Ala Gln Tyr Tyr Cys Ser Lys Thr 20
2510226PRTHomo sapiensNCBP1 Nuclear cap-binding protein
subunit 1; Q09161_C44MOD_RES(4)..(4)Site of chemical conjugation
102Lys Ser Ala Cys Ser Leu Glu Ser Asn Leu Glu Gly Leu Ala Gly Val1
5 10 15Leu Glu Ala Asp Leu Pro
Asn Tyr Lys Ser 20 2510319PRTHomo
sapiensDCTN4 Dynactin subunit 4; Q9UJW0_C258MOD_RES(11)..(11)Site of
chemical conjugation 103Arg Leu Leu Gln Pro Asp Phe Gln Pro Val Cys Ala
Ser Gln Leu Tyr1 5 10
15Pro Arg His10437PRTHomo sapiensOGT UDP-N-acetylglucosamine--peptide
N-acetylglucosamine; O15294_C758MOD_RES(2)..(2)Site of chemical
conjugation 104Lys Cys Pro Asp Gly Gly Asp Asn Ala Asp Ser Ser Asn Thr
Ala Leu1 5 10 15Asn Met
Pro Val Ile Pro Met Asn Thr Ile Ala Glu Ala Val Ile Glu 20
25 30Met Ile Asn Arg Gly
3510528PRTHomo sapiensHSPA9 Stress-70 protein, mitochondrial;
P38646_C317MOD_RES(4)..(4)Site of chemical conjugation 105Lys Ala Lys Cys
Glu Leu Ser Ser Ser Val Gln Thr Asp Ile Asn Leu1 5
10 15Pro Tyr Leu Thr Met Asp Ser Ser Gly Pro
Lys His 20 2510616PRTHomo sapiensFN3KRP
Ketosamine-3-kinase; Q9HA64_C24MOD_RES(10)..(10)Site of chemical
conjugation 106Arg Ala Thr Gly His Ser Gly Gly Gly Cys Ile Ser Gln Gly
Arg Ser1 5 10
1510755PRTHomo sapiensDYNC1LI1 Cytoplasmic dynein 1 light
intermediate chain 1; Q9Y6G9_C51MOD_RES(46)..(46)Site of chemical
conjugation 107Arg Val Gly Ser Phe Gly Ser Ser Pro Pro Gly Leu Ser Ser
Thr Tyr1 5 10 15Thr Gly
Gly Pro Leu Gly Asn Glu Ile Ala Ser Gly Asn Gly Gly Ala 20
25 30Ala Ala Gly Asp Asp Glu Asp Gly Gln
Asn Leu Trp Ser Cys Ile Leu 35 40
45Ser Glu Val Ser Thr Arg Ser 50 5510815PRTHomo
sapiensHNRNPF Heterogeneous nuclear ribonucleoprotein F;
P52597_C267MOD_RES(6)..(6)Site of chemical conjugation 108Arg Asp Leu Ser
Tyr Cys Leu Ser Gly Met Tyr Asp His Arg Tyr1 5
10 1510925PRTHomo sapiensPARK7 Protein DJ-1;
Q99497_C106MOD_RES(8)..(8)Site of chemical conjugation 109Lys Gly Leu Ile
Ala Ala Ile Cys Ala Gly Pro Thr Ala Leu Leu Ala1 5
10 15His Glu Ile Gly Phe Gly Ser Lys Val
20 2511026PRTHomo sapiensGSDMD Gasdermin-D;
P57764_C268MOD_RES(2)..(2)Site of chemical conjugation 110Arg Cys Leu His
Asn Phe Leu Thr Asp Gly Val Pro Ala Glu Gly Ala1 5
10 15Phe Thr Glu Asp Phe Gln Gly Leu Arg Ala
20 2511112PRTHomo sapiensPGP Phosphoglycolate
phosphatase; A6NDG6_C297MOD_RES(8)..(8)Site of chemical conjugation
111Lys Asn Asn Gln Glu Ser Asp Cys Val Ser Lys Lys1 5
1011216PRTHomo sapiensMCM6 DNA replication licensing factor
MCM6; Q14566_C301MOD_RES(7)..(7)Site of chemical conjugation 112Arg
Leu Val Phe Leu Ala Cys Cys Val Ala Pro Thr Asn Pro Arg Phe1
5 10 1511318PRTHomo sapiensCLTC
Clathrin heavy chain 1; Q00610_C870MOD_RES(6)..(6)Site of chemical
conjugation 113Arg Ile His Glu Gly Cys Glu Glu Pro Ala Thr His Asn Ala
Leu Ala1 5 10 15Lys
Ile11433PRTHomo sapiensMMS19 MMS19 nucleotide excision repair protein
homolog; Q96T76_C848MOD_RES(26)..(26)Site of chemical conjugation 114Arg
Leu Met Gly Leu Leu Ser Asp Pro Glu Leu Gly Pro Ala Ala Ala1
5 10 15Asp Gly Phe Ser Leu Leu Met
Ser Asp Cys Thr Asp Val Leu Thr Arg 20 25
30Ala11512PRTHomo sapiensACTR3 Actin-related protein 3;
P61158_C235MOD_RES(6)..(6)Site of chemical conjugation 115Arg Tyr Ser Tyr
Val Cys Pro Asp Leu Val Lys Glu1 5
1011610PRTHomo sapiensCFL1 Cofilin-1; P23528_C80MOD_RES(8)..(8)Site
of chemical conjugation 116Lys Met Leu Pro Asp Lys Asp Cys Arg Tyr1
5 1011731PRTHomo sapiensTIGAR
Fructose-2,6-bisphosphatase TIGAR; Q9NQ88_C161MOD_RES(17)..(17)Site
of chemical conjugation 117Lys Glu Ala Asp Gln Lys Glu Gln Phe Ser Gln
Gly Ser Pro Ser Asn1 5 10
15Cys Leu Glu Thr Ser Leu Ala Glu Ile Phe Pro Leu Gly Lys Asn
20 25 3011826PRTHomo sapiensCPSF3
Cleavage and polyadenylation specificity factor subunit 3;
Q9UKF6_C498MOD_RES(11)..(11)Site of chemical conjugation 118Arg Asn Phe
Asn Tyr His Ile Leu Ser Pro Cys Asp Leu Ser Asn Tyr1 5
10 15Thr Asp Leu Ala Met Ser Thr Val Lys
Gln 20 2511914PRTHomo sapiensSMARCC2 SWI/SNF
complex subunit SMARCC2; Q8TAQ2_C145MOD_RES(7)..(7)Site of chemical
conjugation 119Arg Pro Asn Ile Phe Leu Cys Pro Glu Ile Glu Pro Lys Leu1
5 1012020PRTHomo sapiensUBE2L3
Ubiquitin-conjugating enzyme E2 L3; P68036_C86MOD_RES(5)..(5)Site of
chemical conjugation 120Lys Gly Gln Val Cys Leu Pro Val Ile Ser Ala Glu
Asn Trp Lys Pro1 5 10
15Ala Thr Lys Thr 2012116PRTHomo sapiensMOB4 MOB-like protein
phocein; Q9Y3A3_C134MOD_RES(9)..(9)Site of chemical conjugation
121Arg His Thr Leu Asp Gly Ala Ala Cys Leu Leu Asn Ser Asn Lys Tyr1
5 10 1512210PRTHomo
sapiensAARS Alanine--tRNA ligase, cytoplasmic;
P49588_C773MOD_RES(2)..(2)Site of chemical conjugation 122Lys Cys Leu Ser
Val Met Glu Ala Lys Val1 5 1012313PRTHomo
sapiensGMPPA Mannose-1-phosphate guanyltransferase alpha;
Q96IJ6_C389MOD_RES(11)..(11)Site of chemical conjugation 123Lys Leu Leu
Pro Ala Ile Thr Ile Leu Gly Cys Arg Val1 5
1012411PRTHomo sapiensRPL10 60S ribosomal protein L10;
P27635_C105MOD_RES(5)..(5)Site of chemical conjugation 124Lys Met Leu Ser
Cys Ala Gly Ala Asp Arg Leu1 5
101259PRTHomo sapiensARPC3 Actin-related protein 2/3 complex subunit
3; O15145_C162MOD_RES(5)..(5)Site of chemical conjugation 125Lys Trp Trp
Thr Cys Phe Val Lys Arg1 512616PRTHomo sapiensCCT5
T-complex protein 1 subunit epsilon; P48643_C253MOD_RES(7)..(7)Site
of chemical conjugation 126Lys Ile Ala Ile Leu Thr Cys Pro Phe Glu Pro
Pro Lys Pro Lys Thr1 5 10
1512744PRTHomo sapiensMTHFD1 C-1-tetrahydrofolate synthase,
cytoplasmic; P11586_C918MOD_RES(27)..(27)Site of chemical conjugation
127Arg Ala Ser Val Gly Ala Gly Phe Leu Tyr Pro Leu Val Gly Thr Met1
5 10 15Ser Thr Met Pro Gly Leu
Pro Thr Arg Pro Cys Phe Tyr Asp Ile Asp 20 25
30Leu Asp Pro Glu Thr Glu Gln Val Asn Gly Leu Phe
35 4012822PRTHomo sapiensATXN7L3B Putative ataxin-7-like
protein 3B; Q96GX2_C75MOD_RES(5)..(5)Site of chemical conjugation
128Arg Leu Pro Leu Cys Ser Leu Pro Gly Glu Pro Gly Asn Gly Pro Asp1
5 10 15Gln Gln Leu Gln Arg Ser
2012913PRTHomo sapiensTCP1 T-complex protein 1 subunit alpha;
P17987_C76MOD_RES(4)..(4)Site of chemical conjugation 129Lys Val Leu
Cys Glu Leu Ala Asp Leu Gln Asp Lys Glu1 5
1013038PRTHomo sapiensTMOD3 Tropomodulin-3;
Q9NYL9_C132MOD_RES(21)..(21)Site of chemical conjugation 130Lys Val Ser
Leu Asp Pro Glu Leu Glu Glu Ala Leu Thr Ser Ala Ser1 5
10 15Asp Thr Glu Leu Cys Asp Leu Ala Ala
Ile Leu Gly Met His Asn Leu 20 25
30Ile Thr Asn Thr Lys Phe 3513113PRTHomo sapiensDNM1L
Dynamin-1-like protein; O00429_C367MOD_RES(3)..(3)Site of chemical
conjugation 131Arg Ile Cys Tyr Ile Phe His Glu Thr Phe Gly Arg Thr1
5 1013211PRTHomo sapiensAIMP2 Aminoacyl tRNA
synthase complex-interacting multifunctional protein 2;
Q13155_C23MOD_RES(7)..(7)Site of chemical conjugation 132Arg Val Glu Leu
Pro Thr Cys Met Tyr Arg Leu1 5
1013325PRTHomo sapiensPDIA3 Protein disulfide-isomerase A3;
P30101_C57MOD_RES(20)..(20)Site of chemical conjugation 133Arg Ile Ser
Asp Thr Gly Ser Ala Gly Leu Met Leu Val Glu Phe Phe1 5
10 15Ala Pro Trp Cys Gly His Cys Lys Arg
20 251348PRTHomo sapiensDNPEP Aspartyl
aminopeptidase; Q9ULA0_C144MOD_RES(2)..(2)Site of chemical
conjugation 134Lys Cys Pro Thr Ser Gly Arg Leu1
513528PRTHomo sapiensACAT2 Acetyl-CoA acetyltransferase, cytosolic;
Q9BWD1_C92MOD_RES(22)..(22)Site of chemical conjugation 135Arg Gln Ala
Ser Val Gly Ala Gly Ile Pro Tyr Ser Val Pro Ala Trp1 5
10 15Ser Cys Gln Met Ile Cys Gly Ser Gly
Leu Lys Ala 20 2513612PRTHomo sapiensTXNDC5
Thioredoxin domain-containing protein 5;
Q8NBS9_C350MOD_RES(7)..(7)Site of chemical conjugation 136Lys Phe Tyr Ala
Pro Trp Cys Gly His Cys Lys Thr1 5
1013722PRTHomo sapiensTUBA4A Tubulin alpha-4A chain;
P68366_C54MOD_RES(15)..(15)Site of chemical conjugation 137Lys Thr Ile
Gly Gly Gly Asp Asp Ser Phe Thr Thr Phe Phe Cys Glu1 5
10 15Thr Gly Ala Gly Lys His
2013812PRTHomo sapiensTK1 Thymidine kinase, cytosolic;
P04183_C66MOD_RES(7)..(7)Site of chemical conjugation 138Arg Tyr Ser Ser
Ser Phe Cys Thr His Asp Arg Asn1 5
1013920PRTHomo sapiensNCAPD2 Condensin complex subunit 1;
Q15021_C439MOD_RES(18)..(18)Site of chemical conjugation 139Lys Asn Ala
Ile Gln Leu Leu Ala Ser Phe Leu Ala Asn Asn Pro Phe1 5
10 15Ser Cys Lys Leu
2014019PRTHomo sapiensPDCD6IP Programmed cell death 6-interacting
protein; Q8WUM4_C250MOD_RES(3)..(3)Site of chemical conjugation 140Lys
His Cys Ile Met Gln Ala Asn Ala Glu Tyr His Gln Ser Ile Leu1
5 10 15Ala Lys Gln14125PRTHomo
sapiensEDC3 Enhancer of mRNA-decapping protein 3;
Q96F86_C413MOD_RES(17)..(17)Site of chemical conjugation 141Lys Asp Leu
Pro Thr Ser Pro Val Asp Leu Val Ile Asn Cys Leu Asp1 5
10 15Cys Pro Glu Asn Val Phe Leu Arg Asp
20 2514215PRTHomo sapiensGLRX Glutaredoxin-1;
P35754_C23MOD_RES(10)..(10)Site of chemical conjugation 142Lys Val Val
Val Phe Ile Lys Pro Thr Cys Pro Tyr Cys Arg Arg1 5
10 1514320PRTHomo sapiensTIGAR
Fructose-2,6-bisphosphatase TIGAR; Q9NQ88_C114MOD_RES(4)..(4)Site of
chemical conjugation 143Arg Glu Glu Cys Pro Val Phe Thr Pro Pro Gly Gly
Glu Thr Leu Asp1 5 10
15Gln Val Lys Met 2014429PRTHomo sapiensIMPDH2
Inosine-5-monophosphate dehydrogenase 2;
P12268_C331MOD_RES(10)..(10)Site of chemical conjugation 144Arg Val Gly
Met Gly Ser Gly Ser Ile Cys Ile Thr Gln Glu Val Leu1 5
10 15Ala Cys Gly Arg Pro Gln Ala Thr Ala
Val Tyr Lys Val 20 2514533PRTHomo
sapiensATP5A1 ATP synthase subunit alpha, mitochondrial;
P25705_C294MOD_RES(25)..(25)Site of chemical conjugation 145Lys Tyr Thr
Ile Val Val Ser Ala Thr Ala Ser Asp Ala Ala Pro Leu1 5
10 15Gln Tyr Leu Ala Pro Tyr Ser Gly Cys
Ser Met Gly Glu Tyr Phe Arg 20 25
30Asp14622PRTHomo sapiensUCHL3 Ubiquitin carboxyl-terminal hydrolase
isozyme L3; P15374_C95MOD_RES(8)..(8)Site of chemical conjugation
146Lys Gln Thr Ile Ser Asn Ala Cys Gly Thr Ile Gly Leu Ile His Ala1
5 10 15Ile Ala Asn Asn Lys Asp
2014712PRTHomo sapiensRPL24 60S ribosomal protein L24;
P83731_C6MOD_RES(5)..(5)Site of chemical conjugation 147Lys Val Glu Leu
Cys Ser Phe Ser Gly Tyr Lys Ile1 5
1014821PRTHomo sapiensNHP2 H/ACA ribonucleoprotein complex subunit 2;
Q9NX24_C18MOD_RES(16)..(16)Site of chemical conjugation 148Lys Ile Lys
Ala Asp Pro Asp Gly Pro Glu Ala Gln Ala Glu Ala Cys1 5
10 15Ser Gly Glu Arg Thr
2014910PRTHomo sapiensILK Integrin-linked protein kinase;
Q13418_C346MOD_RES(6)..(6)Site of chemical conjugation 149Lys Phe Ser Phe
Gln Cys Pro Gly Arg Met1 5 1015026PRTHomo
sapiensTCOF1 Treacle protein; Q13428_C38MOD_RES(2)..(2)Site of
chemical conjugation 150Lys Cys Phe Leu Ala Gln Pro Val Thr Leu Leu Asp
Ile Tyr Thr His1 5 10
15Trp Gln Gln Thr Ser Glu Leu Gly Arg Lys 20
2515111PRTHomo sapiensCDK2 Cyclin-dependent kinase 2;
P24941_C177MOD_RES(9)..(9)Site of chemical conjugation 151Arg Ala Pro Glu
Ile Leu Leu Gly Cys Lys Tyr1 5
1015214PRTHomo sapiensPSMC4 26S protease regulatory subunit 6B;
P43686_C210MOD_RES(11)..(11)Site of chemical conjugation 152Arg Gly Val
Leu Met Tyr Gly Pro Pro Gly Cys Gly Lys Thr1 5
1015321PRTHomo sapiensWAC WW domain-containing adapter protein with
coiled-coil region; Q9BTA9_C553MOD_RES(4)..(4)Site of chemical
conjugation 153Arg Ser Thr Cys Ser Leu Thr Pro Ala Leu Ala Ala His Phe
Ser Glu1 5 10 15Asn Leu
Ile Lys His 2015418PRTHomo sapiensHAT1 Histone
acetyltransferase type B catalytic subunit;
O14929_C101MOD_RES(8)..(8)Site of chemical conjugation 154Lys Val Asp Glu
Asn Phe Asp Cys Val Glu Ala Asp Asp Val Glu Gly1 5
10 15Lys Ile15518PRTHomo sapiensECI2 Enoyl-CoA
delta isomerase 2, mitochondrial; O75521_C380MOD_RES(7)..(7)Site of
chemical conjugation 155Arg Trp Leu Ser Asp Glu Cys Thr Asn Ala Val Val
Asn Phe Leu Ser1 5 10
15Arg Lys15614PRTHomo sapiensMTHFD1 C-1-tetrahydrofolate synthase,
cytoplasmic; P11586_C863MOD_RES(10)..(10)Site of chemical conjugation
156Lys Gln Gly Phe Gly Asn Leu Pro Ile Cys Met Ala Lys Thr1
5 1015717PRTHomo sapiensDCK Deoxycytidine kinase;
P27707_C45MOD_RES(4)..(4)Site of chemical conjugation 157Lys Gln Leu Cys
Glu Asp Trp Glu Val Val Pro Glu Pro Val Ala Arg1 5
10 15Trp15817PRTHomo sapiensUBA6 Ubiquitin-like
modifier-activating enzyme 6; A0AVT1_C347MOD_RES(7)..(7)Site of
chemical conjugation 158Arg Lys Pro Asn Val Gly Cys Gln Gln Asp Ser Glu
Glu Leu Leu Lys1 5 10
15Leu15925PRTHomo sapiensISOC2 Isochorismatase domain-containing
protein 2, mitochondrial; Q96AB3_C114MOD_RES(13)..(13)Site of chemical
conjugation 159Arg Ser Val Leu Leu Cys Gly Ile Glu Ala Gln Ala Cys Ile
Leu Asn1 5 10 15Thr Thr
Leu Asp Leu Leu Asp Arg Gly 20 2516014PRTHomo
sapiensDCTN1 Dynactin subunit 1; Q14203_C1252MOD_RES(6)..(6)Site of
chemical conjugation 160Lys Val Thr Phe Ser Cys Ala Ala Gly Phe Gly Gln
Arg His1 5 1016121PRTHomo sapiensMCMBP
Mini-chromosome maintenance complex- binding protein;
Q9BTE3_C287MOD_RES(12)..(12)Site of chemical conjugation 161Arg Asp Ala
Ser Ala Leu Leu Asp Pro Met Glu Cys Thr Asp Thr Ala1 5
10 15Glu Glu Gln Arg Val
2016218PRTHomo sapiensIPO7 Importin-7;
O95373_C757MOD_RES(6)..(6)Site of chemical conjugation 162Arg Gly Ile Asp
Gln Cys Ile Pro Leu Phe Val Glu Ala Ala Leu Glu1 5
10 15Arg Leu1639PRTHomo sapiensPCMT1
Protein-L-isoaspartate(D-aspartate) O- methyltransferase 1;
P22061_C102MOD_RES(5)..(5)Site of chemical conjugation 163Arg Met Val Gly
Cys Thr Gly Lys Val1 516411PRTHomo sapiensCSE1L Exportin-2;
P55060_C842MOD_RES(4)..(4)Site of chemical conjugation 164Lys Lys
Ile Cys Ala Val Gly Ile Thr Lys Leu1 5
1016512PRTHomo sapiensNAA15 N-alpha-acetyltransferase 15, NatA
auxiliary subunit; Q9BXJ9_C322MOD_RES(3)..(3)Site of chemical conjugation
165Lys Gly Cys Pro Pro Val Phe Asn Thr Leu Arg Ser1 5
1016614PRTHomo sapiensPSMC6 26S protease regulatory subunit
10B; P62333_C170MOD_RES(3)..(3)Site of chemical conjugation 166Lys
Gly Cys Leu Leu Tyr Gly Pro Pro Gly Thr Gly Lys Thr1 5
1016719PRTHomo sapiensDLGAP5 Disks large-associated protein
5; Q15398_C129MOD_RES(8)..(8)Site of chemical conjugation 167Arg Tyr
Arg Pro Asp Met Pro Cys Phe Leu Leu Ser Asn Gln Asn Ala1 5
10 15Val Lys Ala16828PRTHomo
sapiensC2orf49 Ashwin; Q9BVC5_C10MOD_RES(3)..(3)Site of chemical
conjugation 168Arg Ser Cys Thr Asp Ser Glu Leu Leu Leu His Pro Glu Leu
Leu Ser1 5 10 15Gln Glu
Phe Leu Leu Leu Thr Leu Glu Gln Lys Asn 20
2516913PRTHomo sapiensRPS11 40S ribosomal protein S11;
P62280_C116MOD_RES(10)..(10)Site of chemical conjugation 169Lys Asn Met
Ser Val His Leu Ser Pro Cys Phe Arg Asp1 5
1017034PRTHomo sapiensSTAT1 Signal transducer and activator of
transcription 1; P42224_C255MOD_RES(14)..(14)Site of chemical conjugation
170Arg Gln Gln Ser Ala Cys Ile Gly Gly Pro Pro Asn Ala Cys Leu Asp1
5 10 15Gln Leu Gln Asn Trp Phe
Thr Ile Val Ala Glu Ser Leu Gln Gln Val 20 25
30Arg Gln17119PRTHomo sapiensMED15 Mediator of RNA
polymerase II transcription subuni; Q96RN5_C618MOD_RES(6)..(6)Site
of chemical conjugation 171Lys Gln Gln Tyr Leu Cys Gln Pro Leu Leu Asp
Ala Val Leu Ala Asn1 5 10
15Ile Arg Ser17242PRTHomo sapiensMTHFD1L Monofunctional
C1-tetrahydrofolate synthase, mitochondrial;
Q6UB35_C961MOD_RES(27)..(27)Site of chemical conjugation 172Arg Ala Ser
Ile Gly Ala Gly Phe Ile Tyr Pro Leu Val Gly Thr Met1 5
10 15Ser Thr Met Pro Gly Leu Pro Thr Arg
Pro Cys Phe Tyr Asp Ile Asp 20 25
30Leu Asp Thr Glu Thr Glu Gln Val Lys Gly 35
401739PRTHomo sapiensDTYMK Thymidylate kinase;
P23919_C163MOD_RES(2)..(2)Site of chemical conjugation 173Arg Cys Phe His
Gln Leu Met Lys Asp1 517429PRTHomo sapiensGCN1L1
Translational activator GCN1; Q92616_C1692MOD_RES(7)..(7)Site of
chemical conjugation 174Lys Gly Met Gly Glu Ser Cys Phe Glu Asp Leu Leu
Pro Trp Leu Met1 5 10
15Glu Thr Leu Thr Tyr Glu Gln Ser Ser Val Asp Arg Ser 20
2517512PRTHomo sapiensTMPO Lamina-associated polypeptide 2,
isoform alpha; P42166_C341MOD_RES(8)..(8)Site of chemical conjugation
175Lys Ser Gly Ile Gln Pro Leu Cys Pro Glu Arg Ser1 5
1017611PRTHomo sapiensETHE1 Protein ETHE1, mitochondrial;
O95571_C170MOD_RES(8)..(8)Site of chemical conjugation 176Arg Thr Asp
Phe Gln Gln Gly Cys Ala Lys Thr1 5
1017714PRTHomo sapiensNDC80 Kinetochore protein NDC80 homolog;
O14777_C449MOD_RES(10)..(10)Site of chemical conjugation 177Lys Phe Asn
Pro Glu Ala Gly Ala Asn Cys Leu Val Lys Tyr1 5
1017814PRTHomo sapiensATP5C1 ATP synthase subunit gamma,
mitochondrial; P36542_C103MOD_RES(4)..(4)Site of chemical conjugation
178Arg Gly Leu Cys Gly Ala Ile His Ser Ser Ile Ala Lys Gln1
5 1017934PRTHomo sapiensFAM96B Mitotic
spindle-associated MMXD complex subunit MIP18;
Q9Y3D0_C93MOD_RES(22)..(22)Site of chemical conjugation 179Arg Val Gln
Val Ser Asp Pro Glu Ser Thr Val Ala Val Ala Phe Thr1 5
10 15Pro Thr Ile Pro His Cys Ser Met Ala
Thr Leu Ile Gly Leu Ser Ile 20 25
30Lys Val18022PRTHomo sapiensRARS Arginine--tRNA ligase,
cytoplasmic; P54136_C34MOD_RES(5)..(5)Site of chemical conjugation
180Lys Asn Cys Gly Cys Leu Gly Ala Ser Pro Asn Leu Glu Gln Leu Gln1
5 10 15Glu Glu Asn Leu Lys Leu
2018113PRTHomo sapiensMMS19 MMS19 nucleotide excision repair
protein homolog; Q96T76_C819MOD_RES(8)..(8)Site of chemical
conjugation 181Arg Tyr His Pro Leu Ser Ser Cys Leu Thr Ala Arg Leu1
5 1018235PRTHomo sapiensUBA3 NEDD8-activating
enzyme E1 catalytic subunit; Q8TBC4_C237MOD_RES(27)..(27)Site of
chemical conjugation 182Arg Val Ile Leu Pro Gly Met Thr Ala Cys Ile Glu
Cys Thr Leu Glu1 5 10
15Leu Tyr Pro Pro Gln Val Asn Phe Pro Met Cys Thr Ile Ala Ser Met
20 25 30Pro Arg Leu
3518311PRTHomo sapiensSNW1 SNW domain-containing protein 1;
Q13573_C250MOD_RES(5)..(5)Site of chemical conjugation 183Lys Ile Pro Pro
Cys Ile Ser Asn Trp Lys Asn1 5
1018418PRTHomo sapiensMAP2K3 Dual specificity mitogen-activated
protein kinase; P46734_C207MOD_RES(3)..(3)Site of chemical conjugation
184Lys Met Cys Asp Phe Gly Ile Ser Gly Tyr Leu Val Asp Ser Val Ala1
5 10 15Lys Thr18527PRTHomo
sapiensHSPA4 Heat shock 70 kDa protein 4;
P34932_C34MOD_RES(16)..(16)Site of chemical conjugation 185Arg Ala Gly
Gly Ile Glu Thr Ile Ala Asn Glu Tyr Ser Asp Arg Cys1 5
10 15Thr Pro Ala Cys Ile Ser Phe Gly Pro
Lys Asn 20 2518625PRTHomo sapiensCTSZ
Cathepsin Z; Q9UBR2_C92MOD_RES(12)..(12)Site of chemical conjugation
186Arg Asn Gln His Ile Pro Gln Tyr Cys Gly Ser Cys Trp Ala His Ala1
5 10 15Ser Thr Ser Ala Met Ala
Asp Arg Ile 20 2518720PRTHomo sapiensUBE2S
Ubiquitin-conjugating enzyme E2 S; Q16763_C118MOD_RES(2)..(2)Site of
chemical conjugation 187Lys Cys Leu Leu Ile His Pro Asn Pro Glu Ser Ala
Leu Asn Glu Glu1 5 10
15Ala Gly Arg Leu 2018819PRTHomo sapiensPPAT
Amidophosphoribosyltransferase; Q06203_C100MOD_RES(2)..(2)Site of
chemical conjugation 188Lys Cys Glu Leu Glu Asn Cys Gln Pro Phe Val Val
Glu Thr Leu His1 5 10
15Gly Lys Ile18918PRTHomo sapiensALDH5A1 Succinate-semialdehyde
dehydrogenase, mitochondria; P51649_C340MOD_RES(7)..(7)Site of
chemical conjugation 189Arg Asn Thr Gly Gln Thr Cys Val Cys Ser Asn Gln
Phe Leu Val Gln1 5 10
15Arg Gly19022PRTHomo sapiensMSH2 DNA mismatch repair protein Msh2;
P43246_C822MOD_RES(5)..(5)Site of chemical conjugation 190Lys Lys Gly Val
Cys Asp Gln Ser Phe Gly Ile His Val Ala Glu Leu1 5
10 15Ala Asn Phe Pro Lys His
2019130PRTHomo sapiensCAD CAD protein;
P27708_C73MOD_RES(28)..(28)Site of chemical conjugation 191Lys Ala Gln
Ile Leu Val Leu Thr Tyr Pro Leu Ile Gly Asn Tyr Gly1 5
10 15Ile Pro Pro Asp Glu Met Asp Glu Phe
Gly Leu Cys Lys Trp 20 25
3019214PRTHomo sapiensPCK2 Phosphoenolpyruvate carboxykinase;
Q16822_C306MOD_RES(11)..(11)Site of chemical conjugation 192Arg Tyr Val
Ala Ala Ala Phe Pro Ser Ala Cys Gly Lys Thr1 5
1019324PRTHomo sapiensPFKP 6-phosphofructokinase type C;
Q01813_C641MOD_RES(5)..(5)Site of chemical conjugation 193Arg Asn Glu Ser
Cys Ser Glu Asn Tyr Thr Thr Asp Phe Ile Tyr Gln1 5
10 15Leu Tyr Ser Glu Glu Gly Lys Gly
2019419PRTHomo sapiensACSF2 Acyl-CoA synthetase family member 2,
mitochondrial; Q96CM8_C64MOD_RES(16)..(16)Site of chemical conjugation
194Arg Met Val Ser Thr Pro Ile Gly Gly Leu Ser Tyr Val Gln Gly Cys1
5 10 15Thr Lys Lys19546PRTHomo
sapiensMSTO1 Protein misato homolog 1;
Q9BUK6_C403MOD_RES(38)..(38)Site of chemical conjugation 195Lys Val Val
Thr Ala Gly Ala Ile Ile Pro Phe Pro Leu Ala Pro Gly1 5
10 15Gln Ser Leu Pro Asp Ser Leu Met Gln
Phe Gly Gly Ala Thr Pro Trp 20 25
30Thr Pro Leu Ser Ala Cys Gly Glu Pro Ser Gly Thr Arg Cys 35
40 4519636PRTHomo sapiensPAICS
Multifunctional protein ADE2; P22234_C374MOD_RES(8)..(8)Site of
chemical conjugation 196Arg Leu Pro Ser Gly Leu Gly Cys Ser Thr Val Leu
Ser Pro Glu Gly1 5 10
15Ser Ala Gln Phe Ala Ala Gln Ile Phe Gly Leu Ser Asn His Leu Val
20 25 30Trp Ser Lys Leu
3519727PRTHomo sapiensDESI1 Desumoylating isopeptidase 1;
Q6ICB0_C108MOD_RES(12)..(12)Site of chemical conjugation 197Arg Gly Glu
Ala Tyr Asn Leu Phe Glu His Asn Cys Asn Thr Phe Ser1 5
10 15Asn Glu Val Ala Gln Phe Leu Thr Gly
Arg Lys 20 2519814PRTHomo sapiensPSMC6 26S
protease regulatory subunit 10B; P62333_C193MOD_RES(9)..(9)Site of
chemical conjugation 198Arg Ala Val Ala Ser Gln Leu Asp Cys Asn Phe Leu
Lys Val1 5 1019929PRTHomo sapiensTRAPPC5
Trafficking protein particle complex subunit 5;
Q8IUR0_C139MOD_RES(8)..(8)Site of chemical conjugation 199Lys Glu Asn Ser
Thr Leu Asn Cys Ala Ser Phe Thr Ala Gly Ile Val1 5
10 15Glu Ala Val Leu Thr His Ser Gly Phe Pro
Ala Lys Val 20 2520034PRTHomo sapiensPFKP
6-phosphofructokinase type C; Q01813_C179MOD_RES(19)..(19)Site of
chemical conjugation 200Lys Tyr Ala Tyr Leu Asn Val Val Gly Met Val Gly
Ser Ile Asp Asn1 5 10
15Asp Phe Cys Gly Thr Asp Met Thr Ile Gly Thr Asp Ser Ala Leu His
20 25 30Arg Ile2019PRTHomo
sapiensATP6V1A V-type proton ATPase catalytic subunit A;
P38606_C138MOD_RES(7)..(7)Site of chemical conjugation 201Lys Trp Asp Phe
Thr Pro Cys Lys Asn1 520215PRTHomo sapiensMRPS6 28S
ribosomal protein S6, mitochondrial; P82932_C105MOD_RES(3)..(3)Site
of chemical conjugation 202Lys Glu Cys Glu Gly Ile Val Pro Val Pro Leu
Ala Glu Lys Leu1 5 10
1520319PRTHomo sapiensLANCL2 LanC-like protein 2;
Q9NS86_C187MOD_RES(5)..(5)Site of chemical conjugation 203Arg Ser Val Val
Cys Gln Glu Ser Asp Leu Pro Asp Glu Leu Leu Tyr1 5
10 15Gly Arg Ala20414PRTHomo sapiensCORO1C
Coronin-1C; Q9ULV4_C456MOD_RES(8)..(8)Site of chemical conjugation
204Lys Ser Ile Lys Asp Thr Ile Cys Asn Gln Asp Glu Arg Ile1
5 1020522PRTHomo sapiensACAA1 3-ketoacyl-CoA thiolase,
peroxisomal; P09110_C381MOD_RES(17)..(17)Site of chemical
conjugation 205Lys Val Asn Pro Leu Gly Gly Ala Val Ala Leu Gly His Pro
Leu Gly1 5 10 15Cys Thr
Gly Ala Arg Gln 2020620PRTHomo sapiensGRPEL1 GrpE protein
homolog 1, mitochondrial; Q9HAV7_C124MOD_RES(5)..(5)Site of chemical
conjugation 206Lys Ala Thr Gln Cys Val Pro Lys Glu Glu Ile Lys Asp Asp
Asn Pro1 5 10 15His Leu
Lys Asn 2020712PRTHomo sapiensPPP2R5D Serine/threonine-protein
phosphatase 2A 56 kDa regulatory subunit;
Q14738_C17MOD_RES(2)..(2)Site of chemical conjugation 207Lys Cys Thr Ala
Lys Pro Ser Ser Ser Gly Lys Asp1 5
1020823PRTHomo sapiensTXN2 Thioredoxin, mitochondrial;
Q99757_C90MOD_RES(18)..(18)Site of chemical conjugation 208Arg Val Val
Asn Ser Glu Thr Pro Val Val Val Asp Phe His Ala Gln1 5
10 15Trp Cys Gly Pro Cys Lys Ile
2020931PRTHomo sapiensGALK1 Galactokinase;
P51570_C182MOD_RES(17)..(17)Site of chemical conjugation 209Arg Ala Gln
Val Cys Gln Gln Ala Glu His Ser Phe Ala Gly Met Pro1 5
10 15Cys Gly Ile Met Asp Gln Phe Ile Ser
Leu Met Gly Gln Lys Gly 20 25
3021013PRTHomo sapiensRBPJ Recombining binding protein suppressor of
hairless; Q06330_C313MOD_RES(10)..(10)Site of chemical conjugation
210Arg Ile Ile Gln Phe Gln Ala Thr Pro Cys Pro Lys Glu1 5
1021114PRTHomo sapiensMED17 Mediator of RNA polymerase II
transcription subunit; Q9NVC6_C649MOD_RES(12)..(12)Site of chemical
conjugation 211Lys Met Glu Leu Leu Met Ser Ala Leu Ser Pro Cys Leu Leu1
5 1021224PRTHomo sapiensCCT7 T-complex
protein 1 subunit eta; Q99832_C511MOD_RES(13)..(13)Site of chemical
conjugation 212Arg Ile Asn Ala Leu Thr Ala Ala Ser Glu Ala Ala Cys Leu
Ile Val1 5 10 15Ser Val
Asp Glu Thr Ile Lys Asn 2021320PRTHomo sapiensMCMBP
Mini-chromosome maintenance complex- binding protein;
Q9BTE3_C325MOD_RES(12)..(12)Site of chemical conjugation 213Lys Leu Gln
His Ile Asn Pro Leu Leu Pro Ala Cys Leu Asn Lys Glu1 5
10 15Glu Ser Lys Thr
2021416PRTHomo sapiensTK1 Thymidine kinase, cytosolic;
P04183_C230MOD_RES(12)..(12)Site of chemical conjugation 214Arg Lys Leu
Phe Ala Pro Gln Gln Ile Leu Gln Cys Ser Pro Ala Asn1 5
10 1521523PRTHomo sapiensWRNIP1 ATPase
WRNIP1; Q96S55_C272MOD_RES(20)..(20)Site of chemical conjugation
215Arg Ser Leu Leu Glu Thr Asn Glu Ile Pro Ser Leu Ile Leu Trp Gly1
5 10 15Pro Pro Gly Cys Gly Lys
Thr 2021645PRTHomo sapiensC7orf59 UPF0539 protein C7orf59;
Q0VGL1_C51MOD_RES(41)..(41)Site of chemical conjugation 216Arg Ile Pro
Asp Gln Leu Gly Tyr Leu Val Leu Ser Glu Gly Ala Val1 5
10 15Leu Ala Ser Ser Gly Asp Leu Glu Asn
Asp Glu Gln Ala Ala Ser Ala 20 25
30Ile Ser Glu Leu Val Ser Thr Ala Cys Gly Phe Arg Leu 35
40 4521717PRTHomo sapiensTHOC6 THO complex
subunit 6 homolog; Q86W42_C35MOD_RES(14)..(14)Site of chemical
conjugation 217Arg Leu His Met Thr Ile Phe Ser Gln Ser Val Ser Pro Cys
Gly Lys1 5 10
15Phe21812PRTHomo sapiensXPO1 Exportin-1;
O14980_C528MOD_RES(7)..(7)Site of chemical conjugation 218Lys Asp Leu Leu
Gly Leu Cys Glu Gln Lys Arg Gly1 5
102199PRTHomo sapiensMSH2 DNA mismatch repair protein Msh2;
P43246_C843MOD_RES(6)..(6)Site of chemical conjugation 219Lys His Val Ile
Glu Cys Ala Lys Gln1 52209PRTHomo sapiensHNRNPU
Heterogeneous nuclear ribonucleoprotein U;
Q00839_C562MOD_RES(5)..(5)Site of chemical conjugation 220Arg Ala Pro Gln
Cys Leu Gly Lys Phe1 522120PRTHomo sapiensTSEN15
tRNA-splicing endonuclease subunit Sen15;
Q8WW01_C13MOD_RES(10)..(10)Site of chemical conjugation 221Arg Gly Asp
Ser Glu Pro Thr Pro Gly Cys Ser Gly Leu Gly Pro Gly1 5
10 15Gly Val Arg Gly
2022218PRTHomo sapiensDIAPH1 Protein diaphanous homolog 1;
O60610_C1227MOD_RES(5)..(5)Site of chemical conjugation 222Arg Lys Ala
Gly Cys Ala Val Thr Ser Leu Leu Ala Ser Glu Leu Thr1 5
10 15Lys Asp22324PRTHomo sapiensAsun
Protein asunder homolog; Q9NVM9_C349MOD_RES(14)..(14)Site of
chemical conjugation 223Arg Ile Ser Pro Val Asp Val Asn Ser Arg Pro Ser
Ser Cys Leu Thr1 5 10
15Asn Phe Leu Leu Asn Gly Arg Ser 2022413PRTHomo sapiensPFAS
Phosphoribosylformylglycinamidine synthase;
O15067_C270MOD_RES(3)..(3)Site of chemical conjugation 224Lys Phe Cys Asp
Asn Ser Ser Ala Ile Gln Gly Lys Glu1 5
1022526PRTHomo sapiensATP6V1A V-type proton ATPase catalytic subunit
A; P38606_C254MOD_RES(23)..(23)Site of chemical conjugation 225Arg Val
Leu Asp Ala Leu Phe Pro Cys Val Gln Gly Gly Thr Thr Ala1 5
10 15Ile Pro Gly Ala Phe Gly Cys Gly
Lys Thr 20 2522612PRTHomo sapiensTXNDC5
Thioredoxin domain-containing protein 5;
Q8NBS9_C217MOD_RES(7)..(7)Site of chemical conjugation 226Lys Phe Phe Ala
Pro Trp Cys Gly His Cys Lys Ala1 5
1022726PRTHomo sapiensADD1 Alpha-adducin;
P35611_C68MOD_RES(12)..(12)Site of chemical conjugation 227Arg Val Ser
Met Ile Leu Gln Ser Pro Ala Phe Cys Glu Glu Leu Glu1 5
10 15Ser Met Ile Gln Glu Gln Phe Lys Lys
Gly 20 2522814PRTHomo sapiensSTAT1 Signal
transducer and activator of transcription 1;
P42224_C492MOD_RES(11)..(11)Site of chemical conjugation 228Arg Asn Leu
Ser Phe Phe Leu Thr Pro Pro Cys Ala Arg Trp1 5
1022916PRTHomo sapiensIQGAP1 Ras GTPase-activating-like protein
IQGAP1; P46940_C781MOD_RES(9)..(9)Site of chemical conjugation 229Lys Lys
Gln Ile Pro Ala Ile Thr Cys Ile Gln Ser Gln Trp Arg Gly1 5
10 1523027PRTHomo sapiensZWINT ZW10
interactor; O95229_C54MOD_RES(6)..(6)Site of chemical conjugation
230Lys Asp Lys Leu Leu Cys Ser Gln Leu Gln Val Ala Asp Phe Leu Gln1
5 10 15Asn Ile Leu Ala Gln Glu
Asp Thr Ala Lys Gly 20 2523112PRTHomo
sapiensRPL36AL 60S ribosomal protein L36a-like;
Q969Q0_C88MOD_RES(2)..(2)Site of chemical conjugation 231Arg Cys Lys His
Phe Glu Leu Gly Gly Asp Lys Lys1 5
1023223PRTHomo sapiensPDS5A Sister chromatid cohesion protein PDS5
homolog A; Q29RF7_C242MOD_RES(9)..(9)Site of chemical conjugation 232Arg
Thr Val Gln Thr Ile Glu Ala Cys Ile Ala Asn Phe Phe Asn Gln1
5 10 15Val Leu Val Leu Gly Arg Ser
2023324PRTHomo sapiensTSR1 Pre-rRNA-processing protein TSR1
homolog; Q2NL82_C126MOD_RES(21)..(21)Site of chemical conjugation
233Arg Asp Thr Gly Thr Val His Leu Asn Glu Leu Gly Asn Thr Gln Asn1
5 10 15Phe Met Leu Leu Cys Pro
Arg Leu 2023411PRTHomo sapiensNUP50 Nuclear pore complex
protein Nup50; Q9UKX7_C151MOD_RES(3)..(3)Site of chemical
conjugation 234Lys Ala Cys Val Gly Asn Ala Tyr His Lys Gln1
5 1023520PRTHomo sapiensRPL17 60S ribosomal protein
L17; P18621_C144MOD_RES(10)..(10)Site of chemical conjugation 235Arg
Ile Asn Pro Tyr Met Ser Ser Pro Cys His Ile Glu Met Ile Leu1
5 10 15Thr Glu Lys Glu
2023621PRTHomo sapiensALDH9A1 4-trimethylaminobutyraldehyde
dehydrogenase; P49189_C288MOD_RES(15)..(15)Site of chemical conjugation
236Lys Gly Ala Leu Met Ala Asn Phe Leu Thr Gln Gly Gln Val Cys Cys1
5 10 15Asn Gly Thr Arg Val
2023718PRTHomo sapiensMAP2K4 Dual specificity mitogen-activated
protein kinase; P45985_C246MOD_RES(3)..(3)Site of chemical conjugation
237Lys Leu Cys Asp Phe Gly Ile Ser Gly Gln Leu Val Asp Ser Ile Ala1
5 10 15Lys Thr23841PRTHomo
sapiensTCOF1 Treacle protein; Q13428_C1298MOD_RES(21)..(21)Site of
chemical conjugation 238Lys Lys Gly Ala Gly Asn Pro Gln Ala Ser Thr Leu
Ala Leu Gln Ser1 5 10
15Asn Ile Thr Gln Cys Leu Leu Gly Gln Pro Trp Pro Leu Asn Glu Ala
20 25 30Gln Val Gln Ala Ser Val Val
Lys Val 35 4023921PRTHomo sapiensCPPED1
Calcineurin-like phosphoesterase domain-containing;
Q9BRF8_C54MOD_RES(8)..(8)Site of chemical conjugation 239Lys Ala Trp Ser
Thr Gly Asp Cys Asp Asn Gly Gly Asp Glu Trp Glu1 5
10 15Gln Glu Ile Arg Leu
2024011PRTHomo sapiensAIP AH receptor-interacting protein;
O00170_C122MOD_RES(4)..(4)Site of chemical conjugation 240Arg His Cys Cys
Gly Val Ala Gln Met Arg Glu1 5
1024133PRTHomo sapiensDUSP12 Dual specificity protein phosphatase 12;
Q9UNI6_C265MOD_RES(5)..(5)Site of chemical conjugation 241Arg Gln Ala
Gln Cys Thr Ser Tyr Phe Ile Glu Pro Val Gln Trp Met1 5
10 15Glu Ser Ala Leu Leu Gly Val Met Asp
Gly Gln Leu Leu Cys Pro Lys 20 25
30Cys24218PRTHomo sapiensGTF2A2 Transcription initiation factor IIA
subunit 2; P52657_C68MOD_RES(3)..(3)Site of chemical conjugation
242Arg Phe Cys Asp Asn Val Trp Thr Phe Val Leu Asn Asp Val Glu Phe1
5 10 15Arg Glu24340PRTHomo
sapiensPFAS Phosphoribosylformylglycinamidine synthase;
O15067_C1287MOD_RES(36)..(36)Site of chemical conjugation 243Arg Gly Leu
Ala Pro Leu His Trp Ala Asp Asp Asp Gly Asn Pro Thr1 5
10 15Glu Gln Tyr Pro Leu Asn Pro Asn Gly
Ser Pro Gly Gly Val Ala Gly 20 25
30Ile Cys Ser Cys Asp Gly Arg His 35
4024413PRTHomo sapiensHPCAL1 Hippocalcin-like protein 1;
P37235_C185MOD_RES(5)..(5)Site of chemical conjugation 244Arg Leu Leu Gln
Cys Asp Pro Ser Ser Ala Ser Gln Phe1 5
1024514PRTHomo sapiensMVD Diphosphomevalonate decarboxylase;
P53602_C108MOD_RES(12)..(12)Site of chemical conjugation 245Arg Asp Gly
Asp Pro Leu Pro Ser Ser Leu Ser Cys Lys Val1 5
1024619PRTHomo sapiensEFHD2 EF-hand domain-containing protein D2;
Q96C19_C172MOD_RES(14)..(14)Site of chemical conjugation 246Lys Ala Ala
Ala Gly Glu Leu Gln Glu Asp Ser Gly Leu Cys Val Leu1 5
10 15Ala Arg Leu24716PRTHomo sapiensRTN4
Reticulon-4; Q9NQC3_C1101MOD_RES(12)..(12)Site of chemical
conjugation 247Lys Tyr Ser Asn Ser Ala Leu Gly His Val Asn Cys Thr Ile
Lys Glu1 5 10
1524818PRTHomo sapiensPHGDH D-3-phosphoglycerate dehydrogenase;
O43175_C369MOD_RES(6)..(6)Site of chemical conjugation 248Lys Asn Ala Gly
Asn Cys Leu Ser Pro Ala Val Ile Val Gly Leu Leu1 5
10 15Lys Glu24910PRTHomo sapiensRAD9A Cell
cycle checkpoint control protein RAD9A;
Q99638_C114MOD_RES(8)..(8)Site of chemical conjugation 249Arg Leu Val Val
Gln Leu His Cys Lys Phe1 5 1025026PRTHomo
sapiensPRDX1 Peroxiredoxin-1; Q06830_C83MOD_RES(16)..(16)Site of
chemical conjugation 250Lys Leu Asn Cys Gln Val Ile Gly Ala Ser Val Asp
Ser His Phe Cys1 5 10
15His Leu Ala Trp Val Asn Thr Pro Lys Lys 20
2525120PRTHomo sapiensSORD Sorbitol dehydrogenase;
Q00796_C45MOD_RES(8)..(8)Site of chemical conjugation 251Arg Met His Ser
Val Gly Ile Cys Gly Ser Asp Val His Tyr Trp Glu1 5
10 15Tyr Gly Arg Ile 2025217PRTHomo
sapiensCHRAC1 Chromatin accessibility complex protein 1;
Q9NRG0_C55MOD_RES(9)..(9)Site of chemical conjugation 252Lys Ala Thr Glu
Leu Phe Val Gln Cys Leu Ala Thr Tyr Ser Tyr Arg1 5
10 15His25334PRTHomo sapiensS100A11 Protein
S100-A11; P31949_C91MOD_RES(27)..(27)Site of chemical conjugation
253Lys Lys Leu Asp Thr Asn Ser Asp Gly Gln Leu Asp Phe Ser Glu Phe1
5 10 15Leu Asn Leu Ile Gly Gly
Leu Ala Met Ala Cys His Asp Ser Phe Leu 20 25
30Lys Ala25415PRTHomo sapiensTUBA8 Tubulin alpha-8
chain; Q9NY65_C347MOD_RES(9)..(9)Site of chemical conjugation 254Arg
Thr Ile Gln Phe Val Asp Trp Cys Pro Thr Gly Phe Lys Val1 5
10 1525512PRTHomo sapiensCDKN2AIP
CDKN2A-interacting protein; Q9NXV6_C516MOD_RES(9)..(9)Site of
chemical conjugation 255Lys Ser Val Tyr Leu Gly Thr Gly Cys Gly Lys Ser1
5 1025623PRTHomo sapiensPRDX4
Peroxiredoxin-4; Q13162_C51MOD_RES(7)..(7)Site of chemical
conjugation 256Arg Thr Arg Glu Glu Glu Cys His Phe Tyr Ala Gly Gly Gln
Val Tyr1 5 10 15Pro Gly
Glu Ala Ser Arg Val 2025723PRTHomo sapiensPSME4 Proteasome
activator complex subunit 4; Q14997_C1840MOD_RES(20)..(20)Site of
chemical conjugation 257Lys Gln Gln Phe Thr Asp Asp Gln Leu Leu Val Leu
Thr Asp Leu Leu1 5 10
15Val Ser Pro Cys Tyr Tyr Ala 2025821PRTHomo sapiensNCAPH
Condensin complex subunit 2; Q15003_C418MOD_RES(4)..(4)Site of
chemical conjugation 258Arg Thr Met Cys Pro Leu Leu Ser Met Lys Pro Gly
Glu Tyr Ser Tyr1 5 10
15Phe Ser Pro Arg Thr 2025912PRTHomo sapiensAMPD2 AMP
deaminase 2; Q01433_C107MOD_RES(9)..(9)Site of chemical conjugation
259Arg Ser Leu Pro Gly Pro Ala Pro Cys Leu Lys His1 5
1026012PRTHomo sapiensIDH1 Isocitrate dehydrogenase;
O75874_C269MOD_RES(10)..(10)Site of chemical conjugation 260Lys Ser Glu
Gly Gly Phe Ile Trp Ala Cys Lys Asn1 5
1026123PRTHomo sapiensPDS5A Sister chromatid cohesion protein PDS5
homolog A; Q29RF7_C532MOD_RES(16)..(16)Site of chemical conjugation
261Arg Glu Leu Leu Asp Leu His Lys Gln Pro Thr Ser Glu Ala Asn Cys1
5 10 15Ser Ala Met Phe Gly Lys
Leu 2026210PRTHomo sapiensPC Pyruvate carboxylase,
mitochondrial; P11498_C622MOD_RES(6)..(6)Site of chemical
conjugation 262Arg Phe Leu Tyr Glu Cys Pro Trp Arg Arg1 5
1026329PRTHomo sapiensTUFM Elongation factor Tu,
mitochondrial; P49411_C147MOD_RES(13)..(13)Site of chemical
conjugation 263Lys Asn Met Ile Thr Gly Thr Ala Pro Leu Asp Gly Cys Ile
Leu Val1 5 10 15Val Ala
Ala Asn Asp Gly Pro Met Pro Gln Thr Arg Glu 20
2526412PRTHomo sapiensILKAP Integrin-linked kinase-associated
serine/threonine; Q9H0C8_C301MOD_RES(2)..(2)Site of chemical conjugation
264Arg Cys Gly Val Thr Ser Val Pro Asp Ile Arg Arg1 5
1026516PRTHomo sapiensALDH6A1 Methylmalonate-semialdehyde
dehydrogenase; Q02252_C317MOD_RES(2)..(2)Site of chemical conjugation
265Arg Cys Met Ala Leu Ser Thr Ala Val Leu Val Gly Glu Ala Lys Lys1
5 10 1526618PRTHomo
sapiensPSMD14 26S proteasome non-ATPase regulatory subunit 14;
O00487_C238MOD_RES(16)..(16)Site of chemical conjugation 266Lys Ser Trp
Met Glu Gly Leu Thr Leu Gln Asp Tyr Ser Glu His Cys1 5
10 15Lys His26711PRTHomo sapiensDTYMK
Thymidylate kinase; P23919_C117MOD_RES(9)..(9)Site of chemical
conjugation 267Lys Glu Asn Phe Ser Leu Asp Trp Cys Lys Gln1
5 1026821PRTHomo sapiensZNF217 Zinc finger protein 217;
O75362_C286MOD_RES(2)..(2)Site of chemical conjugation 268Arg Cys
Ile Pro Gln Leu Asp Pro Phe Thr Thr Phe Gln Ala Trp Gln1 5
10 15Leu Ala Thr Lys Gly
2026910PRTHomo sapiensPRKAR1A cAMP-dependent protein kinase type
I-alpha regulatory subunit; P10644_C18MOD_RES(3)..(3)Site of chemical
conjugation 269Arg Glu Cys Glu Leu Tyr Val Gln Lys His1 5
1027011PRTHomo sapiensHCFC1 Host cell factor 1;
P51610_C1872MOD_RES(8)..(8)Site of chemical conjugation 270Arg Val Ala
Gly Ile Asn Ala Cys Gly Arg Gly1 5
1027114PRTHomo sapiensFAM203B Protein FAM203B;
P0CB43_C51MOD_RES(9)..(9)Site of chemical conjugation 271Arg His Val Leu
Ala Leu Thr Gly Cys Gly Pro Gly Arg Ala1 5
1027224PRTHomo sapiensPFKFB2 6-phosphofructo-2-kinase/fructose-2,6-
bisphosphata; O60825_C158MOD_RES(9)..(9)Site of chemical conjugation
272Lys Val Phe Phe Val Glu Ser Val Cys Asp Asp Pro Asp Val Ile Ala1
5 10 15Ala Asn Ile Leu Glu Val
Lys Val 2027312PRTHomo sapiensTBCE Tubulin-specific chaperone
E; Q15813_C141MOD_RES(7)..(7)Site of chemical conjugation 273Arg Asn
Cys Ala Val Ser Cys Ala Gly Glu Lys Gly1 5
1027418PRTHomo sapiensPRDX5 Peroxiredoxin-5, mitochondrial;
P30044_C100MOD_RES(15)..(15)Site of chemical conjugation 274Lys Gly Val
Leu Phe Gly Val Pro Gly Ala Phe Thr Pro Gly Cys Ser1 5
10 15Lys Thr27522PRTHomo sapiensNUP93
Nuclear pore complex protein Nup93; Q8N1F7_C522MOD_RES(19)..(19)Site
of chemical conjugation 275Lys Ser Ser Gly Gln Ser Ala Gln Leu Leu Ser
His Glu Pro Gly Asp1 5 10
15Pro Pro Cys Leu Arg Arg 2027624PRTHomo sapiensPDP1;
Q9P0J1_C149MOD_RES(14)..(14)Site of chemical conjugation 276Arg Gly Met
Leu Leu Gly Val Phe Asp Gly His Ala Gly Cys Ala Cys1 5
10 15Ser Gln Ala Val Ser Glu Arg Leu
2027719PRTHomo sapiensCTSB Cathepsin B;
P07858_C319MOD_RES(6)..(6)Site of chemical conjugation 277Arg Gly Gln Asp
His Cys Gly Ile Glu Ser Glu Val Val Ala Gly Ile1 5
10 15Pro Arg Thr27819PRTHomo sapiensACTN1
Alpha-actinin-1; P12814_C370MOD_RES(12)..(12)Site of chemical
conjugation 278Arg Met Val Ser Asp Ile Asn Asn Ala Trp Gly Cys Leu Glu
Gln Val1 5 10 15Glu Lys
Gly27921PRTHomo sapiensOGT UDP-N-acetylglucosamine--peptide N-
acetylglucosamine; O15294_C620MOD_RES(17)..(17)Site of chemical
conjugation 279Lys Val Met Ala Glu Ala Asn His Phe Ile Asp Leu Ser Gln
Ile Pro1 5 10 15Cys Asn
Gly Lys Ala 2028019PRTHomo sapiensTMPRSS12 Transmembrane
protease serine 12; Q86WS5_C64MOD_RES(12)..(12)Site of chemical
conjugation 280Arg Arg Arg Glu Gly Gly Ala His Ala Glu Asp Cys Gly Thr
Ala Pro1 5 10 15Leu Lys
Asp28113PRTHomo sapiensANLN Actin-binding protein anillin;
Q9NQW6_C712MOD_RES(7)..(7)Site of chemical conjugation 281Lys Asn Asn Ala
Phe Pro Cys Gln Val Asn Ile Lys Gln1 5
1028217PRTHomo sapiensPPP4R2 Serine/threonine-protein phosphatase 4
regulatory; Q9NY27_C22MOD_RES(4)..(4)Site of chemical conjugation 282Lys
Glu Val Cys Pro Val Leu Asp Gln Phe Leu Cys His Val Ala Lys1
5 10 15Thr28334PRTHomo sapiensSTAT3
Signal transducer and activator of transcription 3;
P40763_C259MOD_RES(14)..(14)Site of chemical conjugation 283Arg Gln Gln
Ile Ala Cys Ile Gly Gly Pro Pro Asn Ile Cys Leu Asp1 5
10 15Arg Leu Glu Asn Trp Ile Thr Ser Leu
Ala Glu Ser Gln Leu Gln Thr 20 25
30Arg Gln28433PRTHomo sapiensUGDH UDP-glucose 6-dehydrogenase;
O60701_C276MOD_RES(10)..(10)Site of chemical conjugation 284Lys Ala Ser
Val Gly Phe Gly Gly Ser Cys Phe Gln Lys Asp Val Leu1 5
10 15Asn Leu Val Tyr Leu Cys Glu Ala Leu
Asn Leu Pro Glu Val Ala Arg 20 25
30Tyr28523PRTHomo sapiensAHNAK Neuroblast differentiation-associated
protein AHNAK; Q09666_C108MOD_RES(7)..(7)Site of chemical
conjugation 285Arg Glu Val Phe Ser Ser Cys Ser Ser Glu Val Val Leu Ser
Gly Asp1 5 10 15Asp Glu
Glu Tyr Gln Arg Ile 2028611PRTHomo sapiensHINT1 Histidine
triad nucleotide-binding protein 1; P49773_C84MOD_RES(2)..(2)Site of
chemical conjugation 286Lys Cys Ala Ala Asp Leu Gly Leu Asn Lys Gly1
5 1028729PRTHomo sapiensUBR5 E3
ubiquitin-protein ligase UBR5; O95071_C2314MOD_RES(22)..(22)Site of
chemical conjugation 287Arg Ser Phe Tyr Thr Ala Ile Ala Gln Ala Phe Leu
Ser Asn Glu Lys1 5 10
15Leu Pro Asn Leu Glu Cys Ile Gln Asn Ala Asn Lys Gly 20
2528813PRTHomo sapiensSAMHD1 SAM domain and HD
domain-containing protein 1; Q9Y3Z3_C522MOD_RES(11)..(11)Site of
chemical conjugation 288Lys Asn Pro Ile Asp His Val Ser Phe Tyr Cys Lys
Thr1 5 102899PRTHomo sapiensWDHD1 WD
repeat and HMG-box DNA-binding protein 1;
O75717_C773MOD_RES(7)..(7)Site of chemical conjugation 289Lys Met Leu Ala
Leu Ser Cys Lys Leu1 529023PRTHomo sapiensOSGEP Probable
tRNA threonylcarbamoyladenosine biosynthe;
Q9NPF4_C265MOD_RES(19)..(19)Site of chemical conjugation 290Arg Ala Met
Ala His Cys Gly Ser Gln Glu Ala Leu Ile Val Gly Gly1 5
10 15Val Gly Cys Asn Val Arg Leu
2029120PRTHomo sapiensMGMT Methylated-DNA--protein-cysteine
methyltransferase; P16455_C150MOD_RES(4)..(4)Site of chemical conjugation
291Arg Val Val Cys Ser Ser Gly Ala Val Gly Asn Tyr Ser Gly Gly Leu1
5 10 15Ala Val Lys Glu
2029224PRTHomo sapiensFASN Fatty acid synthase;
P49327_C1558MOD_RES(7)..(7)Site of chemical conjugation 292Arg His Ala
Gln Pro Thr Cys Pro Gly Ala Gln Leu Cys Thr Val Tyr1 5
10 15Tyr Ala Ser Leu Asn Phe Arg Asp
2029311PRTHomo sapiensDCUN1D1 DCN1-like protein 1;
Q96GG9_C115MOD_RES(6)..(6)Site of chemical conjugation 293Arg Ala Ala Thr
Gln Cys Glu Phe Ser Lys Gln1 5
1029433PRTHomo sapiensPLS3 Plastin-3;
P13797_C33MOD_RES(23)..(23)Site of chemical conjugation 294Lys Asp Glu
Leu Asp Glu Leu Lys Glu Ala Phe Ala Lys Val Asp Leu1 5
10 15Asn Ser Asn Gly Phe Ile Cys Asp Tyr
Glu Leu His Glu Leu Phe Lys 20 25
30Glu29526PRTHomo sapiensNUP62 Nuclear pore glycoprotein p62;
P37198_C475MOD_RES(24)..(24)Site of chemical conjugation 295Lys Asp Ile
Ile Glu His Leu Asn Thr Ser Gly Ala Pro Ala Asp Thr1 5
10 15Ser Asp Pro Leu Gln Gln Ile Cys Lys
Ile 20 2529614PRTHomo sapiensADA Adenosine
deaminase; P00813_C75MOD_RES(12)..(12)Site of chemical conjugation
296Lys Phe Asp Tyr Tyr Met Pro Ala Ile Ala Gly Cys Arg Glu1
5 1029725PRTHomo sapiensKIAA0664 Clustered mitochondria
protein homolog; O75153_C333MOD_RES(22)..(22)Site of chemical
conjugation 297Arg Ile Ala Thr Pro Phe Gln Val Tyr Ser Trp Thr Ala Pro
Gln Ala1 5 10 15Glu His
Ala Met Asp Cys Val Arg Ala 20 2529820PRTHomo
sapiensC5orf51 UPF0600 protein C5orf51;
A6NDU8_C179MOD_RES(2)..(2)Site of chemical conjugation 298Arg Cys Pro Ile
Gln Leu Asn Glu Gly Val Ser Phe Gln Asp Leu Asp1 5
10 15Thr Ala Lys Leu 2029934PRTHomo
sapiensCDK19 Cyclin-dependent kinase 19;
Q9BWU1_C349MOD_RES(27)..(27)Site of chemical conjugation 299Arg Ile Thr
Ser Glu Gln Ala Leu Gln Asp Pro Tyr Phe Gln Glu Asp1 5
10 15Pro Leu Pro Thr Leu Asp Val Phe Ala
Gly Cys Gln Ile Pro Tyr Pro 20 25
30Lys Arg30013PRTHomo sapiensSTK38 Serine/threonine-protein kinase
38; Q15208_C234MOD_RES(8)..(8)Site of chemical conjugation 300Lys
Leu Ser Asp Phe Gly Leu Cys Thr Gly Leu Lys Lys1 5
1030120PRTHomo sapiensACIN1 Apoptotic chromatin condensation
inducer in the nucleus; Q9UKV3_C1223MOD_RES(5)..(5)Site of chemical
conjugation 301Lys Ala Ala Pro Cys Ile Tyr Trp Leu Pro Leu Thr Asp Ser
Gln Ile1 5 10 15Val Gln
Lys Glu 2030217PRTHomo sapiensMAPK9 Mitogen-activated protein
kinase 9; P45984_C177MOD_RES(4)..(4)Site of chemical conjugation
302Arg Thr Ala Cys Thr Asn Phe Met Met Thr Pro Tyr Val Val Thr Arg1
5 10 15Tyr30314PRTHomo
sapiensDYNC1H1 Cytoplasmic dynein 1 heavy chain 1;
Q14204_C1999MOD_RES(8)..(8)Site of chemical conjugation 303Lys Thr Ser
Ala Pro Ile Thr Cys Glu Leu Leu Asn Lys Gln1 5
1030414PRTHomo sapiensPML Protein PML;
P29590_C389MOD_RES(8)..(8)Site of chemical conjugation 304Arg Leu Gln Asp
Leu Ser Ser Cys Ile Thr Gln Gly Lys Asp1 5
1030520PRTHomo sapiensPSMC6 26S protease regulatory subunit 10B;
P62333_C228MOD_RES(6)..(6)Site of chemical conjugation 305Arg Asp His Gln
Pro Cys Ile Ile Phe Met Asp Glu Ile Asp Ala Ile1 5
10 15Gly Gly Arg Arg 2030628PRTHomo
sapiensLCP1 Plastin-2; P13796_C101MOD_RES(5)..(5)Site of chemical
conjugation 306Lys Glu Gly Ile Cys Ala Ile Gly Gly Thr Ser Glu Gln Ser
Ser Val1 5 10 15Gly Thr
Gln His Ser Tyr Ser Glu Glu Glu Lys Tyr 20
2530724PRTHomo sapiensBABAM1 BRISC and BRCA1-A complex member 1;
Q9NWV8_C222MOD_RES(11)..(11)Site of chemical conjugation 307Arg Thr Ile
Leu Val Tyr Ser Arg Pro Pro Cys Gln Pro Gln Phe Ser1 5
10 15Leu Thr Glu Pro Met Lys Lys Met
2030828PRTHomo sapiensIFIT3 Interferon-induced protein with
tetratricopeptide; O14879_C343MOD_RES(20)..(20)Site of chemical
conjugation 308Lys Gly Leu Asn Pro Leu Asn Ala Tyr Ser Asp Leu Ala Glu
Phe Leu1 5 10 15Glu Thr
Glu Cys Tyr Gln Thr Pro Phe Asn Lys Glu 20
2530913PRTHomo sapiensRASSF2 Ras association domain-containing
protein 2; P50749_C251MOD_RES(7)..(7)Site of chemical conjugation 309Arg
Ile Leu Gln Gly Pro Cys Glu Gln Ile Ser Lys Val1 5
103109PRTHomo sapiensTXNL1 Thioredoxin-like protein 1;
O43396_C34MOD_RES(3)..(3)Site of chemical conjugation 310Arg Gly Cys Gly
Pro Cys Leu Arg Ile1 531114PRTHomo sapiensNUP214 Nuclear
pore complex protein Nup214; P35658_C728MOD_RES(3)..(3)Site of
chemical conjugation 311Lys Ala Cys Phe Gln Val Gly Thr Ser Glu Glu Met
Lys Met1 5 1031213PRTHomo sapiensTMPO
Lamina-associated polypeptide 2, isoform alpha;
P42166_C684MOD_RES(11)..(11)Site of chemical conjugation 312Lys Gly Gly
Thr Leu Phe Gly Gly Glu Val Cys Lys Val1 5
1031315PRTHomo sapiensTRMT61A tRNA (adenine(58)-N(1))-
methyltransferase catalyti; Q96FX7_C209MOD_RES(8)..(8)Site of chemical
conjugation 313Arg Phe Cys Ser Phe Ser Pro Cys Ile Glu Gln Val Gln Arg
Thr1 5 10 1531450PRTHomo
sapiensTUBA1A Tubulin alpha-1A chain;
Q71U36_C200MOD_RES(35)..(35)Site of chemical conjugation 314Lys Leu Glu
Phe Ser Ile Tyr Pro Ala Pro Gln Val Ser Thr Ala Val1 5
10 15Val Glu Pro Tyr Asn Ser Ile Leu Thr
Thr His Thr Thr Leu Glu His 20 25
30Ser Asp Cys Ala Phe Met Val Asp Asn Glu Ala Ile Tyr Asp Ile Cys
35 40 45Arg Arg 5031514PRTHomo
sapiensEIF3J Eukaryotic translation initiation factor 3 subunit;
O75822_C207MOD_RES(10)..(10)Site of chemical conjugation 315Lys Ile Thr
Asn Ser Leu Thr Val Leu Cys Ser Glu Lys Gln1 5
1031645PRTHomo sapiensGRHPR Glyoxylate reductase/hydroxypyruvate
reductase; Q9UBQ7_C216MOD_RES(32)..(32)Site of chemical conjugation
316Arg Gln Pro Arg Pro Glu Glu Ala Ala Glu Phe Gln Ala Glu Phe Val1
5 10 15Ser Thr Pro Glu Leu Ala
Ala Gln Ser Asp Phe Ile Val Val Ala Cys 20 25
30Ser Leu Thr Pro Ala Thr Glu Gly Leu Cys Asn Lys Asp
35 40 4531716PRTHomo sapiensCCNH
Cyclin-H; P51946_C244MOD_RES(6)..(6)Site of chemical conjugation
317Lys Glu Asn Arg Thr Cys Leu Ser Gln Leu Leu Asp Ile Met Lys Ser1
5 10 1531837PRTHomo
sapiensALDH2 Aldehyde dehydrogenase, mitochondrial;
P05091_C319MOD_RES(31)..(31)Site of chemical conjugation 318Lys Ser Pro
Asn Ile Ile Met Ser Asp Ala Asp Met Asp Trp Ala Val1 5
10 15Glu Gln Ala His Phe Ala Leu Phe Phe
Asn Gln Gly Gln Cys Cys Cys 20 25
30Ala Gly Ser Arg Thr 3531913PRTHomo sapiensHSPBP1
Hsp70-binding protein 1; Q9NZL4_C204MOD_RES(8)..(8)Site of chemical
conjugation 319Arg Leu Leu Asp Arg Asp Ala Cys Asp Thr Val Arg Val1
5 1032013PRTHomo sapiensPMPCB
Mitochondrial-processing peptidase subunit beta;
O75439_C265MOD_RES(9)..(9)Site of chemical conjugation 320Lys Phe His Phe
Gly Asp Ser Leu Cys Thr His Lys Gly1 5
1032111PRTHomo sapiensCSRP2 Cysteine and glycine-rich protein 2;
Q16527_C33MOD_RES(2)..(2)Site of chemical conjugation 321Arg Cys Cys Phe
Leu Cys Met Val Cys Arg Lys1 5
1032211PRTHomo sapiensTBC1D15 TBC1 domain family member 15;
Q8TC07_C197MOD_RES(7)..(7)Site of chemical conjugation 322Arg Thr Leu Leu
Val Asn Cys Gln Asn Lys Ser1 5
1032313PRTHomo sapiensCTSB Cathepsin B;
P07858_C211MOD_RES(3)..(3)Site of chemical conjugation 323Lys Ile Cys Glu
Pro Gly Tyr Ser Pro Thr Tyr Lys Gln1 5
1032411PRTHomo sapiensPLEC Plectin; Q15149_C3110MOD_RES(5)..(5)Site
of chemical conjugation 324Lys Gly Arg Leu Cys Phe Glu Gly Leu Arg Ser1
5 1032513PRTHomo sapiensLIN7C Protein lin-7
homolog C; Q9NUP9_C47MOD_RES(8)..(8)Site of chemical conjugation
325Arg Val Leu Gln Ser Glu Phe Cys Asn Ala Val Arg Glu1 5
1032636PRTHomo sapiensSMC1A Structural maintenance of
chromosomes protein 1A; Q14683_C987MOD_RES(13)..(13)Site of chemical
conjugation 326Arg Glu Ala Leu Ile Glu Ile Asp Tyr Gly Asp Leu Cys Glu
Asp Leu1 5 10 15Lys Asp
Ala Gln Ala Glu Glu Glu Ile Lys Gln Glu Met Asn Thr Leu 20
25 30Gln Gln Lys Leu 3532720PRTHomo
sapiensACAA1 3-ketoacyl-CoA thiolase, peroxisomal;
P09110_C177MOD_RES(5)..(5)Site of chemical conjugation 327Lys Ala Arg Asp
Cys Leu Ile Pro Met Gly Ile Thr Ser Glu Asn Val1 5
10 15Ala Glu Arg Phe 2032819PRTHomo
sapiensMIF4GD MIF4G domain-containing protein;
A9UHW6_C49MOD_RES(14)..(14)Site of chemical conjugation 328Lys Val Ala
Asn Val Ile Val Asp His Ser Leu Gln Asp Cys Val Phe1 5
10 15Ser Lys Glu32935PRTHomo sapiensACP6
Lysophosphatidic acid phosphatase type 6;
Q9NPH0_C267MOD_RES(30)..(30)Site of chemical conjugation 329Arg Met Gly
Ile Asp Ser Ser Asp Lys Val Asp Phe Phe Ile Leu Leu1 5
10 15Asp Asn Val Ala Ala Glu Gln Ala His
Asn Leu Pro Ser Cys Pro Met 20 25
30Leu Lys Arg 3533012PRTHomo sapiensRAD50 DNA repair protein
RAD50; Q92878_C1302MOD_RES(2)..(2)Site of chemical conjugation
330Lys Cys Ser Val Ser Ser Leu Gly Phe Asn Val His1 5
1033129PRTHomo sapiensUBE2L6 Ubiquitin/ISG15-conjugating
enzyme E2 L6; O14933_C98MOD_RES(26)..(26)Site of chemical
conjugation 331Lys Ile Tyr His Pro Asn Val Asp Glu Asn Gly Gln Ile Cys
Leu Pro1 5 10 15Ile Ile
Ser Ser Glu Asn Trp Lys Pro Cys Thr Lys Thr 20
2533213PRTHomo sapiensRPL3 60S ribosomal protein L3;
P39023_C253MOD_RES(4)..(4)Site of chemical conjugation 332Lys Val Ala Cys
Ile Gly Ala Trp His Pro Ala Arg Val1 5
1033320PRTHomo sapiensSEPT9 Septin-9;
Q9UHD8_C531MOD_RES(13)..(13)Site of chemical conjugation 333Lys Trp Gly
Thr Ile Glu Val Glu Asn Thr Thr His Cys Glu Phe Ala1 5
10 15Tyr Leu Arg Asp
2033420PRTHomo sapiensTMPO Lamina-associated polypeptide 2, isoforms
beta/gam; P42167_C363MOD_RES(18)..(18)Site of chemical conjugation 334Lys
Glu Met Phe Pro Tyr Glu Ala Ser Thr Pro Thr Gly Ile Ser Ala1
5 10 15Ser Cys Arg Arg
2033516PRTHomo sapiensCASP8 Caspase-8;
Q14790_C360MOD_RES(8)..(8)Site of chemical conjugation 335Lys Val Phe Phe
Ile Gln Ala Cys Gln Gly Asp Asn Tyr Gln Lys Gly1 5
10 153368PRTHomo sapiensPLEC Plectin;
Q15149_C4574MOD_RES(5)..(5)Site of chemical conjugation 336Lys Tyr Leu
Thr Cys Pro Lys Thr1 533723PRTHomo sapiensGGA2
ADP-ribosylation factor-binding protein GGA2;
Q9UJY4_C429MOD_RES(14)..(14)Site of chemical conjugation 337Arg Asn Leu
Leu Asp Leu Leu Ser Ala Gln Pro Ala Pro Cys Pro Leu1 5
10 15Asn Tyr Val Ser Gln Lys Ser
2033824PRTHomo sapiensIRF4 Interferon regulatory factor 4;
Q15306_C194MOD_RES(16)..(16)Site of chemical conjugation 338Arg Asp Tyr
Val Pro Asp Gln Pro His Pro Glu Ile Pro Tyr Gln Cys1 5
10 15Pro Met Thr Phe Gly Pro Arg Gly
2033929PRTHomo sapiensPDE12 2,5-phosphodiesterase 12;
Q6L8Q7_C108MOD_RES(12)..(12)Site of chemical conjugation 339Lys Ser Arg
Pro Asn Ala Ser Gly Gly Ala Ala Cys Ser Gly Pro Gly1 5
10 15Pro Glu Pro Ala Val Phe Cys Glu Pro
Val Val Lys Leu 20 2534026PRTHomo
sapiensRNASEH2C Ribonuclease H2 subunit C;
Q8TDP1_C34MOD_RES(13)..(13)Site of chemical conjugation 340Arg Asp Ala
Val Pro Ala Thr Leu His Leu Leu Pro Cys Glu Val Ala1 5
10 15Val Asp Gly Pro Ala Pro Val Gly Arg
Phe 20 2534117PRTHomo sapiensFLII Protein
flightless-1 homolog; Q13045_C46MOD_RES(5)..(5)Site of chemical
conjugation 341Arg Thr Gly Leu Cys Tyr Leu Pro Glu Glu Leu Ala Ala Leu
Gln Lys1 5 10
15Leu3429PRTHomo sapiensTXNDC12 Thioredoxin domain-containing protein
12; O95881_C66MOD_RES(4)..(4)Site of chemical conjugation 342Lys Ser Trp
Cys Gly Ala Cys Lys Ala1 534311PRTHomo sapiensTRAPPC4
Trafficking protein particle complex subunit 4;
Q9Y296_C195MOD_RES(2)..(2)Site of chemical conjugation 343Arg Cys Glu Leu
Phe Asp Gln Asn Leu Lys Leu1 5
1034411PRTHomo sapiensREEP5 Receptor expression-enhancing protein 5;
Q00765_C18MOD_RES(3)..(3)Site of chemical conjugation 344Lys Asn Cys Met
Thr Asp Leu Leu Ala Lys Leu1 5
1034538PRTHomo sapiensNIT1 Nitrilase homolog 1;
Q86X76_C165MOD_RES(5)..(5)Site of chemical conjugation 345Lys Thr His Leu
Cys Asp Val Glu Ile Pro Gly Gln Gly Pro Met Cys1 5
10 15Glu Ser Asn Ser Thr Met Pro Gly Pro Ser
Leu Glu Ser Pro Val Ser 20 25
30Thr Pro Ala Gly Lys Ile 3534610PRTHomo sapiensHNRNPF
Heterogeneous nuclear ribonucleoprotein F;
P52597_C122MOD_RES(7)..(7)Site of chemical conjugation 346Arg Gly Leu Pro
Phe Gly Cys Thr Lys Glu1 5 1034723PRTHomo
sapiensERO1L ERO1-like protein alpha; Q96HE7_C37MOD_RES(4)..(4)Site
of chemical conjugation 347Arg Cys Phe Cys Gln Val Ser Gly Tyr Leu Asp
Asp Cys Thr Cys Asp1 5 10
15Val Glu Thr Ile Asp Arg Phe 2034814PRTHomo sapiensC9orf142
Uncharacterized protein C9orf142; Q9BUH6_C180MOD_RES(2)..(2)Site of
chemical conjugation 348Arg Cys Pro Gly Glu Ser Leu Ile Asn Pro Gly Phe
Lys Ser1 5 1034935PRTHomo sapiensFADD
Protein FADD; Q13158_C98MOD_RES(22)..(22)Site of chemical
conjugation 349Arg Arg Val Asp Asp Phe Glu Ala Gly Ala Ala Ala Gly Ala
Ala Pro1 5 10 15Gly Glu
Glu Asp Leu Cys Ala Ala Phe Asn Val Ile Cys Asp Asn Val 20
25 30Gly Lys Asp 3535010PRTHomo
sapiensSAE1 SUMO-activating enzyme subunit 1;
Q9UBE0_C214MOD_RES(6)..(6)Site of chemical conjugation 350Lys Lys Val Val
Phe Cys Pro Val Lys Glu1 5 1035112PRTHomo
sapiensLARS Leucine--tRNA ligase, cytoplasmic;
Q9P2J5_C554MOD_RES(7)..(7)Site of chemical conjugation 351Lys Asn Leu Glu
Thr Phe Cys Glu Glu Thr Arg Arg1 5
1035219PRTHomo sapiensQKI Protein quaking;
Q96PU8_C35MOD_RES(10)..(10)Site of chemical conjugation 352Lys Leu Met
Ser Ser Leu Pro Asn Phe Cys Gly Ile Phe Asn His Leu1 5
10 15Glu Arg Leu35316PRTHomo sapiensDNMBP
Dynamin-binding protein; Q6XZF7_C691MOD_RES(9)..(9)Site of chemical
conjugation 353Arg Ser Leu Asp Gln Thr Ser Pro Cys Pro Leu Val Leu Val
Arg Ile1 5 10
1535424PRTHomo sapiensTELO2 Telomere length regulation protein TEL2
homolog; Q9Y4R8_C628MOD_RES(20)..(20)Site of chemical conjugation 354Arg
Met Asp Ile Leu Asp Val Leu Thr Leu Ala Ala Gln Glu Leu Ser1
5 10 15Arg Pro Gly Cys Leu Gly Arg
Thr 2035514PRTHomo sapiensSPC25 Kinetochore protein Spc25;
Q9HBM1_C27MOD_RES(7)..(7)Site of chemical conjugation 355Lys Ser Thr Asp
Thr Ser Cys Gln Met Ala Gly Leu Arg Asp1 5
1035618PRTHomo sapiensTGM2 Protein-glutamine gamma-
glutamyltransferase 2; P21980_C277MOD_RES(5)..(5)Site of chemical
conjugation 356Lys Tyr Gly Gln Cys Trp Val Phe Ala Ala Val Ala Cys Thr
Val Leu1 5 10 15Arg
Cys35720PRTHomo sapiensUNC45A Protein unc-45 homolog A;
Q9H3U1_C426MOD_RES(14)..(14)Site of chemical conjugation 357Arg Ala Ile
Gln Thr Val Ser Cys Leu Leu Gln Gly Pro Cys Asp Ala1 5
10 15Gly Asn Arg Ala
2035816PRTHomo sapiensAPOBEC3C Probable DNA dC- dU-editing enzyme
APOBEC-3C; Q9NRW3_C130MOD_RES(9)..(9)Site of chemical conjugation 358Arg
Leu Tyr Tyr Phe Gln Tyr Pro Cys Tyr Gln Glu Gly Leu Arg Ser1
5 10 153598PRTHomo sapiensADAR
Double-stranded RNA-specific adenosine deaminase;
P55265_C1224MOD_RES(6)..(6)Site of chemical conjugation 359Lys Asn Phe
Tyr Leu Cys Pro Val1 536012PRTHomo sapiensIDH2 Isocitrate
dehydrogenase; P48735_C308MOD_RES(10)..(10)Site of chemical
conjugation 360Lys Ser Ser Gly Gly Phe Val Trp Ala Cys Lys Asn1
5 1036124PRTHomo sapiensPLIN3 Perilipin-3;
O60664_C39MOD_RES(12)..(12)Site of chemical conjugation 361Arg Val Ala
Ser Met Pro Leu Ile Ser Ser Thr Cys Asp Met Val Ser1 5
10 15Ala Ala Tyr Ala Ser Thr Lys Glu
2036223PRTHomo sapiensTMOD3 Tropomodulin-3;
Q9NYL9_C150MOD_RES(3)..(3)Site of chemical conjugation 362Lys Phe Cys Asn
Ile Met Gly Ser Ser Asn Gly Val Asp Gln Glu His1 5
10 15Phe Ser Asn Val Val Lys Gly
2036332PRTHomo sapiensMCCC2 Methylcrotonoyl-CoA carboxylase beta
chain, mitoch; Q9HCC0_C216MOD_RES(13)..(13)Site of chemical conjugation
363Lys Asn Ile Ala Gln Ile Ala Val Val Met Gly Ser Cys Thr Ala Gly1
5 10 15Gly Ala Tyr Val Pro Ala
Met Ala Asp Glu Asn Ile Ile Val Arg Lys 20 25
3036428PRTHomo sapiensUPP1 Uridine phosphorylase 1;
Q16831_C162MOD_RES(25)..(25)Site of chemical conjugation 364Arg Ile Gly
Thr Ser Gly Gly Ile Gly Leu Glu Pro Gly Thr Val Val1 5
10 15Ile Thr Glu Gln Ala Val Asp Thr Cys
Phe Lys Ala 20 2536520PRTHomo sapiensATOX1
Copper transport protein ATOX1; O00244_C12MOD_RES(10)..(10)Site of
chemical conjugation 365Lys His Glu Phe Ser Val Asp Met Thr Cys Gly Gly
Cys Ala Glu Ala1 5 10
15Val Ser Arg Val 2036611PRTHomo sapiensPYGB Glycogen
phosphorylase, brain form; P11216_C326MOD_RES(3)..(3)Site of
chemical conjugation 366Arg Thr Cys Phe Glu Thr Phe Pro Asp Lys Val1
5 103679PRTHomo sapiensTMPO Lamina-associated
polypeptide 2, isoform alpha; P42166_C280MOD_RES(7)..(7)Site of
chemical conjugation 367Arg Asn Leu Phe Ile Ser Cys Lys Ser1
536820PRTHomo sapiensEIF3H Eukaryotic translation initiation factor
3 subunit; O15372_C327MOD_RES(15)..(15)Site of chemical conjugation
368Arg Met Asp Ser Leu Leu Ile Ala Gly Gln Ile Asn Thr Tyr Cys Gln1
5 10 15Asn Ile Lys Glu
2036920PRTHomo sapiensPDCD2L Programmed cell death protein 2-like;
Q9BRP1_C82MOD_RES(10)..(10)Site of chemical conjugation 369Arg Leu Leu
His Val Phe Ala Cys Ala Cys Pro Gly Cys Ser Thr Gly1 5
10 15Gly Ala Arg Ser
2037010PRTHomo sapiensNCAPD2 Condensin complex subunit 1;
Q15021_C767MOD_RES(5)..(5)Site of chemical conjugation 370Lys Val Ala Cys
Cys Pro Leu Glu Arg Cys1 5 1037123PRTHomo
sapiensC21orf33 ES1 protein homolog, mitochondrial;
P30042_C177MOD_RES(14)..(14)Site of chemical conjugation 371Lys Glu Phe
His Gln Ala Gly Lys Pro Ile Gly Leu Cys Cys Ile Ala1 5
10 15Pro Val Leu Ala Ala Lys Val
2037211PRTHomo sapiensSNTB2 Beta-2-syntrophin;
Q13425_C391MOD_RES(9)..(9)Site of chemical conjugation 372Arg Leu Val His
Ser Gly Ser Gly Cys Arg Ser1 5
1037314PRTHomo sapiensMYO1C Unconventional myosin-Ic;
O00159_C802MOD_RES(2)..(2)Site of chemical conjugation 373Arg Cys Pro Glu
Asn Ala Phe Phe Leu Asp His Val Arg Thr1 5
1037412PRTHomo sapiensUBR5 E3 ubiquitin-protein ligase UBR5;
O95071_C2267MOD_RES(2)..(2)Site of chemical conjugation 374Arg Cys Ala
Thr Thr Pro Met Ala Val His Arg Val1 5
1037513PRTHomo sapiensPLOD1 Procollagen-lysine,2-oxoglutarate 5-
dioxygenase 1; Q02809_C680MOD_RES(11)..(11)Site of chemical conjugation
375Arg Val Gly Val Asp Tyr Glu Gly Gly Gly Cys Arg Phe1 5
1037614PRTHomo sapiensNACC1 Nucleus accumbens-associated
protein 1; Q96RE7_C416MOD_RES(8)..(8)Site of chemical conjugation
376Arg Asn Thr Leu Ala Asn Ser Cys Gly Thr Gly Ile Arg Ser1
5 1037728PRTHomo sapiensLIMCH1 LIM and calponin
homology domains- containing protein; Q9UPQ0_C140MOD_RES(6)..(6)Site
of chemical conjugation 377Lys Ala Ala Asn Ser Cys Thr Ser Tyr Ser Gly
Thr Thr Leu Asn Leu1 5 10
15Lys Glu Phe Glu Gly Leu Leu Ala Gln Met Arg Lys 20
2537813PRTHomo sapiensTCERG1 Transcription elongation regulator
1; O14776_C1062MOD_RES(7)..(7)Site of chemical conjugation 378Arg
Tyr Leu Val Leu Asp Cys Val Pro Glu Glu Arg Arg1 5
1037919PRTHomo sapiensDSN1 Kinetochore-associated protein DSN1
homolog; Q9H410_C287MOD_RES(5)..(5)Site of chemical conjugation 379Lys
Val Phe Asp Cys Met Glu Leu Val Met Asp Glu Leu Gln Gly Ser1
5 10 15Val Lys Gln38024PRTHomo
sapiensDLG1 Disks large homolog 1; Q12959_C378MOD_RES(9)..(9)Site of
chemical conjugation 380Lys Leu Leu Ala Val Asn Asn Val Cys Leu Glu Glu
Val Thr His Glu1 5 10
15Glu Ala Val Thr Ala Leu Lys Asn 2038110PRTHomo sapiensUSP48
Ubiquitin carboxyl-terminal hydrolase 48;
Q86UV5_C39MOD_RES(7)..(7)Site of chemical conjugation 381Arg Ile Trp Leu
Glu Pro Cys Ile Arg Gly1 5 1038211PRTHomo
sapiensSERPINH1 Serpin H1; P50454_C156MOD_RES(6)..(6)Site of
chemical conjugation 382Lys Gln His Tyr Asn Cys Glu His Ser Lys Ile1
5 1038322PRTHomo sapiensACO2 Aconitate
hydratase, mitochondrial; Q99798_C451MOD_RES(15)..(15)Site of
chemical conjugation 383Arg Asp Leu Gly Gly Ile Val Leu Ala Asn Ala Cys
Gly Pro Cys Ile1 5 10
15Gly Gln Trp Asp Arg Lys 2038414PRTHomo sapiensGMPR2 GMP
reductase 2; Q9P2T1_C186MOD_RES(10)..(10)Site of chemical
conjugation 384Lys Val Gly Ile Gly Pro Gly Ser Val Cys Thr Thr Arg Lys1
5 1038534PRTHomo sapiensTBCD
Tubulin-specific chaperone D; Q9BTW9_C850MOD_RES(19)..(19)Site of
chemical conjugation 385Lys Ala Gly Ala Pro Asp Glu Ala Val Cys Gly Glu
Asn Val Ser Gln1 5 10
15Ile Tyr Cys Ala Leu Leu Gly Cys Met Asp Asp Tyr Thr Thr Asp Ser
20 25 30Arg Gly38617PRTHomo
sapiensNUP35 Nucleoporin NUP53; Q8NFH5_C255MOD_RES(2)..(2)Site of
chemical conjugation 386Arg Cys Ala Leu Ser Ser Pro Ser Leu Ala Phe Thr
Pro Pro Ile Lys1 5 10
15Thr38712PRTHomo sapiensPYCR1 Pyrroline-5-carboxylate reductase 1,
mitochondrial; P32322_C120MOD_RES(2)..(2)Site of chemical conjugation
387Arg Cys Met Thr Asn Thr Pro Val Val Val Arg Glu1 5
1038839PRTHomo sapiensLRBA Lipopolysaccharide-responsive and
beige- like ancho; P50851_C1704MOD_RES(22)..(22)Site of chemical
conjugation 388Arg Ser Leu Val Asn Ile Pro Ala Asp Gly Val Thr Val Asp
Pro Ala1 5 10 15Leu Leu
Pro Pro Ala Cys Leu Gly Ala Leu Gly Asp Leu Ser Val Glu 20
25 30Gln Pro Val Gln Phe Arg Ser
3538919PRTHomo sapiensCTSZ Cathepsin Z;
Q9UBR2_C173MOD_RES(14)..(14)Site of chemical conjugation 389Lys Asp Gln
Glu Cys Asp Lys Phe Asn Gln Cys Gly Thr Cys Asn Glu1 5
10 15Phe Lys Glu39010PRTHomo sapiensUBR2 E3
ubiquitin-protein ligase UBR2; Q8IWV8_C1717MOD_RES(8)..(8)Site of
chemical conjugation 390Arg Gly Asn Pro Leu His Leu Cys Lys Glu1
5 1039142PRTHomo sapiensFLNB Filamin-B;
O75369_C1087MOD_RES(4)..(4)Site of chemical conjugation 391Lys Ile Glu
Cys Ser Asp Asn Gly Asp Gly Thr Cys Ser Val Ser Tyr1 5
10 15Leu Pro Thr Lys Pro Gly Glu Tyr Phe
Val Asn Ile Leu Phe Glu Glu 20 25
30Val His Ile Pro Gly Ser Pro Phe Lys Ala 35
4039223PRTHomo sapiensATG4B Cysteine protease ATG4B;
Q9Y4P1_C74MOD_RES(19)..(19)Site of chemical conjugation 392Lys Asn Phe
Pro Ala Ile Gly Gly Thr Gly Pro Thr Ser Asp Thr Gly1 5
10 15Trp Gly Cys Met Leu Arg Cys
2039310PRTHomo sapiensNEK9 Serine/threonine-protein kinase Nek9;
Q8TD19_C623MOD_RES(7)..(7)Site of chemical conjugation 393Arg Leu Leu Thr
Phe Gly Cys Asn Lys Cys1 5 1039418PRTHomo
sapiensNFU1 NFU1 iron-sulfur cluster scaffold homolog, mitocho;
Q9UMS0_C213MOD_RES(9)..(9)Site of chemical conjugation 394Lys Leu Gln Gly
Ser Cys Thr Ser Cys Pro Ser Ser Ile Ile Thr Leu1 5
10 15Lys Asn39524PRTHomo sapiensKDM4B
Lysine-specific demethylase 4B; O94953_C694MOD_RES(16)..(16)Site of
chemical conjugation 395Arg Thr Glu Pro Tyr Cys Ala Ile Cys Thr Leu Phe
Tyr Pro Tyr Cys1 5 10
15Gln Ala Leu Gln Thr Glu Lys Glu 2039633PRTHomo sapiensPLEC
Plectin; Q15149_C992MOD_RES(16)..(16)Site of chemical conjugation
396Lys Val Leu Ser Ser Ser Gly Ser Glu Ala Ala Val Pro Ser Val Cys1
5 10 15Phe Leu Val Pro Pro Pro
Asn Gln Glu Ala Gln Glu Ala Val Thr Arg 20 25
30Leu39741PRTHomo sapiensIDE Insulin-degrading enzyme;
P14735_C974MOD_RES(14)..(14)Site of chemical conjugation 397Arg Glu
Met Asp Ser Cys Pro Val Val Gly Glu Phe Pro Cys Gln Asn1 5
10 15Asp Ile Asn Leu Ser Gln Ala Pro
Ala Leu Pro Gln Pro Glu Val Ile 20 25
30Gln Asn Met Thr Glu Phe Lys Arg Gly 35
4039814PRTHomo sapiensTBC1D13 TBC1 domain family member 13;
Q9NVG8_C282MOD_RES(8)..(8)Site of chemical conjugation 398Lys Ser Leu Asp
Asp Ser Gln Cys Gly Ile Thr Tyr Lys Met1 5
1039921PRTHomo sapiensDPP9 Dipeptidyl peptidase 9;
Q86TI2_C844MOD_RES(2)..(2)Site of chemical conjugation 399Arg Cys Pro Glu
Ser Gly Glu His Tyr Glu Val Thr Leu Leu His Phe1 5
10 15Leu Gln Glu Tyr Leu
2040038PRTHomo sapiensHEXIM1 Protein HEXIM1;
O94992_C84MOD_RES(35)..(35)Site of chemical conjugation 400Arg Ala Phe
Pro Gln Leu Gly Gly Arg Pro Gly Pro Glu Gly Glu Gly1 5
10 15Ser Leu Glu Ser Gln Pro Pro Pro Leu
Gln Thr Gln Ala Cys Pro Glu 20 25
30Ser Ser Cys Leu Arg Glu 3540117PRTHomo sapiensPDSS2
Decaprenyl-diphosphate synthase subunit 2;
Q86YH6_C71MOD_RES(2)..(2)Site of chemical conjugation 401Arg Cys Leu Leu
Ser Asp Glu Leu Ser Asn Ile Ala Met Gln Val Arg1 5
10 15Lys40214PRTHomo sapiensERCC3 TFIIH basal
transcription factor complex helicase;
P19447_C342MOD_RES(9)..(9)Site of chemical conjugation 402Arg Ser Gly Val
Ile Val Leu Pro Cys Gly Ala Gly Lys Ser1 5
1040330PRTHomo sapiensMSRB2 Methionine-R-sulfoxide reductase B2,
mitochondrial; Q9Y3D2_C105MOD_RES(3)..(3)Site of chemical conjugation
403Lys Tyr Cys Ser Gly Thr Gly Trp Pro Ser Phe Ser Glu Ala His Gly1
5 10 15Thr Ser Gly Ser Asp Glu
Ser His Thr Gly Ile Leu Arg Arg 20 25
3040427PRTHomo sapiensVPS18 Vacuolar protein sorting-associated
protein 18 hom; Q9P253_C22MOD_RES(9)..(9)Site of chemical conjugation
404Arg Ser Ala Val Leu Gln Pro Gly Cys Pro Ser Val Gly Ile Pro His1
5 10 15Ser Gly Tyr Val Asn Ala
Gln Leu Glu Lys Glu 20 2540512PRTHomo
sapiensRNF40 E3 ubiquitin-protein ligase BRE1B;
O75150_C890MOD_RES(6)..(6)Site of chemical conjugation 405Arg Glu Ile Gln
Pro Cys Leu Ala Glu Ser Arg Ala1 5
1040632PRTHomo sapiensTYMS Thymidylate synthase;
P04818_C199MOD_RES(15)..(15)Site of chemical conjugation 406Arg Asp Leu
Pro Leu Met Ala Leu Pro Pro Cys His Ala Leu Cys Gln1 5
10 15Phe Tyr Val Val Asn Ser Glu Leu Ser
Cys Gln Leu Tyr Gln Arg Ser 20 25
3040723PRTHomo sapiensCDK5 Cyclin-dependent kinase 5;
Q00535_C157MOD_RES(2)..(2)Site of chemical conjugation 407Arg Cys Tyr Ser
Ala Glu Val Val Thr Leu Trp Tyr Arg Pro Pro Asp1 5
10 15Val Leu Phe Gly Ala Lys Leu
2040811PRTHomo sapiensDOCK7 Dedicator of cytokinesis protein 7;
Q96N67_C2125MOD_RES(8)..(8)Site of chemical conjugation 408Lys Ala Val
Leu Pro Val Thr Cys His Arg Asp1 5
1040910PRTHomo sapiensPAPSS2 Bifunctional 3-phosphoadenosine 5-
phosphosulfate; O95340_C350MOD_RES(7)..(7)Site of chemical conjugation
409Arg Val Trp Gly Thr Thr Cys Thr Lys His1 5
1041029PRTHomo sapiensEDC3 Enhancer of mRNA-decapping protein 3;
Q96F86_C499MOD_RES(27)..(27)Site of chemical conjugation 410Arg Ile Tyr
Leu Cys Asp Ile Gly Ile Pro Gln Gln Val Phe Gln Glu1 5
10 15Val Gly Ile Asn Tyr His Ser Pro Phe
Gly Cys Lys Phe 20 2541111PRTHomo
sapiensBLOC1S3 Biogenesis of lysosome-related organelles complex;
Q6QNY0_C168MOD_RES(5)..(5)Site of chemical conjugation 411Arg Gly Asp Leu
Cys Ala Leu Ala Glu Arg Leu1 5
1041225PRTHomo sapiensNUP54 Nucleoporin p54;
Q7Z3B4_C180MOD_RES(7)..(7)Site of chemical conjugation 412Lys Ala Val Gly
Tyr Ser Cys Met Pro Ser Asn Lys Asp Glu Asp Gly1 5
10 15Leu Val Val Leu Val Phe Asn Lys Lys
20 2541313PRTHomo sapiensUSP22 Ubiquitin
carboxyl-terminal hydrolase 22; Q9UPT9_C171MOD_RES(7)..(7)Site of
chemical conjugation 413Arg Lys Ile Thr Ser Asn Cys Thr Ile Gly Leu Arg
Gly1 5 1041421PRTHomo sapiensACADSB
Short/branched chain specific acyl-CoA dehydrogena;
P45954_C175MOD_RES(6)..(6)Site of chemical conjugation 414Lys Val Gly Ser
Phe Cys Leu Ser Glu Ala Gly Ala Gly Ser Asp Ser1 5
10 15Phe Ala Leu Lys Thr
2041515PRTHomo sapiensIKZF3 Zinc finger protein Aiolos;
Q9UKT9_C434MOD_RES(12)..(12)Site of chemical conjugation 415Arg Ser Tyr
Glu Leu Leu Lys Pro Pro Pro Ile Cys Pro Arg Asp1 5
10 1541620PRTHomo sapiensEIF2B3 Translation
initiation factor eIF-2B subunit gamma;
Q9NR50_C281MOD_RES(14)..(14)Site of chemical conjugation 416Lys Glu Ala
Asn Thr Leu Asn Leu Ala Pro Tyr Asp Ala Cys Trp Asn1 5
10 15Ala Cys Arg Gly
2041714PRTHomo sapiensRRAGC Ras-related GTP-binding protein C;
Q9HB90_C377MOD_RES(3)..(3)Site of chemical conjugation 417Arg Ser Cys Gly
His Gln Thr Ser Ala Ser Ser Leu Lys Ala1 5
1041823PRTHomo sapiensFNBP1L Formin-binding protein 1-like;
Q5T0N5_C69MOD_RES(5)..(5)Site of chemical conjugation 418Arg Phe Thr Ser
Cys Val Ala Phe Phe Asn Ile Leu Asn Glu Leu Asn1 5
10 15Asp Tyr Ala Gly Gln Arg Glu
2041913PRTHomo sapiensCTSD Cathepsin D;
P07339_C117MOD_RES(7)..(7)Site of chemical conjugation 419Lys Leu Leu Asp
Ile Ala Cys Trp Ile His His Lys Tyr1 5
1042026PRTHomo sapiensHUWE1 E3 ubiquitin-protein ligase HUWE1;
Q7Z6Z7_C3372MOD_RES(3)..(3)Site of chemical conjugation 420Lys Ala Cys
Ser Pro Cys Ser Ser Gln Ser Ser Ser Ser Gly Ile Cys1 5
10 15Thr Asp Phe Trp Asp Leu Leu Val Lys
Leu 20 2542110PRTHomo sapiensSUPT5H
Transcription elongation factor SPT5; O00267_C626MOD_RES(8)..(8)Site
of chemical conjugation 421Arg Ser Phe Ala Phe Leu His Cys Lys Lys1
5 1042210PRTHomo sapiensSMC2 Structural
maintenance of chromosomes protein 2;
O95347_C1174MOD_RES(5)..(5)Site of chemical conjugation 422Arg Phe Thr
Gln Cys Gln Asn Gly Lys Ile1 5
1042318PRTHomo sapiensGCN1L1 Translational activator GCN1;
Q92616_C648MOD_RES(8)..(8)Site of chemical conjugation 423Arg Val Leu Gln
Glu Ala Leu Cys Val Ile Ser Gly Val Pro Gly Leu1 5
10 15Lys Gly42426PRTHomo sapiensFOXK1 Forkhead
box protein K1; P85037_C254MOD_RES(20)..(20)Site of chemical
conjugation 424Arg Ser Met Val Ser Pro Val Pro Ser Pro Thr Gly Thr Ile
Ser Val1 5 10 15Pro Asn
Ser Cys Pro Ala Ser Pro Arg Gly 20
2542514PRTHomo sapiensCAPN2 Calpain-2 catalytic subunit;
P17655_C640MOD_RES(4)..(4)Site of chemical conjugation 425Lys Met Pro Cys
Gln Leu His Gln Val Ile Val Ala Arg Phe1 5
1042618PRTHomo sapiensLRBA Lipopolysaccharide-responsive and beige-
like ancho; P50851_C2675MOD_RES(2)..(2)Site of chemical conjugation
426Lys Cys Ser Gly Ile Gly Asp Asn Pro Gly Ser Glu Thr Ala Ala Pro1
5 10 15Arg Ala42726PRTHomo
sapiensMAP2K7 Dual specificity mitogen-activated protein kinase;
O14733_C131MOD_RES(20)..(20)Site of chemical conjugation 427Arg Tyr Gln
Ala Glu Ile Asn Asp Leu Glu Asn Leu Gly Glu Met Gly1 5
10 15Ser Gly Thr Cys Gly Gln Val Trp Lys
Met 20 2542837PRTHomo sapiensRCCD1 RCC1
domain-containing protein 1; A6NED2_C139MOD_RES(34)..(34)Site of
chemical conjugation 428Arg Gly Glu Pro Leu Trp Ala Gln Asn Val Val Pro
Glu Ala Glu Gly1 5 10
15Glu Asp Asp Pro Ala Gly Glu Ala Gln Ala Gly Arg Leu Pro Leu Leu
20 25 30Pro Cys Ala Arg Ala
3542916PRTHomo sapiensNUMA1 Nuclear mitotic apparatus protein 1;
Q14980_C961MOD_RES(4)..(4)Site of chemical conjugation 429Arg Gln Phe Cys
Ser Thr Gln Ala Ala Leu Gln Ala Met Glu Arg Glu1 5
10 1543022PRTHomo sapiensMLTK Mitogen-activated
protein kinase kinase kinase MLTK; Q9NYL2_C22MOD_RES(11)..(11)Site
of chemical conjugation 430Lys Phe Asp Asp Leu Gln Phe Phe Glu Asn Cys
Gly Gly Gly Ser Phe1 5 10
15Gly Ser Val Tyr Arg Ala 2043113PRTHomo sapiensBLMH
Bleomycin hydrolase; Q13867_C73MOD_RES(2)..(2)Site of chemical
conjugation 431Arg Cys Trp Ile Phe Ser Cys Leu Asn Val Met Arg Leu1
5 1043212PRTHomo sapiensNUDT16L1 Protein
syndesmos; Q9BRJ7_C88MOD_RES(9)..(9)Site of chemical conjugation
432Arg Val Leu Gly Leu Gly Leu Gly Cys Leu Arg Leu1 5
1043317PRTHomo sapiensARAP1 Arf-GAP with Rho-GAP domain, ANK
repeat and PH domain; Q96P48_C900MOD_RES(9)..(9)Site of chemical
conjugation 433Arg Ala Val Phe Pro Glu Gly Pro Cys Glu Glu Pro Leu Gln
Leu Arg1 5 10
15Lys43413PRTHomo sapiensWDR45L WD repeat domain phosphoinositide-
interacting protein; Q5MNZ6_C63MOD_RES(2)..(2)Site of chemical
conjugation 434Arg Cys Asn Tyr Leu Ala Leu Val Gly Gly Gly Lys Lys1
5 1043526PRTHomo sapiensGRAP GRB2-related
adapter protein; Q13588_C161MOD_RES(6)..(6)Site of chemical
conjugation 435Lys Ser Pro Gly Ala Cys Phe Ala Gln Ala Gln Phe Asp Phe
Ser Ala1 5 10 15Gln Asp
Pro Ser Gln Leu Ser Phe Arg Arg 20
2543617PRTHomo sapiensUncharacterized protein;
H0Y2S0_C31MOD_RES(13)..(13)Site of chemical conjugation 436Arg Tyr Thr
Gln Gln Gly Phe Gly Asn Leu Pro Ile Cys Met Ala Lys1 5
10 15Thr43714PRTHomo sapiensTPX2 Targeting
protein for Xklp2; Q9ULW0_C536MOD_RES(6)..(6)Site of chemical
conjugation 437Arg Thr Val Glu Ile Cys Pro Phe Ser Phe Asp Ser Arg Asp1
5 1043817PRTHomo sapiensXPO5 Exportin-5;
Q9HAV4_C646MOD_RES(2)..(2)Site of chemical conjugation 438Lys Cys Ala
Leu Met Glu Ala Leu Val Leu Ile Ser Asn Gln Phe Lys1 5
10 15Asn43910PRTHomo sapiensDDX59 Probable
ATP-dependent RNA helicase DDX59; Q5T1V6_C414MOD_RES(5)..(5)Site of
chemical conjugation 439Lys Asn Leu Pro Cys Ala Asn Val Arg Gln1
5 1044015PRTHomo sapiensPOLA2 DNA polymerase alpha
subunit B; Q14181_C198MOD_RES(5)..(5)Site of chemical conjugation
440Lys Val Leu Gly Cys Pro Glu Ala Leu Thr Gly Ser Tyr Lys Ser1
5 10 1544126PRTHomo sapiensRPUSD2
RNA pseudouridylate synthase domain- containing protein;
Q8IZ73_C246MOD_RES(23)..(23)Site of chemical conjugation 441Arg Leu Leu
Ala Glu Asn Glu Asp Val Val Val Val Asp Lys Pro Ser1 5
10 15Ser Ile Pro Val His Pro Cys Gly Arg
Phe 20 2544219PRTHomo sapiensKTN1 Kinectin;
Q86UP2_C303MOD_RES(12)..(12)Site of chemical conjugation 442Lys Thr
Met Met Phe Ser Glu Asp Glu Ala Leu Cys Val Val Asp Leu1 5
10 15Leu Lys Glu44316PRTHomo
sapiensLIMD1 LIM domain-containing protein 1;
Q9UGP4_C305MOD_RES(13)..(13)Site of chemical conjugation 443Arg Thr Pro
Ser Val Ser Ala Pro Leu Ala Leu Ser Cys Pro Arg Gln1 5
10 1544425PRTHomo sapiensCTH Cystathionine
gamma-lyase; P32929_C229MOD_RES(18)..(18)Site of chemical
conjugation 444Lys Tyr Met Asn Gly His Ser Asp Val Val Met Gly Leu Val
Ser Val1 5 10 15Asn Cys
Glu Ser Leu His Asn Arg Leu 20 2544518PRTHomo
sapiensHUWE1 E3 ubiquitin-protein ligase HUWE1;
Q7Z6Z7_C4367MOD_RES(11)..(11)Site of chemical conjugation 445Arg His Met
Leu Leu Leu Ala Ile Gln Glu Cys Ser Glu Gly Phe Gly1 5
10 15Leu Ala44622PRTHomo sapiensMAT2A
S-adenosylmethionine synthase isoform type-2;
P31153_C214MOD_RES(16)..(16)Site of chemical conjugation 446Arg Val His
Thr Ile Val Ile Ser Val Gln His Asp Glu Glu Val Cys1 5
10 15Leu Asp Glu Met Arg Asp
2044718PRTHomo sapiensCLIC6 Chloride intracellular channel protein 6;
Q96NY7_C487MOD_RES(12)..(12)Site of chemical conjugation 447Arg Ala Gly
Tyr Asp Gly Glu Ser Ile Gly Asn Cys Pro Phe Ser Gln1 5
10 15Arg Leu44811PRTHomo sapiensCCNB1
G2/mitotic-specific cyclin-B1; P14635_C238MOD_RES(7)..(7)Site of
chemical conjugation 448Arg Phe Met Gln Asn Asn Cys Val Pro Lys Lys1
5 1044944PRTHomo sapiensKCNAB2 Voltage-gated
potassium channel subunit beta-2; Q13303_C248MOD_RES(27)..(27)Site
of chemical conjugation 449Arg Glu Lys Val Glu Val Gln Leu Pro Glu Leu
Phe His Lys Ile Gly1 5 10
15Val Gly Ala Met Thr Trp Ser Pro Leu Ala Cys Gly Ile Val Ser Gly
20 25 30Lys Tyr Asp Ser Gly Ile Pro
Pro Tyr Ser Arg Ala 35 4045012PRTHomo sapiensXPO5
Exportin-5; Q9HAV4_C44MOD_RES(2)..(2)Site of chemical conjugation
450Lys Cys Pro Ile Cys Val Pro Cys Gly Leu Arg Leu1 5
1045111PRTHomo sapiensRNF214 RING finger protein 214;
Q8ND24_C655MOD_RES(9)..(9)Site of chemical conjugation 451Arg Ser Ser His
Ala Pro Ala Thr Cys Lys Leu1 5
1045214PRTHomo sapiensRRAGC Ras-related GTP-binding protein C;
Q9HB90_C358MOD_RES(11)..(11)Site of chemical conjugation 452Arg Lys Gly
Leu Ile Asp Tyr Asn Phe His Cys Phe Arg Lys1 5
1045312PRTHomo sapiensRAD50 DNA repair protein RAD50;
Q92878_C1296MOD_RES(6)..(6)Site of chemical conjugation 453Lys Asn Ile
Asp Gln Cys Ser Glu Ile Val Lys Cys1 5
104549PRTHomo sapiensMORC3 MORC family CW-type zinc finger protein
3; Q14149_C15MOD_RES(6)..(6)Site of chemical conjugation 454Arg Leu Ser
Ala Leu Cys Pro Lys Phe1 545546PRTHomo sapiensAPEH
Acylamino-acid-releasing enzyme; P13798_C641MOD_RES(32)..(32)Site of
chemical conjugation 455Arg Asn Pro Val Ile Asn Ile Ala Ser Met Leu Gly
Ser Thr Asp Ile1 5 10
15Pro Asp Trp Cys Val Val Glu Ala Gly Phe Pro Phe Ser Ser Asp Cys
20 25 30Leu Pro Asp Leu Ser Val Trp
Ala Glu Met Leu Asp Lys Ser 35 40
4545645PRTHomo sapiensPFKP 6-phosphofructokinase type C;
Q01813_C563MOD_RES(40)..(40)Site of chemical conjugation 456Lys His Glu
Glu Phe Cys Val Pro Met Val Met Val Pro Ala Thr Val1 5
10 15Ser Asn Asn Val Pro Gly Ser Asp Phe
Ser Ile Gly Ala Asp Thr Ala 20 25
30Leu Asn Thr Ile Thr Asp Thr Cys Asp Arg Ile Lys Gln 35
40 4545719PRTHomo sapiensMMS19 MMS19
nucleotide excision repair protein homolog;
Q96T76_C549MOD_RES(16)..(16)Site of chemical conjugation 457Arg Val Gly
Glu Ser Asn Leu Thr Asn Gly Asp Glu Pro Thr Gln Cys1 5
10 15Ser Arg His45829PRTHomo sapiensAARSD1
Alanyl-tRNA editing protein Aarsd1; Q9BTE6_C209MOD_RES(13)..(13)Site
of chemical conjugation 458Arg Val Val Asn Ile Glu Gly Val Asp Ser Asn
Met Cys Cys Gly Thr1 5 10
15His Val Ser Asn Leu Ser Asp Leu Gln Val Ile Lys Ile 20
2545918PRTHomo sapiensMTMR12 Myotubularin-related protein 12;
Q9C0I1_C694MOD_RES(14)..(14)Site of chemical conjugation 459Arg His
His Ser Gln Gln Ala Pro Gln Ala Glu Ala Pro Cys Leu Leu1 5
10 15Arg Asn46024PRTHomo sapiensCDA
Cytidine deaminase; P32320_C14MOD_RES(11)..(11)Site of chemical
conjugation 460Lys Arg Pro Ala Cys Thr Leu Lys Pro Glu Cys Val Gln Gln
Leu Leu1 5 10 15Val Cys
Ser Gln Glu Ala Lys Lys 2046116PRTHomo sapiensRRBP1
Ribosome-binding protein 1; Q9P2E9_C1323MOD_RES(14)..(14)Site of
chemical conjugation 461Lys Leu Thr Ala Glu Phe Glu Glu Ala Gln Thr Ser
Ala Cys Arg Leu1 5 10
1546215PRTHomo sapiensPOLR2B DNA-directed RNA polymerase II subunit
RPB2; P30876_C1093MOD_RES(3)..(3)Site of chemical conjugation 462Arg Asp
Cys Gln Ile Ala His Gly Ala Ala Gln Phe Leu Arg Glu1 5
10 1546317PRTHomo sapiensP4HB Protein
disulfide-isomerase; P07237_C53MOD_RES(12)..(12)Site of chemical
conjugation 463Lys Tyr Leu Leu Val Glu Phe Tyr Ala Pro Trp Cys Gly His
Cys Lys1 5 10
15Ala46443PRTHomo sapiensRPUSD2 RNA pseudouridylate synthase domain-
containing protein; Q8IZ73_C431MOD_RES(10)..(10)Site of chemical
conjugation 464Lys Gln Ser Leu Asp Val Leu Asp Leu Cys Glu Gly Asp Leu
Ser Pro1 5 10 15Gly Leu
Thr Asp Ser Thr Ala Pro Ser Ser Glu Leu Gly Lys Asp Asp 20
25 30Leu Glu Glu Leu Ala Ala Ala Ala Gln
Lys Met 35 4046515PRTHomo sapiensZCCHC8 Zinc
finger CCHC domain-containing protein 8;
Q6NZY4_C393MOD_RES(11)..(11)Site of chemical conjugation 465Arg Ile Phe
Gly Ser Ile Pro Met Gln Ala Cys Gln Gln Lys Asp1 5
10 1546614PRTHomo sapiensELP4 Elongator complex
protein 4; Q96EB1_C218MOD_RES(5)..(5)Site of chemical conjugation
466Lys Val Glu Pro Cys Ser Leu Thr Pro Gly Tyr Thr Lys Leu1
5 1046712PRTHomo sapiensPLOD3
Procollagen-lysine,2-oxoglutarate 5- dioxygenase 3;
O60568_C691MOD_RES(10)..(10)Site of chemical conjugation 467Lys Gly Leu
Asp Tyr Glu Gly Gly Gly Cys Arg Phe1 5
1046818PRTHomo sapiensNUDT8 Nucleoside diphosphate-linked moiety X
motif 8, mi; Q8WV74_C207MOD_RES(7)..(7)Site of chemical conjugation
468Arg Leu Ala Gly Leu Thr Cys Ser Gly Ala Glu Gly Leu Ala Arg Pro1
5 10 15Lys Gln46927PRTHomo
sapiensHUWE1 E3 ubiquitin-protein ligase HUWE1;
Q7Z6Z7_C4341MOD_RES(12)..(12)Site of chemical conjugation 469Arg Ser Thr
Asp Arg Leu Pro Ser Ala His Thr Cys Phe Asn Gln Leu1 5
10 15Asp Leu Pro Ala Tyr Glu Ser Phe Glu
Lys Leu 20 2547014PRTHomo sapiensMGMT
Methylated-DNA--protein-cysteine methyltransferase;
P16455_C145MOD_RES(11)..(11)Site of chemical conjugation 470Arg Gly Asn
Pro Val Pro Ile Leu Ile Pro Cys His Arg Val1 5
1047112PRTHomo sapiensNIT1 Nitrilase homolog 1;
Q86X76_C203MOD_RES(7)..(7)Site of chemical conjugation 471Lys Ile Gly Leu
Ala Val Cys Tyr Asp Met Arg Phe1 5
1047216PRTHomo sapiensACBD6 Acyl-CoA-binding domain-containing
protein 6; Q9BR61_C267MOD_RES(14)..(14)Site of chemical conjugation
472Arg Asp Gln Asp Gly Cys Leu Pro Glu Glu Val Thr Gly Cys Lys Thr1
5 10 1547332PRTHomo
sapiensIRF2BP1 Interferon regulatory factor 2-binding protein 1;
Q8IU81_C363MOD_RES(22)..(22)Site of chemical conjugation 473Arg Glu Pro
Ala Pro Ala Glu Ala Leu Pro Gln Gln Tyr Pro Glu Pro1 5
10 15Ala Pro Ala Ala Leu Cys Gly Pro Pro
Pro Arg Ala Pro Ser Arg Asn 20 25
3047427PRTHomo sapiensUSP16 Ubiquitin carboxyl-terminal hydrolase
16; Q9Y5T5_C205MOD_RES(10)..(10)Site of chemical conjugation 474Lys
Gly Leu Ser Asn Leu Gly Asn Thr Cys Phe Phe Asn Ala Val Met1
5 10 15Gln Asn Leu Ser Gln Thr Pro
Val Leu Arg Glu 20 2547515PRTHomo
sapiensANKHD1 Ankyrin repeat and KH domain-containing protein 1;
Q8IWZ3_C615MOD_RES(6)..(6)Site of chemical conjugation 475Arg Ala Gly His
Leu Cys Thr Val Gln Phe Leu Ile Ser Lys Gly1 5
10 1547639PRTHomo sapiensFADD Protein FADD;
Q13158_C105MOD_RES(29)..(29)Site of chemical conjugation 476Arg Arg Val
Asp Asp Phe Glu Ala Gly Ala Ala Ala Gly Ala Ala Pro1 5
10 15Gly Glu Glu Asp Leu Cys Ala Ala Phe
Asn Val Ile Cys Asp Asn Val 20 25
30Gly Lys Asp Trp Arg Arg Leu 3547723PRTHomo sapiensPML
Protein PML; P29590_C213MOD_RES(9)..(9)Site of chemical conjugation
477Arg Gly Cys Ser Lys Pro Leu Cys Cys Ser Cys Ala Leu Leu Asp Ser1
5 10 15Ser His Ser Glu Leu Lys
Cys 2047820PRTHomo sapiensNFKB2 Nuclear factor NF-kappa-B p100
subunit; Q00653_C57MOD_RES(4)..(4)Site of chemical conjugation
478Arg Tyr Gly Cys Glu Gly Pro Ser His Gly Gly Leu Pro Gly Ala Ser1
5 10 15Ser Glu Lys Gly
2047934PRTHomo sapiensTUBB2B Tubulin beta-2B chain;
Q9BVA1_C129MOD_RES(8)..(8)Site of chemical conjugation 479Lys Glu Ser Glu
Ser Cys Asp Cys Leu Gln Gly Phe Gln Leu Thr His1 5
10 15Ser Leu Gly Gly Gly Thr Gly Ser Gly Met
Gly Thr Leu Leu Ile Ser 20 25
30Lys Ile48018PRTHomo sapiensCLIC5 Chloride intracellular channel
protein 5; Q9NZA1_C191MOD_RES(12)..(12)Site of chemical conjugation
480Lys Ala Gly Ile Asp Gly Glu Ser Ile Gly Asn Cys Pro Phe Ser Gln1
5 10 15Arg Leu48115PRTHomo
sapiensNMT2 Glycylpeptide N-tetradecanoyltransferase 2;
O60551_C104MOD_RES(9)..(9)Site of chemical conjugation 481Arg Ala Met Glu
Leu Leu Ser Ala Cys Gln Gly Pro Ala Arg Asn1 5
10 1548218PRTHomo sapiensCDKN3 Cyclin-dependent
kinase inhibitor 3; Q16667_C39MOD_RES(4)..(4)Site of chemical
conjugation 482Arg Val Asn Cys Ser Gln Phe Leu Gly Leu Cys Ala Leu Pro
Gly Cys1 5 10 15Lys
Phe48314PRTHomo sapiensSBNO1 Protein strawberry notch homolog 1;
A3KN83_C445MOD_RES(4)..(4)Site of chemical conjugation 483Lys Asn Leu Cys
Pro Val Gly Ser Ser Lys Pro Thr Lys Thr1 5
1048410PRTHomo sapiensC12orf29 Uncharacterized protein C12orf29;
Q8N999_C302MOD_RES(2)..(2)Site of chemical conjugation 484Lys Cys Leu Phe
Asn His Phe Leu Lys Ile1 5 1048511PRTHomo
sapiensHSDL2 Hydroxysteroid dehydrogenase-like protein 2;
Q6YN16_C166MOD_RES(4)..(4)Site of chemical conjugation 485Lys Gln His Cys
Ala Tyr Thr Ile Ala Lys Tyr1 5
1048624PRTHomo sapiensPLEKHA2 Pleckstrin homology domain-containing
family A mem; Q9HB19_C332MOD_RES(22)..(22)Site of chemical conjugation
486Arg Ser Ile Ser Leu Thr Arg Pro Gly Ser Ser Ser Leu Ser Ser Gly1
5 10 15Pro Asn Ser Ile Leu Cys
Arg Gly 2048728PRTHomo sapiensHNRPLL Heterogeneous nuclear
ribonucleoprotein L-like; Q8WVV9_C464MOD_RES(19)..(19)Site of
chemical conjugation 487Lys Asn Ile Ile Gln Pro Pro Ser Cys Val Leu His
Tyr Tyr Asn Val1 5 10
15Pro Leu Cys Val Thr Glu Glu Thr Phe Thr Lys Leu 20
2548843PRTHomo sapiensCNOT3 CCR4-NOT transcription complex subunit
3; O75175_C600MOD_RES(33)..(33)Site of chemical conjugation 488Arg
Asp Ile Ile Leu Ser Ser Thr Ser Ala Pro Pro Ala Ser Ala Gln1
5 10 15Pro Pro Leu Gln Leu Ser Glu
Val Asn Ile Pro Leu Ser Leu Gly Val 20 25
30Cys Pro Leu Gly Pro Val Pro Leu Thr Lys Glu 35
4048910PRTHomo sapiensMEPCE 7SK snRNA methylphosphate capping
enzyme; Q7L2J0_C419MOD_RES(8)..(8)Site of chemical conjugation
489Lys Phe Gln Tyr Gly Asn Tyr Cys Lys Tyr1 5
1049026PRTHomo sapiensARL3 ADP-ribosylation factor-like protein 3;
P36405_C174MOD_RES(24)..(24)Site of chemical conjugation 490Arg Val Trp
Gln Ile Gln Ser Cys Ser Ala Leu Thr Gly Glu Gly Val1 5
10 15Gln Asp Gly Met Asn Trp Val Cys Lys
Asn 20 2549118PRTHomo sapiensAMMECR1 AMME
syndrome candidate gene 1 protein; Q9Y4X0_C175MOD_RES(3)..(3)Site of
chemical conjugation 491Arg Gly Cys Ile Gly Thr Phe Ser Ala Met Asn Leu
His Ser Gly Leu1 5 10
15Arg Glu49221PRTHomo sapiensFAM120A Constitutive coactivator of
PPAR-gamma- like protein; Q9NZB2_C531MOD_RES(13)..(13)Site of
chemical conjugation 492Lys Gly Ser Gln Met Gly Thr Val Gln Pro Ile Pro
Cys Leu Leu Ser1 5 10
15Met Pro Thr Arg Asn 2049312PRTHomo sapiensDNMT1 DNA
(cytosine-5)-methyltransferase 1; P26358_C1478MOD_RES(6)..(6)Site of
chemical conjugation 493Arg Gly Val Cys Ser Cys Val Glu Ala Gly Lys Ala1
5 104949PRTHomo sapiensCXorf38
Uncharacterized protein CXorf38; Q8TB03_C12MOD_RES(4)..(4)Site of
chemical conjugation 494Arg Leu Asn Cys Ala Glu Tyr Lys Asn1
549526PRTHomo sapiensMRPS18B 28S ribosomal protein S18b,
mitochondrial; Q9Y676_C128MOD_RES(8)..(8)Site of chemical conjugation
495Lys Leu Leu Glu Gln Phe Val Cys Ala His Thr Gly Ile Ile Phe Tyr1
5 10 15Ala Pro Tyr Thr Gly Val
Cys Val Lys Gln 20 2549614PRTHomo
sapiensFHOD1 FH1/FH2 domain-containing protein 1;
Q9Y613_C502MOD_RES(9)..(9)Site of chemical conjugation 496Arg Thr Pro Gln
Ser Pro Ala Pro Cys Val Leu Leu Arg Ala1 5
1049735PRTHomo sapiensVRK1 Serine/threonine-protein kinase VRK1;
Q99986_C50MOD_RES(13)..(13)Site of chemical conjugation 497Lys Val Gly
Leu Pro Ile Gly Gln Gly Gly Phe Gly Cys Ile Tyr Leu1 5
10 15Ala Asp Met Asn Ser Ser Glu Ser Val
Gly Ser Asp Ala Pro Cys Val 20 25
30Val Lys Val 3549844PRTHomo sapiensSEC24C Protein transport
protein Sec24C; P53992_C78MOD_RES(18)..(18)Site of chemical
conjugation 498Arg Ala Pro Pro Ser Ser Gly Ala Pro Pro Ala Ser Thr Ala
Gln Ala1 5 10 15Pro Cys
Gly Gln Ala Ala Tyr Gly Gln Phe Gly Gln Gly Asp Val Gln 20
25 30Asn Gly Pro Ser Ser Thr Val Gln Met
Gln Arg Leu 35 4049924PRTHomo sapiensMED15
Mediator of RNA polymerase II transcription subuni;
Q96RN5_C660MOD_RES(21)..(21)Site of chemical conjugation 499Arg Thr Phe
Val Pro Ala Met Thr Ala Ile His Gly Pro Pro Ile Thr1 5
10 15Ala Pro Val Val Cys Thr Arg Lys
2050020PRTHomo sapiensINF2 Inverted formin-2 L;
Q27J81_C332MOD_RES(12)..(12)Site of chemical conjugation 500Arg Ala Val
Leu Leu Ala Ser Asp Ala Gln Glu Cys Thr Leu Glu Glu1 5
10 15Val Val Glu Arg
2050117PRTHomo sapiensINF2 Inverted formin-2;
Q27J81_C971MOD_RES(6)..(6)Site of chemical conjugation 501Lys Gln Glu Glu
Val Cys Val Ile Asp Ala Leu Leu Ala Asp Ile Arg1 5
10 15Lys50215PRTHomo sapiensATXN10 Ataxin-10;
Q9UBB4_C356MOD_RES(12)..(12)Site of chemical conjugation 502Lys Glu
Thr Thr Asn Ile Phe Ser Asn Cys Gly Cys Val Arg Ala1 5
10 1550342PRTHomo sapiensGOLGA3 Golgin
subfamily A member 3; Q08378_C1403MOD_RES(18)..(18)Site of chemical
conjugation 503Lys Gly Glu Ala Ser Ser Ser Asn Pro Ala Thr Pro Ile Lys
Ile Pro1 5 10 15Asp Cys
Pro Val Pro Ala Ser Leu Leu Glu Glu Leu Leu Arg Pro Pro 20
25 30Pro Ala Val Ser Lys Glu Pro Leu Lys
Asn 35 4050414PRTHomo sapiensLMO7 LIM domain only
protein 7; Q8WWI1_C228MOD_RES(10)..(10)Site of chemical conjugation
504Arg Asp Ser Gly Tyr Gly Asp Ile Trp Cys Pro Glu Arg Gly1
5 1050517PRTHomo sapiensARAF Serine/threonine-protein
kinase A-Raf; P10398_C597MOD_RES(10)..(10)Site of chemical
conjugation 505Arg Thr Gln Ala Asp Glu Leu Pro Ala Cys Leu Leu Ser Ala
Ala Arg1 5 10
15Leu50622PRTHomo sapiensACTR10 Actin-related protein 10;
Q9NZ32_C388MOD_RES(6)..(6)Site of chemical conjugation 506Arg Ile Pro Asp
Trp Cys Ser Leu Asn Asn Pro Pro Leu Glu Met Met1 5
10 15Phe Asp Val Gly Lys Thr
2050718PRTHomo sapiensNOP58 Nucleolar protein 58;
Q9Y2X3_C106MOD_RES(6)..(6)Site of chemical conjugation 507Lys Leu Asn Leu
Ser Cys Ile His Ser Pro Val Val Asn Glu Leu Met1 5
10 15Arg Gly50842PRTHomo sapiensACLY
ATP-citrate synthase; P53396_C764MOD_RES(30)..(30)Site of chemical
conjugation 508Arg Leu Thr Lys Pro Ile Val Cys Trp Cys Ile Gly Thr Cys
Ala Thr1 5 10 15Met Phe
Ser Ser Glu Val Gln Phe Gly His Ala Gly Ala Cys Ala Asn 20
25 30Gln Ala Ser Glu Thr Ala Val Ala Lys
Asn 35 4050920PRTHomo sapiensRPL7 60S ribosomal
protein L7; P18124_C186MOD_RES(6)..(6)Site of chemical conjugation
509Lys Tyr Gly Ile Ile Cys Met Glu Asp Leu Ile His Glu Ile Tyr Thr1
5 10 15Val Gly Lys Arg
2051026PRTHomo sapiensMSTO1 Protein misato homolog 1;
Q9BUK6_C485MOD_RES(13)..(13)Site of chemical conjugation 510Arg Val Ala
Pro Pro Tyr Pro His Leu Phe Ser Ser Cys Ser Pro Pro1 5
10 15Gly Met Val Leu Asp Gly Ser Pro Lys
Gly 20 2551118PRTHomo sapiensPES1 Pescadillo
homolog; O00541_C272MOD_RES(14)..(14)Site of chemical conjugation
511Lys Ala Gly Glu Gly Thr Tyr Ala Leu Asp Ser Glu Ser Cys Met Glu1
5 10 15Lys Leu51213PRTHomo
sapiensVDAC3 Voltage-dependent anion-selective channel protein;
Q9Y277_C65MOD_RES(3)..(3)Site of chemical conjugation 512Lys Val Cys Asn
Tyr Gly Leu Thr Phe Thr Gln Lys Trp1 5
1051313PRTHomo sapiensIRF8 Interferon regulatory factor 8;
Q02556_C306MOD_RES(11)..(11)Site of chemical conjugation 513Arg Val Phe
Cys Ser Gly Asn Ala Val Val Cys Lys Gly1 5
1051411PRTHomo sapiensSPTBN1 Spectrin beta chain, non-erythrocytic 1;
Q01082_C112MOD_RES(4)..(4)Site of chemical conjugation 514Arg Ile His
Cys Leu Glu Asn Val Asp Lys Ala1 5
1051526PRTHomo sapiensZC3H12D Probable ribonuclease ZC3H12D;
A2A288_C367MOD_RES(20)..(20)Site of chemical conjugation 515Arg Leu Ala
Phe Ser Asp Asp Leu Gly Pro Leu Gly Pro Pro Leu Pro1 5
10 15Val Pro Ala Cys Ser Leu Thr Pro Arg
Leu 20 2551623PRTHomo sapiensCASK Peripheral
plasma membrane protein CASK; O14936_C914MOD_RES(11)..(11)Site of
chemical conjugation 516Arg His Leu Glu Glu Ala Val Glu Leu Val Cys Thr
Ala Pro Gln Trp1 5 10
15Val Pro Val Ser Trp Val Tyr 2051714PRTHomo sapiensSART1
U4/U6.U5 tri-snRNP-associated protein 1;
O43290_C645MOD_RES(10)..(10)Site of chemical conjugation 517Arg Gly Leu
Ala Ala Ala Leu Leu Leu Cys Gln Asn Lys Gly1 5
1051827PRTHomo sapiensPEF1 Peflin;
Q9UBV8_C146MOD_RES(7)..(7)Site of chemical conjugation 518Lys Gln Ala Leu
Val Asn Cys Asn Trp Ser Ser Phe Asn Asp Glu Thr1 5
10 15Cys Leu Met Met Ile Asn Met Phe Asp Lys
Thr 20 2551921PRTHomo sapiensRSBN1L Round
spermatid basic protein 1-like protein;
Q6PCB5_C280MOD_RES(7)..(7)Site of chemical conjugation 519Lys Ser Ile Gln
Thr Ile Cys Ser Gly Leu Leu Thr Asp Val Glu Asp1 5
10 15Gln Ala Ala Lys Gly
2052018PRTHomo sapiensLGALS3BP Galectin-3-binding protein;
Q08380_C561MOD_RES(8)..(8)Site of chemical conjugation 520Lys Ser Thr Ser
Ser Phe Pro Cys Pro Ala Gly His Phe Asn Gly Phe1 5
10 15Arg Thr52110PRTHomo sapiensCORO7
Coronin-7; P57737_C34MOD_RES(8)..(8)Site of chemical conjugation
521Arg Ala Gly Thr Ala Pro Ser Cys Arg Asn1 5
1052211PRTHomo sapiensGCN1L1 Translational activator GCN1;
Q92616_C1235MOD_RES(2)..(2)Site of chemical conjugation 522Arg Cys Gly
Leu Ala Leu Ala Leu Asn Lys Leu1 5
1052311PRTHomo sapiensNR2F2 COUP transcription factor 2;
P24468_C326MOD_RES(2)..(2)Site of chemical conjugation 523Arg Cys Gly Leu
Ala Leu Ala Leu Asn Lys Leu1 5
1052426PRTHomo sapiensGFM1 Elongation factor G, mitochondrial;
Q96RP9_C153MOD_RES(18)..(18)Site of chemical conjugation 524Arg Val Leu
Asp Gly Ala Val Leu Val Leu Cys Ala Val Gly Gly Val1 5
10 15Gln Cys Gln Thr Met Thr Val Asn Arg
Gln 20 2552521PRTHomo sapiensPOLE3 DNA
polymerase epsilon subunit 3; Q9NRF9_C51MOD_RES(13)..(13)Site of
chemical conjugation 525Arg Ala Ala Ser Val Phe Val Leu Tyr Ala Thr Ser
Cys Ala Asn Asn1 5 10
15Phe Ala Met Lys Gly 2052625PRTHomo sapiensALDH1B1 Aldehyde
dehydrogenase X, mitochondrial; P30837_C179MOD_RES(8)..(8)Site of
chemical conjugation 526Arg His Glu Pro Val Gly Val Cys Gly Gln Ile Ile
Pro Trp Asn Phe1 5 10
15Pro Leu Val Met Gln Gly Trp Lys Leu 20
2552717PRTHomo sapiensPDCD6IP Programmed cell death 6-interacting
protein; Q8WUM4_C90MOD_RES(10)..(10)Site of chemical conjugation 527Lys
Phe Pro Phe Ser Glu Asn Gln Ile Cys Leu Thr Phe Thr Trp Lys1
5 10 15Asp52821PRTHomo sapiensALG13
UDP-N-acetylglucosamine transferase subunit ALG13;
Q9NP73_C86MOD_RES(14)..(14)Site of chemical conjugation 528Lys Ala Asp
Leu Val Ile Ser His Ala Gly Ala Gly Ser Cys Leu Glu1 5
10 15Thr Leu Glu Lys Gly
2052938PRTHomo sapiensPDIA4 Protein disulfide-isomerase A4;
P13667_C91MOD_RES(33)..(33)Site of chemical conjugation 529Lys Glu Glu
Asn Gly Val Leu Val Leu Asn Asp Ala Asn Phe Asp Asn1 5
10 15Phe Val Ala Asp Lys Asp Thr Val Leu
Leu Glu Phe Tyr Ala Pro Trp 20 25
30Cys Gly His Cys Lys Gln 3553013PRTHomo sapiensPOLA1 DNA
polymerase alpha catalytic subunit; P09884_C1403MOD_RES(8)..(8)Site
of chemical conjugation 530Arg Tyr Ile Phe Asp Ala Glu Cys Ala Leu Glu
Lys Leu1 5 1053118PRTHomo sapiensCLIC3
Chloride intracellular channel protein 3;
O95833_C22MOD_RES(12)..(12)Site of chemical conjugation 531Lys Ala Ser
Glu Asp Gly Glu Ser Val Gly His Cys Pro Ser Cys Gln1 5
10 15Arg Leu53223PRTHomo sapiensECHDC1
Ethylmalonyl-CoA decarboxylase; Q9NTX5_C133MOD_RES(13)..(13)Site of
chemical conjugation 532Lys Ser Leu Gly Thr Pro Glu Asp Gly Met Ala Val
Cys Met Phe Met1 5 10
15Gln Asn Thr Leu Thr Arg Phe 2053318PRTHomo sapiensPTK2B
Protein-tyrosine kinase 2-beta; Q14289_C899MOD_RES(5)..(5)Site of
chemical conjugation 533Lys Asn Glu Leu Cys Gln Leu Pro Pro Glu Gly Tyr
Val Val Val Val1 5 10
15Lys Asn53413PRTHomo sapiensRANBP2 E3 SUMO-protein ligase RanBP2;
P49792_C815MOD_RES(4)..(4)Site of chemical conjugation 534Lys Met Ile Cys
Gln Gln Val Glu Ala Ile Lys Lys Glu1 5
1053516PRTHomo sapiensBOP1 Ribosome biogenesis protein BOP1;
Q14137_C404MOD_RES(9)..(9)Site of chemical conjugation 535Arg Asp Leu Gln
Pro Phe Pro Thr Cys Gln Ala Leu Val Tyr Arg Gly1 5
10 1553611PRTHomo sapiensLRRC41 Leucine-rich
repeat-containing protein 41; Q15345_C297MOD_RES(2)..(2)Site of
chemical conjugation 536Arg Cys Ala Ala Ala Leu Met Ala Ser Arg Arg1
5 1053713PRTHomo sapiensLGMN Legumain;
Q99538_C219MOD_RES(7)..(7)Site of chemical conjugation 537Arg Glu Ser Ser
Tyr Ala Cys Tyr Tyr Asp Glu Lys Arg1 5
1053819PRTHomo sapiensTHAP11 THAP domain-containing protein 11;
Q96EK4_C48MOD_RES(7)..(7)Site of chemical conjugation 538Arg Ala Gly Val
Ser Gly Cys Phe Ser Thr Phe Gln Pro Thr Thr Gly1 5
10 15His Arg Leu53915PRTHomo sapiensACSL4
Long-chain-fatty-acid--CoA ligase 4; O60488_C420MOD_RES(8)..(8)Site
of chemical conjugation 539Lys Gly Tyr Asp Ala Pro Leu Cys Asn Leu Leu
Leu Phe Lys Lys1 5 10
1554018PRTHomo sapiensFAM125A Multivesicular body subunit 12A;
Q96EY5_C231MOD_RES(3)..(3)Site of chemical conjugation 540Lys Ser Cys Ser
Pro Leu Ala Phe Ser Ala Phe Gly Asp Leu Thr Ile1 5
10 15Lys Ser54144PRTHomo sapiensSCP2
Non-specific lipid-transfer protein;
P22307_C307MOD_RES(17)..(17)Site of chemical conjugation 541Lys Ser Gly
Leu Thr Pro Asn Asp Ile Asp Val Ile Glu Leu His Asp1 5
10 15Cys Phe Ser Thr Asn Glu Leu Leu Thr
Tyr Glu Ala Leu Gly Leu Cys 20 25
30Pro Glu Gly Gln Gly Ala Thr Leu Val Asp Arg Gly 35
4054224PRTHomo sapiensTBRG4 Protein TBRG4;
Q969Z0_C335MOD_RES(21)..(21)Site of chemical conjugation 542Arg Leu Ala
Thr Asp Leu Leu Ser Leu Met Pro Ser Leu Thr Ser Gly1 5
10 15Glu Val Ala His Cys Ala Lys Ser
2054318PRTHomo sapiensMAP2K3 Dual specificity mitogen-activated
protein kinase; P46734_C29MOD_RES(4)..(4)Site of chemical conjugation
543Arg Ile Ser Cys Met Ser Lys Pro Pro Ala Pro Asn Pro Thr Pro Pro1
5 10 15Arg Asn54424PRTHomo
sapiensC3orf38 Uncharacterized protein C3orf38;
Q5JPI3_C308MOD_RES(17)..(17)Site of chemical conjugation 544Lys Phe Glu
Gln Ser Asp Leu Glu Ala Phe Tyr Asn Val Ile Thr Val1 5
10 15Cys Gly Thr Asn Glu Val Arg His
2054545PRTHomo sapiensEIF4ENIF1 Eukaryotic translation initiation
factor 4E transp; Q9NRA8_C318MOD_RES(26)..(26)Site of chemical
conjugation 545Arg Asp Ala Val Leu Pro Glu Gln Ser Pro Gly Asp Phe Asp
Phe Asn1 5 10 15Glu Phe
Phe Asn Leu Asp Lys Val Pro Cys Leu Ala Ser Met Ile Glu 20
25 30Asp Val Leu Gly Glu Gly Ser Val Ser
Ala Ser Arg Phe 35 40
4554621PRTHomo sapiensMED1 Mediator of RNA polymerase II
transcription subuni; Q15648_C135MOD_RES(12)..(12)Site of chemical
conjugation 546Lys Val Ala His His Gly Glu Asn Pro Val Ser Cys Pro Glu
Leu Val1 5 10 15Gln Gln
Leu Arg Glu 2054717PRTHomo sapiensTRMT2A tRNA
(uracil-5-)-methyltransferase homolog A;
Q8IZ69_C463MOD_RES(8)..(8)Site of chemical conjugation 547Arg Val Ile Gly
Val Glu Leu Cys Pro Glu Ala Val Glu Asp Ala Arg1 5
10 15Val5488PRTHomo sapiensRELA Transcription
factor p65; Q04206_C38MOD_RES(4)..(4)Site of chemical conjugation
548Arg Tyr Lys Cys Glu Gly Arg Ser1 554915PRTHomo
sapiensTAPBP Tapasin; O15533_C115MOD_RES(12)..(12)Site of chemical
conjugation 549Lys Trp Ala Ser Gly Leu Thr Pro Ala Gln Asn Cys Pro Arg
Ala1 5 10 1555031PRTHomo
sapiensDUSP12 Dual specificity protein phosphatase 12;
Q9UNI6_C23MOD_RES(4)..(4)Site of chemical conjugation 550Arg Val Ser Cys
Ala Gly Gln Met Leu Glu Val Gln Pro Gly Leu Tyr1 5
10 15Phe Gly Gly Ala Ala Ala Val Ala Glu Pro
Asp His Leu Arg Glu 20 25
3055115PRTHomo sapiensHTATIP2 Oxidoreductase HTATIP2;
Q9BUP3_C172MOD_RES(12)..(12)Site of chemical conjugation 551Arg Tyr Ser
Val Phe Arg Pro Gly Val Leu Leu Cys Asp Arg Gln1 5
10 1555219PRTHomo sapiensNUBP1 Cytosolic Fe-S
cluster assembly factor NUBP1; P53384_C235MOD_RES(16)..(16)Site of
chemical conjugation 552Lys Leu Pro Ile Ile Gly Val Val Glu Asn Met Ser
Gly Phe Ile Cys1 5 10
15Pro Lys Cys55325PRTHomo sapiensWAPAL Wings apart-like protein homolog;
Q7Z5K2_C293MOD_RES(23)..(23)Site of chemical conjugation 553Arg Leu
Glu Asn Leu Asn Glu Ala Ile Glu Glu Asp Ile Val Gln Ser1 5
10 15Val Leu Arg Pro Thr Asn Cys Arg
Thr 20 2555421PRTHomo sapiensCDC42BPB
Serine/threonine-protein kinase MRCK beta;
Q9Y5S2_C1517MOD_RES(16)..(16)Site of chemical conjugation 554Arg Ile Arg
Pro Leu Asn Ser Glu Gly Thr Leu Asn Leu Leu Asn Cys1 5
10 15Glu Pro Pro Arg Leu
205559PRTHomo sapiensEXOSC7 Exosome complex component RRP42;
Q15024_C238MOD_RES(6)..(6)Site of chemical conjugation 555Lys Gly Val Val
Thr Cys Met Arg Lys1 555618PRTHomo sapiensPEX19 Peroxisomal
biogenesis factor 19; P40855_C128MOD_RES(15)..(15)Site of chemical
conjugation 556Arg Val Gly Ser Asp Met Thr Ser Gln Gln Glu Phe Thr Ser
Cys Leu1 5 10 15Lys
Glu5579PRTHomo sapiensEZH2 Histone-lysine N-methyltransferase EZH2;
Q15910_C503MOD_RES(7)..(7)Site of chemical conjugation 557Arg Leu Trp Ala
Ala His Cys Arg Lys1 555819PRTHomo sapiensPLEC Plectin;
Q15149_C4071MOD_RES(8)..(8)Site of chemical conjugation 558Lys Phe Leu
Glu Gly Thr Ser Cys Ile Ala Gly Val Phe Val Asp Ala1 5
10 15Thr Lys Glu55933PRTHomo sapiensSCP2
Non-specific lipid-transfer protein; P22307_C94MOD_RES(21)..(21)Site
of chemical conjugation 559Arg Ala Ile Tyr His Ser Leu Gly Met Thr Gly
Ile Pro Ile Ile Asn1 5 10
15Val Asn Asn Asn Cys Ala Thr Gly Ser Thr Ala Leu Phe Met Ala Arg
20 25 30Gln56020PRTHomo
sapiensGTF3C1 General transcription factor 3C polypeptide 1;
Q12789_C42MOD_RES(12)..(12)Site of chemical conjugation 560Arg Val Pro
Pro Phe Pro Leu Pro Leu Glu Pro Cys Thr Gln Glu Phe1 5
10 15Leu Trp Arg Ala
2056138PRTHomo sapiensSMAD2 Mothers against decapentaplegic homolog
2; Q15796_C81MOD_RES(9)..(9)Site of chemical conjugation 561Lys Cys Val
Thr Ile Pro Ser Thr Cys Ser Glu Ile Trp Gly Leu Ser1 5
10 15Thr Pro Asn Thr Ile Asp Gln Trp Asp
Thr Thr Gly Leu Tyr Ser Phe 20 25
30Ser Glu Gln Thr Arg Ser 3556224PRTHomo sapiensBAG5 BAG
family molecular chaperone regulator 5;
Q9UL15_C327MOD_RES(21)..(21)Site of chemical conjugation 562Lys Thr Glu
Leu Gln Gly Leu Ile Gly Gln Leu Asp Glu Val Ser Leu1 5
10 15Glu Lys Asn Pro Cys Ile Arg Glu
2056317PRTHomo sapiensKRI1 Protein KRI1 homolog;
Q8N9T8_C673MOD_RES(12)..(12)Site of chemical conjugation 563Arg Leu Leu
Gly Pro Thr Val Met Leu Gly Gly Cys Glu Phe Ser Arg1 5
10 15Gln56427PRTHomo sapiensAUP1 Ancient
ubiquitous protein 1; Q9Y679_C391MOD_RES(4)..(4)Site of chemical
conjugation 564Lys Thr Gly Cys Val Asp Leu Thr Ile Thr Asn Leu Leu Glu
Gly Ala1 5 10 15Val Ala
Phe Met Pro Glu Asp Ile Thr Lys Gly 20
2556511PRTHomo sapiensMAP2K7 Dual specificity mitogen-activated
protein kinase; O14733_C260MOD_RES(3)..(3)Site of chemical conjugation
565Lys Leu Cys Asp Phe Gly Ile Ser Gly Arg Leu1 5
1056615PRTHomo sapiensANKRD17 Ankyrin repeat domain-containing
protein 17; O75179_C644MOD_RES(6)..(6)Site of chemical conjugation
566Arg Ala Gly His Val Cys Thr Val Gln Phe Leu Ile Ser Lys Gly1
5 10 1556712PRTHomo sapiensPRKAR2B
cAMP-dependent protein kinase type II-beta regulat;
P31323_C388MOD_RES(6)..(6)Site of chemical conjugation 567Arg Leu Leu Gly
Pro Cys Met Glu Ile Met Lys Arg1 5
1056818PRTHomo sapiensNMRAL1 NmrA-like family domain-containing
protein 1; Q9HBL8_C154MOD_RES(4)..(4)Site of chemical conjugation 568Arg
Leu Pro Cys Tyr Phe Glu Asn Leu Leu Ser His Phe Leu Pro Gln1
5 10 15Lys Ala56924PRTHomo
sapiensUSP28 Ubiquitin carboxyl-terminal hydrolase 28;
Q96RU2_C171MOD_RES(7)..(7)Site of chemical conjugation 569Lys Asn Val Gly
Asn Thr Cys Trp Phe Ser Ala Val Ile Gln Ser Leu1 5
10 15Phe Gln Leu Pro Glu Phe Arg Arg
2057022PRTHomo sapiensMMS19 MMS19 nucleotide excision repair protein
homolog; Q96T76_C750MOD_RES(12)..(12)Site of chemical conjugation 570Arg
Glu Leu Leu Glu Leu Ser Cys Cys His Ser Cys Pro Phe Ser Ser1
5 10 15Thr Ala Ala Ala Lys Cys
205719PRTHomo sapiensPFKL 6-phosphofructokinase, liver type;
P17858_C89MOD_RES(2)..(2)Site of chemical conjugation 571Arg Cys Lys Ala
Phe Thr Thr Arg Glu1 557216PRTHomo sapiensCYFIP2
Cytoplasmic FMR1-interacting protein 2;
Q96F07_C1112MOD_RES(4)..(4)Site of chemical conjugation 572Arg Leu Cys
Cys Gly Leu Ser Met Phe Glu Val Ile Leu Thr Arg Ile1 5
10 155739PRTHomo sapiensNCAPD3 Condensin-2
complex subunit D3; P42695_C541MOD_RES(2)..(2)Site of chemical
conjugation 573Arg Cys Val Met Ala Met Leu Arg Arg1
557410PRTHomo sapiensPOLDIP2 Polymerase delta-interacting protein 2;
Q9Y2S7_C143MOD_RES(3)..(3)Site of chemical conjugation 574Arg Asp Cys Pro
His Ile Ser Gln Arg Ser1 5 1057521PRTHomo
sapiensNUP98 Nuclear pore complex protein Nup98-Nup96;
P52948_C1312MOD_RES(5)..(5)Site of chemical conjugation 575Arg Trp Leu
Ser Cys Thr Ala Thr Pro Gln Ile Glu Glu Glu Val Ser1 5
10 15Leu Thr Gln Lys Asn
2057625PRTHomo sapiensDAXX Death domain-associated protein 6;
Q9UER7_C664MOD_RES(3)..(3)Site of chemical conjugation 576Lys Ile Cys Thr
Leu Pro Ser Pro Pro Ser Pro Leu Ala Ser Leu Ala1 5
10 15Pro Val Ala Asp Ser Ser Thr Arg Val
20 2557714PRTHomo sapiensSMARCAD1 SWI/SNF-related
matrix-associated actin-dependent; Q9H4L7_C772MOD_RES(6)..(6)Site of
chemical conjugation 577Lys Asn Thr Glu Met Cys Asn Val Met Met Gln Leu
Arg Lys1 5 1057816PRTHomo sapiensPPME1
Protein phosphatase methylesterase 1;
Q9Y570_C381MOD_RES(11)..(11)Site of chemical conjugation 578Arg Phe Ala
Glu Pro Ile Gly Gly Phe Gln Cys Val Phe Pro Gly Cys1 5
10 1557927PRTHomo sapiensMSH6 DNA mismatch
repair protein Msh6; P52701_C1117MOD_RES(17)..(17)Site of chemical
conjugation 579Lys Thr Phe Phe Gly Asp Asp Phe Ile Pro Asn Asp Ile Leu
Ile Gly1 5 10 15Cys Glu
Glu Glu Glu Gln Glu Asn Gly Lys Ala 20
2558015PRTHomo sapiensMCM5 DNA replication licensing factor MCM5;
P33992_C482MOD_RES(2)..(2)Site of chemical conjugation 580Arg Cys Ser Val
Leu Ala Ala Ala Asn Ser Val Phe Gly Arg Trp1 5
10 1558110PRTHomo sapiensC15orf38-AP3S2 Protein
C15orf38-AP3S2; E2QRD5_C183MOD_RES(2)..(2)Site of chemical
conjugation 581Lys Cys Asn Phe Thr Gly Asp Gly Lys Thr1 5
1058224PRTHomo sapiensPFKFB4
6-phosphofructo-2-kinase/fructose-2,6- bisphosphata;
Q16877_C159MOD_RES(9)..(9)Site of chemical conjugation 582Lys Thr Phe Phe
Val Glu Ser Ile Cys Val Asp Pro Glu Val Ile Ala1 5
10 15Ala Asn Ile Val Gln Val Lys Leu
2058330PRTHomo sapiensCCM2 Malcavernin;
Q9BSQ5_C211MOD_RES(9)..(9)Site of chemical conjugation 583Lys Val Ala Ala
Glu Glu Leu Cys Cys Leu Leu Gly Gln Val Phe Gln1 5
10 15Val Val Tyr Thr Glu Ser Thr Ile Asp Phe
Leu Asp Arg Ala 20 25
3058410PRTHomo sapiensMPP1 55 kDa erythrocyte membrane protein;
Q00013_C179MOD_RES(8)..(8)Site of chemical conjugation 584Lys Lys Asp Asn
Leu Ile Pro Cys Lys Glu1 5 1058536PRTHomo
sapiensDHRS11 Dehydrogenase/reductase SDR family member 11;
Q6UWP2_C226MOD_RES(2)..(2)Site of chemical conjugation 585Lys Cys Leu Lys
Pro Glu Asp Val Ala Glu Ala Val Ile Tyr Val Leu1 5
10 15Ser Thr Pro Ala His Ile Gln Ile Gly Asp
Ile Gln Met Arg Pro Thr 20 25
30Glu Gln Val Thr 3558613PRTHomo sapiensAFAP1 Actin
filament-associated protein 1; Q8N556_C713MOD_RES(11)..(11)Site of
chemical conjugation 586Lys Ser Gln Ala Ala Pro Gly Ser Ser Pro Cys Arg
Gly1 5 1058719PRTHomo sapiensPGPEP1
Pyroglutamyl-peptidase 1; Q9NXJ5_C149MOD_RES(4)..(4)Site of chemical
conjugation 587Arg Tyr Leu Cys Asp Phe Thr Tyr Tyr Thr Ser Leu Tyr Gln
Ser His1 5 10 15Gly Arg
Ser58818PRTHomo sapiensPM20D2 Peptidase M20 domain-containing protein
2; Q8IYS1_C14MOD_RES(14)..(14)Site of chemical conjugation 588Met Arg
Pro Gly Gly Glu Arg Pro Val Glu Gly Gly Ala Cys Asn Gly1 5
10 15Arg Ser58910PRTHomo sapiensRAD50
DNA repair protein RAD50; Q92878_C48MOD_RES(7)..(7)Site of chemical
conjugation 589Lys Thr Thr Ile Ile Glu Cys Leu Lys Tyr1 5
1059022PRTHomo sapiensTNS3 Tensin-3;
Q68CZ2_C888MOD_RES(3)..(3)Site of chemical conjugation 590Arg Ser Cys Pro
Glu Thr Leu Thr His Ala Val Gly Met Ser Glu Ser1 5
10 15Pro Ile Gly Pro Lys Ser
2059117PRTHomo sapiensMYCBP2 Probable E3 ubiquitin-protein ligase
MYCBP2; O75592_C1131MOD_RES(2)..(2)Site of chemical conjugation 591Arg
Cys Ser Ile Leu Ser Pro Glu Leu Ala Leu Pro Thr Gly Ser Arg1
5 10 15Ala59212PRTHomo sapiensZNF318
Zinc finger protein 318; Q5VUA4_C1860MOD_RES(7)..(7)Site of chemical
conjugation 592Lys Leu Ser Pro Gln Ala Cys Ser Phe Thr Lys Ala1
5 1059312PRTHomo sapiensTXNIP
Thioredoxin-interacting protein; Q9H3M7_C170MOD_RES(4)..(4)Site of
chemical conjugation 593Lys Val Ser Cys Met Phe Ile Pro Asp Gly Arg Val1
5 1059415PRTHomo sapiensHMHA1 Minor
histocompatibility protein HA-1; Q92619_C278MOD_RES(2)..(2)Site of
chemical conjugation 594Arg Cys Glu Gly Gly Val Asp Ala Ala Leu Leu Tyr
Ala Lys Asn1 5 10
1559516PRTHomo sapiensMKI67 Antigen KI-67;
P46013_C903MOD_RES(12)..(12)Site of chemical conjugation 595Lys Ser Glu
Glu Thr Asn Thr Glu Ile Val Glu Cys Ile Leu Lys Arg1 5
10 1559639PRTHomo sapiensTRMT112 tRNA
methyltransferase 112 homolog; Q9UI30_C100MOD_RES(33)..(33)Site of
chemical conjugation 596Lys Gly Pro Val Glu Gly Tyr Glu Glu Asn Glu Glu
Phe Leu Arg Thr1 5 10
15Met His His Leu Leu Leu Glu Val Glu Val Ile Glu Gly Thr Leu Gln
20 25 30Cys Pro Glu Ser Gly Arg Met
3559720PRTHomo sapiensSYMPK Symplekin;
Q92797_C848MOD_RES(17)..(17)Site of chemical conjugation 597Arg Gly Met
Gly Met Asn Ser Pro Glu Leu Leu Leu Leu Val Glu Asn1 5
10 15Cys Pro Lys Gly
205988PRTHomo sapiensCCT8 T-complex protein 1 subunit theta;
P50990_C36MOD_RES(6)..(6)Site of chemical conjugation 598Arg Asn Ile Gln
Ala Cys Lys Glu1 559910PRTHomo sapiensDFFB DNA
fragmentation factor subunit beta; O76075_C194MOD_RES(7)..(7)Site of
chemical conjugation 599Arg Val Leu Gly Ser Met Cys Gln Arg Leu1
5 1060021PRTHomo sapiensNR3C1 Glucocorticoid
receptor; P04150_C302MOD_RES(7)..(7)Site of chemical conjugation
600Lys Leu Gly Thr Val Tyr Cys Gln Ala Ser Phe Pro Gly Ala Asn Ile1
5 10 15Ile Gly Asn Lys Met
2060125PRTHomo sapiensVCPIP1 Deubiquitinating protein VCIP135;
Q96JH7_C219MOD_RES(17)..(17)Site of chemical conjugation 601Lys Ser Gln
Glu Cys Leu Ile Pro Val His Val Asp Gly Asp Gly His1 5
10 15Cys Leu Val His Ala Val Ser Arg Ala
20 2560212PRTHomo sapiensGSTCD Glutathione
S-transferase C-terminal domain-contai;
Q8NEC7_C140MOD_RES(3)..(3)Site of chemical conjugation 602Lys Ala Cys Ala
Glu Val Ser Gln Trp Thr Arg Leu1 5
1060318PRTHomo sapiensIREB2 Iron-responsive element-binding protein
2; P48200_C137MOD_RES(2)..(2)Site of chemical conjugation 603Lys Cys Ala
Ile Gln Asn Ala Pro Asn Pro Gly Gly Gly Asp Leu Gln1 5
10 15Lys Ala60420PRTHomo sapiensCOG1
Conserved oligomeric Golgi complex subunit 1;
Q8WTW3_C513MOD_RES(8)..(8)Site of chemical conjugation 604Lys Ala Gln Ala
Ile Ser Pro Cys Val Gln Asn Phe Cys Ser Ala Leu1 5
10 15Asp Ser Lys Leu 2060517PRTHomo
sapiensTBC1D2 TBC1 domain family member 2A;
Q9BYX2_C469MOD_RES(5)..(5)Site of chemical conjugation 605Arg Thr Gln Asn
Cys Phe Leu Asn Ser Glu Ile His Gln Val Thr Lys1 5
10 15Ile60622PRTHomo sapiensHNRNPA3
Heterogeneous nuclear ribonucleoprotein A3;
P51991_C85MOD_RES(10)..(10)Site of chemical conjugation 606Arg Gly Phe
Gly Phe Val Thr Tyr Ser Cys Val Glu Glu Val Asp Ala1 5
10 15Ala Met Cys Ala Arg Pro
2060737PRTHomo sapiensRIF1 Telomere-associated protein RIF1;
Q5UIP0_C2298MOD_RES(18)..(18)Site of chemical conjugation 607Lys Glu Ser
Ile Pro Cys Pro Thr Glu Ser Val Tyr Pro Pro Leu Val1 5
10 15Asn Cys Val Ala Pro Val Asp Ile Ile
Leu Pro Gln Ile Thr Ser Asn 20 25
30Met Trp Ala Arg Gly 3560822PRTHomo sapiensIBA57 Putative
transferase CAF17, mitochondrial; Q5T440_C170MOD_RES(13)..(13)Site
of chemical conjugation 608Arg Val Trp Ala Val Leu Pro Ser Ser Pro Glu
Ala Cys Gly Ala Ala1 5 10
15Ser Leu Gln Glu Arg Ala 206098PRTHomo sapiensMAP1B
Microtubule-associated protein 1B; P46821_C293MOD_RES(4)..(4)Site of
chemical conjugation 609Arg Lys Ser Cys Phe Trp Lys Leu1
561020PRTHomo sapiensCNPY3 Protein canopy homolog 3;
Q9BT09_C166MOD_RES(3)..(3)Site of chemical conjugation 610Lys Gln Cys Asp
Val Leu Val Glu Glu Phe Glu Glu Val Ile Glu Asp1 5
10 15Trp Tyr Arg Asn 2061138PRTHomo
sapiensDNAJC10 DnaJ homolog subfamily C member 10;
Q8IXB1_C480MOD_RES(33)..(33)Site of chemical conjugation 611Lys Glu Ser
Val Asn Ser His Val Thr Thr Leu Gly Pro Gln Asn Phe1 5
10 15Pro Ala Asn Asp Lys Glu Pro Trp Leu
Val Asp Phe Phe Ala Pro Trp 20 25
30Cys Pro Pro Cys Arg Ala 3561211PRTHomo sapiensCYR61 Protein
CYR61; O00622_C39MOD_RES(2)..(2)Site of chemical conjugation 612Lys
Cys Ala Pro Gly Val Gly Leu Val Arg Asp1 5
1061322PRTHomo sapiensCWF19L1 CWF19-like protein 1;
Q69YN2_C288MOD_RES(12)..(12)Site of chemical conjugation 613Lys Gln Ile
Leu Ala Pro Val Glu Glu Ser Ala Cys Gln Phe Phe Phe1 5
10 15Asp Leu Asn Glu Lys Gln
2061412PRTHomo sapiensIPO4 Importin-4;
Q8TEX9_C708MOD_RES(5)..(5)Site of chemical conjugation 614Lys Leu Leu Glu
Cys Pro His Leu Asn Val Arg Lys1 5
1061531PRTHomo sapiensCAPN7 Calpain-7;
Q9Y6W3_C197MOD_RES(24)..(24)Site of chemical conjugation 615Arg Ala His
Phe Pro Leu Gly Ala Asn Pro Phe Leu Glu Arg Pro Gln1 5
10 15Ser Phe Ile Ser Pro Gln Ser Cys Asp
Ala Gln Gly Gln Arg Tyr 20 25
3061615PRTHomo sapiensSEC23IP SEC23-interacting protein;
Q9Y6Y8_C604MOD_RES(2)..(2)Site of chemical conjugation 616Lys Cys Pro Gly
Pro Leu Ala Val Ala Asn Gly Val Val Lys Gln1 5
10 1561711PRTHomo sapiensAKAP8L A-kinase anchor
protein 8-like; Q9ULX6_C211MOD_RES(4)..(4)Site of chemical
conjugation 617Arg Gly Gln Cys Met Ser Gly Ala Ser Arg Leu1
5 1061815PRTHomo sapiensTSTA3 GDP-L-fucose synthase;
Q13630_C116MOD_RES(9)..(9)Site of chemical conjugation 618Lys Val Val
Ser Cys Leu Ser Thr Cys Ile Phe Pro Asp Lys Thr1 5
10 1561918PRTHomo sapiensPDIA4 Protein
disulfide-isomerase A4; P13667_C94MOD_RES(16)..(16)Site of chemical
conjugation 619Lys Asp Thr Val Leu Leu Glu Phe Tyr Ala Pro Trp Cys Gly
His Cys1 5 10 15Lys
Gln62015PRTHomo sapiensDDX59 Probable ATP-dependent RNA helicase
DDX59; Q5T1V6_C453MOD_RES(13)..(13)Site of chemical conjugation 620Lys
Leu Phe Lys Pro Pro Val Leu Val Phe Val Asp Cys Lys Leu1 5
10 1562124PRTHomo sapiensREXO4 RNA
exonuclease 4; Q9GZR2_C382MOD_RES(13)..(13)Site of chemical
conjugation 621Lys Ile Leu Gly Leu Gln Val Gln Gln Ala Glu His Cys Ser
Ile Gln1 5 10 15Asp Ala
Gln Ala Ala Met Arg Leu 2062213PRTHomo sapiensGEMIN6
Gem-associated protein 6; Q8WXD5_C91MOD_RES(11)..(11)Site of
chemical conjugation 622Lys Leu Met His Leu Phe Thr Ser Gly Asp Cys Lys
Ala1 5 1062315PRTHomo sapiensPDK1;
Q15118_C71MOD_RES(12)..(12)Site of chemical conjugation 623Lys Gln Phe
Leu Asp Phe Gly Ser Val Asn Ala Cys Glu Lys Thr1 5
10 1562412PRTHomo sapiensATRIP ATR-interacting
protein; Q8WXE1_C585MOD_RES(4)..(4)Site of chemical conjugation
624Arg Phe Gln Cys Val Phe Gln Val Leu Pro Lys Cys1 5
1062511PRTHomo sapiensPIAS4 E3 SUMO-protein ligase PIAS4;
Q8N2W9_C326MOD_RES(6)..(6)Site of chemical conjugation 625Arg Val Ser
Leu Ile Cys Pro Leu Val Lys Met1 5
1062621PRTHomo sapiensSEPT6 Septin-6;
Q14141_C269MOD_RES(16)..(16)Site of chemical conjugation 626Arg Gln Tyr
Pro Trp Gly Thr Val Gln Val Glu Asn Glu Ala His Cys1 5
10 15Asp Phe Val Lys Leu
2062725PRTHomo sapiensDNMT1 DNA (cytosine-5)-methyltransferase 1;
P26358_C1071MOD_RES(13)..(13)Site of chemical conjugation 627Arg Cys Thr
Val Glu Tyr Gly Glu Asp Leu Pro Glu Cys Val Gln Val1 5
10 15Tyr Ser Met Gly Gly Pro Asn Arg Phe
20 2562819PRTHomo sapiensTHNSL1 Threonine
synthase-like 1; Q8IYQ7_C324MOD_RES(16)..(16)Site of chemical
conjugation 628Arg Leu Gly Glu Met Ile Glu Thr Ala Tyr Gly Glu Asn Phe
Ala Cys1 5 10 15Ser Lys
Ile62911PRTHomo sapiensALDH7A1 Alpha-aminoadipic semialdehyde
dehydrogenase; P49419_C522MOD_RES(4)..(4)Site of chemical conjugation
629Arg Ser Thr Cys Thr Ile Asn Tyr Ser Lys Asp1 5
1063014PRTHomo sapiensZNF295 Zinc finger protein 295;
Q9ULJ3_C129MOD_RES(10)..(10)Site of chemical conjugation 630Lys Thr Pro
Gln Ala Pro Phe Pro Thr Cys Pro Asn Arg Lys1 5
1063111PRTHomo sapiensPLEKHA2 Pleckstrin homology domain-containing
family A mem; Q9HB19_C232MOD_RES(2)..(2)Site of chemical conjugation
631Lys Cys Glu Gln Asp Arg Glu Pro Leu Arg Thr1 5
1063213PRTHomo sapiensCLPX ATP-dependent Clp protease ATP-binding
subunit clp; O76031_C538MOD_RES(2)..(2)Site of chemical conjugation
632Lys Cys Glu Leu Asn Val Thr Glu Asp Ala Leu Lys Ala1 5
1063348PRTHomo sapiensHMGCS1 Hydroxymethylglutaryl-CoA
synthase, cytoplasmic; Q01581_C129MOD_RES(25)..(25)Site of chemical
conjugation 633Lys Thr Asn Leu Met Gln Leu Phe Glu Glu Ser Gly Asn Thr
Asp Ile1 5 10 15Glu Gly
Ile Asp Thr Thr Asn Ala Cys Tyr Gly Gly Thr Ala Ala Val 20
25 30Phe Asn Ala Val Asn Trp Ile Glu Ser
Ser Ser Trp Asp Gly Arg Tyr 35 40
4563442PRTHomo sapiensNUDCD1 NudC domain-containing protein 1;
Q96RS6_C402MOD_RES(21)..(21)Site of chemical conjugation 634Arg Leu Met
His Leu Thr Ser Glu Glu Leu Asn Pro Asn Pro Asp Lys1 5
10 15Glu Lys Pro Pro Cys Asn Ala Gln Glu
Leu Glu Glu Cys Asp Ile Phe 20 25
30Phe Glu Glu Ser Ser Ser Leu Cys Arg Phe 35
4063520PRTHomo sapiensPLDN Pallidin; Q9UL45_C95MOD_RES(5)..(5)Site
of chemical conjugation 635Lys Phe Lys Glu Cys His Ser Met Leu Asp Ile
Asn Ala Leu Phe Ala1 5 10
15Glu Ala Lys His 2063618PRTHomo sapiensSMURF2 E3
ubiquitin-protein ligase SMURF2; Q9HAU4_C706MOD_RES(11)..(11)Site of
chemical conjugation 636Arg Leu Phe Thr Ile His Gln Ile Asp Ala Cys Thr
Asn Asn Leu Pro1 5 10
15Lys Ala63712PRTHomo sapiensKIAA1524 Protein CIP2A;
Q8TCG1_C337MOD_RES(2)..(2)Site of chemical conjugation 637Lys Cys Leu Glu
Pro Thr Val Ala Leu Leu Arg Trp1 5
106389PRTHomo sapiensRAI14 Ankycorbin;
Q9P0K7_C973MOD_RES(7)..(7)Site of chemical conjugation 638Lys Gln Ile Leu
Thr Met Cys Lys Asn1 563926PRTHomo sapiensEFTUD1 Elongation
factor Tu GTP-binding domain- containing;
Q7Z2Z2_C124MOD_RES(17)..(17)Site of chemical conjugation 639Arg Ile Cys
Asp Gly Cys Ile Ile Val Val Asp Ala Val Glu Gly Val1 5
10 15Cys Pro Gln Thr Gln Ala Val Leu Arg
Gln 20 2564014PRTHomo sapiensTJAP1 Tight
junction-associated protein 1; Q5JTD0_C350MOD_RES(8)..(8)Site of
chemical conjugation 640Arg Asn Ser Pro Leu Pro Asn Cys Thr Tyr Ala Thr
Arg Gln1 5 1064124PRTHomo sapiensSUZ12
Polycomb protein SUZ12; Q15022_C325MOD_RES(19)..(19)Site of chemical
conjugation 641Arg Leu Gln Leu Leu Asp Gly Glu Tyr Glu Val Ala Met Gln
Glu Met1 5 10 15Glu Glu
Cys Pro Ile Ser Lys Lys 2064237PRTHomo sapiensALDH1B1 Aldehyde
dehydrogenase X, mitochondrial; P30837_C320MOD_RES(32)..(32)Site of
chemical conjugation 642Lys Ser Pro Ser Ile Val Leu Ala Asp Ala Asp Met
Glu His Ala Val1 5 10
15Glu Gln Cys His Glu Ala Leu Phe Phe Asn Met Gly Gln Cys Cys Cys
20 25 30Ala Gly Ser Arg Thr
3564313PRTHomo sapiensCSTF2T Cleavage stimulation factor subunit 2
tau variant; Q9H0L4_C150MOD_RES(3)..(3)Site of chemical conjugation
643Lys Leu Cys Val Gln Asn Ser His Gln Glu Ala Arg Asn1 5
1064413PRTHomo sapiensTNFAIP2 Tumor necrosis factor
alpha-induced protein 2; Q03169_C45MOD_RES(8)..(8)Site of chemical
conjugation 644Lys Gly Leu Ala Asn Val Phe Cys Val Phe Thr Lys Gly1
5 1064522PRTHomo sapiensATXN10 Ataxin-10;
Q9UBB4_C354MOD_RES(17)..(17)Site of chemical conjugation 645Arg Val Ile
His Val Ala Gly Lys Glu Thr Thr Asn Ile Phe Ser Asn1 5
10 15Cys Gly Cys Val Arg Ala
2064623PRTHomo sapiensBTK Tyrosine-protein kinase BTK;
Q06187_C481MOD_RES(16)..(16)Site of chemical conjugation 646Lys Gln Arg
Pro Ile Phe Ile Ile Thr Glu Tyr Met Ala Asn Gly Cys1 5
10 15Leu Leu Asn Tyr Leu Arg Glu
2064724PRTHomo sapiensENTHD2 AP-4 complex accessory subunit tepsin;
Q96N21_C302MOD_RES(4)..(4)Site of chemical conjugation 647Arg Ala Leu Cys
Ala Ile Ala Ser Leu Gly Ser Ser Asp Leu Leu Pro1 5
10 15Gln Glu His Ile Leu Leu Arg Thr
206489PRTHomo sapiensRAD50 DNA repair protein RAD50;
Q92878_C102MOD_RES(5)..(5)Site of chemical conjugation 648Arg Ser Met Val
Cys Thr Gln Lys Ser1 564929PRTHomo sapiensDENND4A C-myc
promoter-binding protein; Q7Z401_C117MOD_RES(6)..(6)Site of chemical
conjugation 649Arg Leu Lys Gln Gly Cys Glu Ile Ile Gln Ser Thr Pro Tyr
Gly Arg1 5 10 15Pro Ala
Asn Ile Ser Gly Ser Thr Ser Ser Gln Arg Ile 20
256509PRTHomo sapiensPNN Pinin; Q9H307_C249MOD_RES(3)..(3)Site of
chemical conjugation 650Arg Met Cys Pro Ala Thr Gln Lys Leu1
565116PRTHomo sapiensDGCR14 Protein DGCR14;
Q96DF8_C263MOD_RES(2)..(2)Site of chemical conjugation 651Arg Cys Gln Leu
Gln Gln Ala Ala Ala Leu Asn Ala Gln His Lys Gln1 5
10 1565216PRTHomo sapiensATPBD4 ATP-binding
domain-containing protein 4; Q7L8W6_C88MOD_RES(2)..(2)Site of
chemical conjugation 652Lys Cys Glu Gly Asp Glu Val Glu Asp Leu Tyr Glu
Leu Leu Lys Leu1 5 10
1565318PRTHomo sapiensNME3 Nucleoside diphosphate kinase 3;
Q13232_C158MOD_RES(7)..(7)Site of chemical conjugation 653Arg Ala Asp Glu
Leu Leu Cys Trp Glu Asp Ser Ala Gly His Trp Leu1 5
10 15Tyr Glu65411PRTHomo sapiensMED9 Mediator
of RNA polymerase II transcription subuni;
Q9NWA0_C139MOD_RES(4)..(4)Site of chemical conjugation 654Lys Ser Leu Cys
Met Phe Glu Ile Pro Lys Glu1 5
1065519PRTHomo sapiensFRMD8 FERM domain-containing protein 8;
Q9BZ67_C191MOD_RES(15)..(15)Site of chemical conjugation 655Arg Val Gln
Leu Gly Pro Tyr Gln Pro Gly Arg Pro Ala Ala Cys Asp1 5
10 15Leu Arg Glu65618PRTHomo sapiensIRAK1
Interleukin-1 receptor-associated kinase 1;
P51617_C608MOD_RES(9)..(9)Site of chemical conjugation 656Arg Ser Trp His
Leu Thr Pro Ser Cys Pro Leu Asp Pro Ala Pro Leu1 5
10 15Arg Glu65718PRTHomo sapiensLAS1L Ribosomal
biogenesis protein LAS1L; Q9Y4W2_C140MOD_RES(2)..(2)Site of chemical
conjugation 657Lys Cys Leu Ala Gln Glu Val Asn Ile Pro Asp Trp Ile Val
Asp Leu1 5 10 15Arg
His65816PRTHomo sapiensSDHAF2 Succinate dehydrogenase assembly factor
2, mitocho; Q9NX18_C83MOD_RES(7)..(7)Site of chemical conjugation 658Arg
Gly Met Leu Glu Asn Cys Ile Leu Leu Ser Leu Phe Ala Lys Glu1
5 10 1565920PRTHomo sapiensPOP5
Ribonuclease P/MRP protein subunit POP5;
Q969H6_C146MOD_RES(3)..(3)Site of chemical conjugation 659Arg Ser Cys Leu
Leu Glu Glu Glu Glu Glu Ser Gly Glu Glu Ala Ala1 5
10 15Glu Ala Met Glu 2066015PRTHomo
sapiensNGLY1 Peptide-N(4)-(N-acetyl-beta- glucosaminyl)asparagin;
Q96IV0_C309MOD_RES(2)..(2)Site of chemical conjugation 660Arg Cys Gly Glu
Trp Ala Asn Cys Phe Thr Leu Cys Cys Arg Ala1 5
10 1566113PRTHomo sapiensCASP2 Caspase-2;
P42575_C370MOD_RES(10)..(10)Site of chemical conjugation 661Arg Ser Asp
Met Ile Cys Gly Tyr Ala Cys Leu Lys Gly1 5
1066214PRTHomo sapiensRPS6KA3 Ribosomal protein S6 kinase alpha-3;
P51812_C436MOD_RES(12)..(12)Site of chemical conjugation 662Lys Glu Asp
Ile Gly Val Gly Ser Tyr Ser Val Cys Lys Arg1 5
106638PRTHomo sapiensPOU2F2 POU domain, class 2, transcription
factor 2; P09086_C346MOD_RES(5)..(5)Site of chemical conjugation 663Arg
Val Trp Phe Cys Asn Arg Arg1 566423PRTHomo sapiensUBR1 E3
ubiquitin-protein ligase UBR1; Q8IWV7_C1603MOD_RES(13)..(13)Site of
chemical conjugation 664Arg Asn Ser Leu Ile Glu Leu Pro Asp Asp Tyr Ser
Cys Leu Leu Asn1 5 10
15Gln Ala Ser His Phe Arg Cys 206659PRTHomo sapiensSMG9
Protein SMG9; Q9H0W8_C380MOD_RES(6)..(6)Site of chemical conjugation
665Arg Arg Glu Asp Phe Cys Pro Arg Lys1 566613PRTHomo
sapiensCHEK2 Serine/threonine-protein kinase Chk2;
O96017_C231MOD_RES(8)..(8)Site of chemical conjugation 666Lys Thr Leu Gly
Ser Gly Ala Cys Gly Glu Val Lys Leu1 5
1066717PRTHomo sapiensFNBP1 Formin-binding protein 1;
Q96RU3_C70MOD_RES(15)..(15)Site of chemical conjugation 667Lys Lys Asn
Ser Lys Glu Glu Glu Glu Tyr Lys Tyr Thr Ser Cys Lys1 5
10 15Ala66811PRTHomo sapiensSMC2 Structural
maintenance of chromosomes protein 2; O95347_C326MOD_RES(6)..(6)Site
of chemical conjugation 668Lys Lys Asn Leu Ala Cys Glu Glu Ser Lys Arg1
5 1066915PRTHomo sapiensINPPL1
Phosphatidylinositol 3,4,5-trisphosphate 5-phospha;
O15357_C926MOD_RES(10)..(10)Site of chemical conjugation 669Arg Lys Pro
Ala Phe Thr Glu Ala Ser Cys Pro Leu Ser Arg Leu1 5
10 1567032PRTHomo sapiensRPP25L Ribonuclease P
protein subunit p25-like protein; Q8N5L8_C131MOD_RES(9)..(9)Site of
chemical conjugation 670Arg Asp Pro Leu Asp Pro Asn Glu Cys Gly Tyr Gln
Pro Pro Gly Ala1 5 10
15Pro Pro Gly Leu Gly Ser Met Pro Ser Ser Ser Cys Gly Pro Arg Ser
20 25 306719PRTHomo sapiensMTMR12
Myotubularin-related protein 12; Q9C0I1_C152MOD_RES(6)..(6)Site of
chemical conjugation 671Arg Val Phe Gln Phe Cys Leu Arg Tyr1
567217PRTHomo sapiensEDC3 Enhancer of mRNA-decapping protein 3;
Q96F86_C137MOD_RES(14)..(14)Site of chemical conjugation 672Lys Ser Gln
Asp Val Ala Val Ser Pro Gln Gln Gln Gln Cys Ser Lys1 5
10 15Ser67310PRTHomo sapiensDFNA5
Non-syndromic hearing impairment protein 5;
O60443_C45MOD_RES(4)..(4)Site of chemical conjugation 673Arg Phe Trp Cys
Trp Gln Arg Pro Lys Tyr1 5 1067414PRTHomo
sapiensMDN1 Midasin; Q9NU22_C1011MOD_RES(4)..(4)Site of chemical
conjugation 674Lys Leu Ile Cys Gln His Ile Val Pro Gly Asn Val Lys Ser1
5 1067521PRTHomo sapiensDYNC1H1 Cytoplasmic
dynein 1 heavy chain 1; Q14204_C1059MOD_RES(8)..(8)Site of chemical
conjugation 675Lys Val Trp Leu Gln Tyr Gln Cys Leu Trp Asp Met Gln Ala
Glu Asn1 5 10 15Ile Tyr
Asn Arg Leu 2067614PRTHomo sapiensEP300 Histone
acetyltransferase p300; Q09472_C1738MOD_RES(2)..(2)Site of chemical
conjugation 676Arg Cys Ile Gln Ser Leu Val His Ala Cys Gln Cys Arg Asn1
5 1067718PRTHomo sapiensCKMT1B Creatine
kinase U-type, mitochondrial; P12532_C316MOD_RES(8)..(8)Site of
chemical conjugation 677Arg Leu Gly Tyr Ile Leu Thr Cys Pro Ser Asn Leu
Gly Thr Gly Leu1 5 10
15Arg Ala67816PRTHomo sapiensIMPACT Protein IMPACT;
Q9P2X3_C195MOD_RES(13)..(13)Site of chemical conjugation 678Arg Ser Thr
Phe Gln Ala His Leu Ala Pro Val Val Cys Pro Lys Gln1 5
10 1567914PRTHomo sapiensAKAP1 A-kinase
anchor protein 1, mitochondrial; Q92667_C147MOD_RES(7)..(7)Site of
chemical conjugation 679Lys Ser Ile Pro Leu Glu Cys Pro Leu Ser Ser Pro
Lys Gly1 5 1068022PRTHomo sapiensIFIT3
Interferon-induced protein with tetratricopeptide;
O14879_C283MOD_RES(18)..(18)Site of chemical conjugation 680Arg Val Leu
Glu Ser Thr Pro Asn Asn Gly Tyr Leu Tyr His Gln Ile1 5
10 15Gly Cys Cys Tyr Lys Ala
2068130PRTHomo sapiensRELB Transcription factor RelB;
Q01201_C109MOD_RES(25)..(25)Site of chemical conjugation 681Arg Gly Ala
Ala Ser Leu Ser Thr Val Thr Leu Gly Pro Val Ala Pro1 5
10 15Pro Ala Thr Pro Pro Pro Trp Gly Cys
Pro Leu Gly Arg Leu 20 25
3068216PRTHomo sapiensTRIM33 E3 ubiquitin-protein ligase TRIM33;
Q9UPN9_C582MOD_RES(6)..(6)Site of chemical conjugation 682Arg Gly Asn Met
Asn Cys Gly Ala Phe Gln Ala His Gln Met Arg Leu1 5
10 1568311PRTHomo sapiensNTPCR Cancer-related
nucleoside-triphosphatase; Q9BSD7_C110MOD_RES(3)..(3)Site of
chemical conjugation 683Arg Val Cys Val Ile Asp Glu Ile Gly Lys Met1
5 1068418PRTHomo sapiensDENND1C DENN
domain-containing protein 1C; Q8IV53_C174MOD_RES(9)..(9)Site of
chemical conjugation 684Arg Gly Asn Ser Lys Pro Leu Ser Cys Phe Val Ala
Pro Asp Ser Gly1 5 10
15Arg Leu68519PRTHomo sapiensACO2 Aconitate hydratase, mitochondrial;
Q99798_C385MOD_RES(8)..(8)Site of chemical conjugation 685Arg Val Gly
Leu Ile Gly Ser Cys Thr Asn Ser Ser Tyr Glu Asp Met1 5
10 15Gly Arg Ser68626PRTHomo sapiensDYNC1H1
Cytoplasmic dynein 1 heavy chain 1; Q14204_C3089MOD_RES(2)..(2)Site
of chemical conjugation 686Arg Cys Val Leu Asn Trp Phe Gly Asp Trp Ser
Thr Glu Ala Leu Tyr1 5 10
15Gln Val Gly Lys Glu Phe Thr Ser Lys Met 20
2568712PRTHomo sapiensSEPHS1 Selenide, water dikinase 1;
P49903_C31MOD_RES(10)..(10)Site of chemical conjugation 687Arg Phe Thr
Glu Leu Lys Gly Thr Gly Cys Lys Val1 5
1068819PRTHomo sapiensUSP34 Ubiquitin carboxyl-terminal hydrolase 34;
Q70CQ2_C741MOD_RES(17)..(17)Site of chemical conjugation 688Arg Thr Gly
Asp Phe Leu Gly Glu Thr Ile Gly Asn Glu Leu Phe Asn1 5
10 15Cys Arg Gln68927PRTHomo sapiensCBFA2T2
Protein CBFA2T2; O43439_C111MOD_RES(23)..(23)Site of chemical
conjugation 689Arg Phe Ser Asn Gly Pro Ala Ser Ser Thr Ser Ser Ala Leu
Thr Asn1 5 10 15Gln Gln
Leu Pro Ala Thr Cys Gly Ala Arg Gln 20
2569020PRTHomo sapiensRPAP1 RNA polymerase II-associated protein 1;
Q9BWH6_C1039MOD_RES(2)..(2)Site of chemical conjugation 690Arg Cys Gly
Gln Gly Thr Leu Leu Ala Gln Ala Cys Gln Asp Leu Pro1 5
10 15Ser Ile Arg Asn
2069121PRTHomo sapiensANKRD40 Ankyrin repeat domain-containing
protein 40; Q6AI12_C209MOD_RES(12)..(12)Site of chemical conjugation
691Arg Thr Pro Glu Ser Thr Lys Pro Gly Pro Val Cys Gln Pro Pro Val1
5 10 15Ser Gln Ser Arg Ser
2069228PRTHomo sapiensHERC4 Probable E3 ubiquitin-protein ligase
HERC4; Q5GLZ8_C60MOD_RES(17)..(17)Site of chemical conjugation 692Arg
His Thr Val Phe Val Leu Asp Asp Gly Thr Val Tyr Thr Cys Gly1
5 10 15Cys Asn Asp Leu Gly Gln Leu
Gly His Glu Lys Ser 20 2569312PRTHomo
sapiensNUB1 NEDD8 ultimate buster 1; Q9Y5A7_C52MOD_RES(5)..(5)Site
of chemical conjugation 693Arg Leu Glu Cys Cys Glu Asn Glu Val Glu Lys
Val1 5 1069418PRTHomo sapiensBRAT1
BRCA1-associated ATM activator 1; Q6PJG6_C326MOD_RES(14)..(14)Site
of chemical conjugation 694Arg Thr Gln Ala Phe Gln Val Leu Leu Gln Pro
Leu Ala Cys Val Leu1 5 10
15Lys Ala69525PRTHomo sapiensBRAT1 BRCA1-associated ATM activator 1;
Q6PJG6_C673MOD_RES(4)..(4)Site of chemical conjugation 695Arg Thr His
Cys Pro Tyr Ala Val Ala Leu Pro Glu Val Ala Pro Ala1 5
10 15Gln Pro Leu Thr Glu Ala Leu Arg Ala
20 2569614PRTHomo sapiensSPATA5
Spermatogenesis-associated protein 5;
Q8NB90_C672MOD_RES(11)..(11)Site of chemical conjugation 696Lys Gly Val
Leu Leu Tyr Gly Pro Pro Gly Cys Ser Lys Thr1 5
1069713PRTHomo sapiensHECTD1 E3 ubiquitin-protein ligase HECTD1;
Q9ULT8_C1855MOD_RES(7)..(7)Site of chemical conjugation 697Lys Leu Leu
Gln Leu Ser Cys Asn Gly Asn Val Lys Ser1 5
1069811PRTHomo sapiensDECR2 Peroxisomal 2,4-dienoyl-CoA reductase;
Q9NUI1_C22MOD_RES(5)..(5)Site of chemical conjugation 698Arg His Leu Phe
Cys Pro Asp Leu Leu Arg Asp1 5
1069921PRTHomo sapiensSEPT10 Septin-10;
Q9P0V9_C293MOD_RES(16)..(16)Site of chemical conjugation 699Arg Gln Tyr
Pro Trp Gly Val Val Gln Val Glu Asn Glu Asn His Cys1 5
10 15Asp Phe Val Lys Leu
207009PRTHomo sapiensPALLD Palladin; Q8WX93_C964MOD_RES(5)..(5)Site
of chemical conjugation 700Lys Val Ser Ser Cys Glu Gln Arg Leu1
570115PRTHomo sapiensRAD54L2 Helicase ARIP4;
Q9Y4B4_C820MOD_RES(4)..(4)Site of chemical conjugation 701Arg Ala Gly Cys
Leu Gly Val Asn Leu Ile Gly Ala Asn Arg Val1 5
10 1570221PRTHomo sapiensUBA7 Ubiquitin-like
modifier-activating enzyme 7; P41226_C599MOD_RES(17)..(17)Site of
chemical conjugation 702Arg Ala Pro Ala Ser Ala Ala Ala Ser Glu Asp Ala
Pro Tyr Pro Val1 5 10
15Cys Thr Val Arg Tyr 2070311PRTHomo sapiensEDEM3 ER
degradation-enhancing alpha- mannosidase-like 3;
Q9BZQ6_C441MOD_RES(4)..(4)Site of chemical conjugation 703Arg Val Pro Cys
Gly Phe Ala Ala Met Lys Asp1 5
1070430PRTHomo sapiensKIF11 Kinesin-like protein KIF11;
P52732_C87MOD_RES(5)..(5)Site of chemical conjugation 704Arg Ser Val Val
Cys Pro Ile Leu Asp Glu Val Ile Met Gly Tyr Asn1 5
10 15Cys Thr Ile Phe Ala Tyr Gly Gln Thr Gly
Thr Gly Lys Thr 20 25
307058PRTHomo sapiensNCF1 Neutrophil cytosol factor 1;
P14598_C378MOD_RES(2)..(2)Site of chemical conjugation 705Arg Cys Ser Glu
Ser Thr Lys Arg1 570613PRTHomo sapiensZNF346 Zinc finger
protein 346; Q9UL40_C68MOD_RES(4)..(4)Site of chemical conjugation
706Lys Asn Gln Cys Leu Phe Thr Asn Thr Gln Cys Lys Val1 5
1070713PRTHomo sapiensUBE2O Ubiquitin-conjugating enzyme
E2 O; Q9C0C9_C375MOD_RES(3)..(3)Site of chemical conjugation 707Lys
Asn Cys Ala Gln Gly Glu Gly Ser Met Ala Lys Lys1 5
1070814PRTHomo sapiensMAP2K7 Dual specificity mitogen-activated
protein kinase; O14733_C280MOD_RES(5)..(5)Site of chemical
conjugation 708Arg Ser Ala Gly Cys Ala Ala Tyr Met Ala Pro Glu Arg Ile1
5 1070919PRTHomo sapiensKIF4A
Chromosome-associated kinesin KIF4A; O95239_C269MOD_RES(5)..(5)Site
of chemical conjugation 709Arg Gly Leu Leu Cys Leu Gly Asn Val Ile Ser
Ala Leu Gly Asp Asp1 5 10
15Lys Lys Gly71012PRTHomo sapiensAHCTF1 Protein ELYS;
Q8WYP5_C521MOD_RES(2)..(2)Site of chemical conjugation 710Arg Cys Leu Val
Ala Gly Leu Leu Ser Pro Arg Phe1 5
1071121PRTHomo sapiensMED12 Mediator of RNA polymerase II
transcription subuni; Q93074_C1188MOD_RES(8)..(8)Site of chemical
conjugation 711Lys Thr Pro Gln Leu Asn Pro Cys Gln Ser Asp Gly Asn Lys
Pro Thr1 5 10 15Val Gly
Ile Arg Ser 2071230PRTHomo sapiensECM1 Extracellular matrix
protein 1; Q16610_C284MOD_RES(3)..(3)Site of chemical conjugation
712Arg Ala Cys Pro Ser His Gln Pro Asp Ile Ser Ser Gly Leu Glu Leu1
5 10 15Pro Phe Pro Pro Gly Val
Pro Thr Leu Asp Asn Ile Lys Asn 20 25
3071333PRTHomo sapiensCYR61 Protein CYR61;
O00622_C134MOD_RES(17)..(17)Site of chemical conjugation 713Lys His Gln
Cys Thr Cys Ile Asp Gly Ala Val Gly Cys Ile Pro Leu1 5
10 15Cys Pro Gln Glu Leu Ser Leu Pro Asn
Leu Gly Cys Pro Asn Pro Arg 20 25
30Leu71418PRTHomo sapiensCTU2 Cytoplasmic tRNA 2-thiolation protein
2; Q2VPK5_C433MOD_RES(7)..(7)Site of chemical conjugation 714Arg Thr
Pro Pro Gly Pro Cys Cys Ser Pro Gly Val Gly Trp Ala Gln1 5
10 15Arg Cys71520PRTHomo sapiensEIF2B4
Translation initiation factor eIF-2B subunit delta;
Q9UI10_C444MOD_RES(17)..(17)Site of chemical conjugation 715Arg Ala His
Asn Val Pro Val Leu Val Cys Cys Glu Thr Tyr Lys Phe1 5
10 15Cys Glu Arg Val
2071628PRTHomo sapiensAGTPBP1 Cytosolic carboxypeptidase 1;
Q9UPW5_C1164MOD_RES(26)..(26)Site of chemical conjugation 716Arg Leu Thr
Ser Pro Leu Glu Tyr Asn Leu Pro Ser Ser Leu Leu Asp1 5
10 15Phe Glu Asn Asp Leu Ile Glu Ser Ser
Cys Lys Val 20 2571724PRTHomo sapiensMTMR1
Myotubularin-related protein 1; Q13613_C117MOD_RES(7)..(7)Site of
chemical conjugation 717Lys Asp Val Met Tyr Ile Cys Pro Phe Met Gly Ala
Val Ser Gly Thr1 5 10
15Leu Thr Val Thr Asp Phe Lys Leu 2071816PRTHomo sapiensPES1
Pescadillo homolog; O00541_C361MOD_RES(4)..(4)Site of chemical
conjugation 718Lys Ser Leu Cys Ile Gly Ala Thr Tyr Asp Val Thr Asp Ser
Arg Ile1 5 10
1571924PRTHomo sapiensPLA2G5 Calcium-dependent phospholipase A2;
P39877_C46MOD_RES(16)..(16)Site of chemical conjugation 719Lys Val Thr
Gly Lys Asn Ala Leu Thr Asn Tyr Gly Phe Tyr Gly Cys1 5
10 15Tyr Cys Gly Trp Gly Gly Arg Gly
2072024PRTHomo sapiensNPAT Protein NPAT;
Q14207_C1059MOD_RES(21)..(21)Site of chemical conjugation 720Lys Ser Glu
Glu Thr Thr Val Pro Phe Pro Glu Glu Ser Ile Val Pro1 5
10 15Ala Ala Lys Pro Cys His Arg Arg
2072123PRTHomo sapiensLRPPRC Leucine-rich PPR motif-containing
protein, mitocho; P42704_C500MOD_RES(9)..(9)Site of chemical conjugation
721Arg Ala Ile Leu Gln Glu Asn Gly Cys Leu Ser Asp Ser Asp Met Phe1
5 10 15Ser Gln Ala Gly Leu Arg
Ser 2072218PRTHomo sapiensTRAPPC1 Trafficking protein particle
complex subunit 1; Q9Y5R8_C115MOD_RES(5)..(5)Site of chemical
conjugation 722Lys Asn Pro Leu Cys Pro Leu Gly Gln Thr Val Gln Ser Glu
Leu Phe1 5 10 15Arg
Ser72320PRTHomo sapiensBUB1B Mitotic checkpoint serine/threonine-
protein kinase; O60566_C504MOD_RES(17)..(17)Site of chemical conjugation
723Lys Ile Pro Gly Met Thr Leu Ser Ser Ser Val Cys Gln Val Asn Cys1
5 10 15Cys Ala Arg Glu
2072413PRTHomo sapiensIBA57 Putative transferase CAF17,
mitochondrial; Q5T440_C259MOD_RES(3)..(3)Site of chemical conjugation
724Lys Gly Cys Tyr Ile Gly Gln Glu Leu Thr Ala Arg Thr1 5
1072526PRTHomo sapiensAHDC1 AT-hook DNA-binding
motif-containing protein 1; Q5TGY3_C1540MOD_RES(12)..(12)Site of
chemical conjugation 725Arg Gly Pro Ala Ala Ala Ala Ala Gly Tyr Gly Cys
Pro Leu Leu Ser1 5 10
15Asp Leu Thr Leu Ser Pro Val Pro Arg Asp 20
2572617PRTHomo sapiensZAP70 Tyrosine-protein kinase ZAP-70;
P43403_C117MOD_RES(14)..(14)Site of chemical conjugation 726Arg Pro Ser
Gly Leu Glu Pro Gln Pro Gly Val Phe Asp Cys Leu Arg1 5
10 15Asp72715PRTHomo sapiensPSAP
Proactivator polypeptide; P07602_C48MOD_RES(3)..(3)Site of chemical
conjugation 727Lys His Cys Leu Gln Thr Val Trp Asn Lys Pro Thr Val Lys
Ser1 5 10 1572814PRTHomo
sapiensMED12 Mediator of RNA polymerase II transcription subuni;
Q93074_C444MOD_RES(2)..(2)Site of chemical conjugation 728Lys Cys Gln Glu
Ala Thr Ala Gly Phe Thr Ile Gly Arg Val1 5
1072925PRTHomo sapiensPELI1 E3 ubiquitin-protein ligase pellino
homolog 1; Q96FA3_C282MOD_RES(11)..(11)Site of chemical conjugation
729Arg Gln Glu Ile Asn Ala Ala Arg Pro Gln Cys Pro Val Gly Phe Asn1
5 10 15Thr Leu Ala Phe Pro Ser
Met Lys Arg 20 2573010PRTHomo sapiensPNPLA6
Neuropathy target esterase; Q8IY17_C1199MOD_RES(7)..(7)Site of
chemical conjugation 730Arg Leu Ala Tyr Val Ser Cys Val Arg Gln1
5 1073122PRTHomo sapiensSMC5 Structural
maintenance of chromosomes protein 5; Q8IY18_C91MOD_RES(6)..(6)Site
of chemical conjugation 731Lys Ser Ser Ile Val Cys Ala Ile Cys Leu Gly
Leu Ala Gly Lys Pro1 5 10
15Ala Phe Met Gly Arg Ala 2073214PRTHomo sapiensSMC4
Structural maintenance of chromosomes protein 4;
Q9NTJ3_C110MOD_RES(4)..(4)Site of chemical conjugation 732Arg Phe Ser Cys
Ile Ile Gly Pro Asn Gly Ser Gly Lys Ser1 5
1073326PRTHomo sapiensCKAP4 Cytoskeleton-associated protein 4;
Q07065_C100MOD_RES(23)..(23)Site of chemical conjugation 733Lys Ser Ser
Ser Ser Ser Ser Ala Ser Ala Ala Ala Ala Ala Ala Ala1 5
10 15Ala Ser Ser Ser Ala Ser Cys Ser Arg
Arg 20 2573442PRTHomo sapiensRPS6KA3
Ribosomal protein S6 kinase alpha-3; P51812_C599MOD_RES(8)..(8)Site
of chemical conjugation 734Arg Gln Gly Tyr Asp Ala Ala Cys Asp Ile Trp
Ser Leu Gly Val Leu1 5 10
15Leu Tyr Thr Met Leu Thr Gly Tyr Thr Pro Phe Ala Asn Gly Pro Asp
20 25 30Asp Thr Pro Glu Glu Ile Leu
Ala Arg Ile 35 4073522PRTHomo sapiensMETTL3
N6-adenosine-methyltransferase 70 kDa subunit;
Q86U44_C500MOD_RES(13)..(13)Site of chemical conjugation 735Lys Gly Asn
Pro Gln Gly Phe Asn Gln Gly Leu Asp Cys Asp Val Ile1 5
10 15Val Ala Glu Val Arg Ser
2073616PRTHomo sapiensFN3K Fructosamine-3-kinase;
Q9H479_C24MOD_RES(10)..(10)Site of chemical conjugation 736Arg Ala Phe
Gly Gly Pro Gly Ala Gly Cys Ile Ser Glu Gly Arg Ala1 5
10 1573717PRTHomo sapiensUSP22 Ubiquitin
carboxyl-terminal hydrolase 22; Q9UPT9_C44MOD_RES(6)..(6)Site of
chemical conjugation 737Arg Ala Ile Tyr Gln Cys Phe Val Trp Ser Gly Thr
Ala Glu Ala Arg1 5 10
15Lys73827PRTHomo sapiensC21orf33 ES1 protein homolog, mitochondrial;
P30042_C244MOD_RES(10)..(10)Site of chemical conjugation 738Lys Val Val
Thr Thr Pro Ala Phe Met Cys Glu Thr Ala Leu His Tyr1 5
10 15Ile His Asp Gly Ile Gly Ala Met Val
Arg Lys 20 2573926PRTHomo sapiensKBTBD8 Kelch
repeat and BTB domain-containing protein 8;
Q8NFY9_C490MOD_RES(20)..(20)Site of chemical conjugation 739Arg Ile Gln
Gly Leu Ala Ala Val Tyr Lys Asp Ser Ile Tyr Tyr Ile1 5
10 15Ala Gly Thr Cys Gly Asn His Gln Arg
Met 20 2574019PRTHomo sapiensNARF Nuclear
prelamin A recognition factor; Q9UHQ1_C99MOD_RES(8)..(8)Site of
chemical conjugation 740Lys Val Leu Val Val Ser Val Cys Pro Gln Ser Leu
Pro Tyr Phe Ala1 5 10
15Ala Lys Phe74118PRTHomo sapiensRAB3GAP1 Rab3 GTPase-activating protein
catalytic subunit; Q15042_C218MOD_RES(4)..(4)Site of chemical
conjugation 741Lys Ile Gly Cys Pro Leu Thr Pro Leu Pro Pro Val Ser Ile
Ala Ile1 5 10 15Arg
Phe74211PRTHomo sapiensBRD8 Bromodomain-containing protein 8;
Q9H0E9_C26MOD_RES(3)..(3)Site of chemical conjugation 742Lys Leu Cys Leu
Ala Ser Ser Val Met Arg Ser1 5
1074310PRTHomo sapiensCASP5 Caspase-5;
P51878_C315MOD_RES(8)..(8)Site of chemical conjugation 743Lys Val Ile Ile
Val Gln Ala Cys Arg Gly1 5 1074421PRTHomo
sapiensTNRC6B Trinucleotide repeat-containing gene 6B protein;
Q9UPQ9_C600MOD_RES(10)..(10)Site of chemical conjugation 744Arg Ser Tyr
Arg Pro Thr His Pro Asp Cys Gln Ala Val Leu Gln Thr1 5
10 15Leu Leu Ser Arg Thr
2074522PRTHomo sapiensARNT Aryl hydrocarbon receptor nuclear
translocator; P27540_C119MOD_RES(16)..(16)Site of chemical conjugation
745Lys Met Thr Ala Tyr Ile Thr Glu Leu Ser Asp Met Val Pro Thr Cys1
5 10 15Ser Ala Leu Ala Arg Lys
2074620PRTHomo sapiensFAM136A Protein FAM136A;
Q96C01_C57MOD_RES(2)..(2)Site of chemical conjugation 746Arg Cys His Val
Pro Leu Ala Gln Ala Gln Ala Leu Val Thr Ser Glu1 5
10 15Leu Glu Lys Phe 2074719PRTHomo
sapiensSTARD7 StAR-related lipid transfer protein 7, mitochondri;
Q9NQZ5_C302MOD_RES(3)..(3)Site of chemical conjugation 747Arg Tyr Cys Val
Ser Trp Met Val Ser Ser Gly Met Pro Asp Phe Leu1 5
10 15Glu Lys Leu74811PRTHomo sapiensL2HGDH
L-2-hydroxyglutarate dehydrogenase, mitochondrial;
Q9H9P8_C376MOD_RES(3)..(3)Site of chemical conjugation 748Lys Ala Cys Phe
Leu Gly Ala Thr Val Lys Tyr1 5
107499PRTHomo sapiensSCCPDH Saccharopine dehydrogenase-like
oxidoreductase; Q8NBX0_C238MOD_RES(7)..(7)Site of chemical conjugation
749Arg Trp Pro Ile Ser Tyr Cys Arg Glu1 575025PRTHomo
sapiensFLAD1 FAD synthase; Q8NFF5_C499MOD_RES(10)..(10)Site of
chemical conjugation 750Arg Thr Asp Pro Tyr Ser Cys Ser Leu Cys Pro Phe
Ser Pro Thr Asp1 5 10
15Pro Gly Trp Pro Ala Phe Met Arg Ile 20
2575114PRTHomo sapiensBRAT1 BRCA1-associated ATM activator 1;
Q6PJG6_C228MOD_RES(2)..(2)Site of chemical conjugation 751Arg Cys Gln Ser
Pro Trp Thr Glu Ala Leu Trp Val Arg Leu1 5
1075240PRTHomo sapiensTNKS1BP1 182 kDa tankyrase-1-binding protein;
Q9C0C2_C749MOD_RES(24)..(24)Site of chemical conjugation 752Lys Asp Leu
Gln Ser Glu Phe Gly Ile Thr Gly Asp Pro Gln Pro Ser1 5
10 15Ser Phe Ser Pro Ser Ser Trp Cys Gln
Gly Ala Ser Gln Asp Tyr Gly 20 25
30Leu Gly Gly Ala Ser Pro Arg Gly 35
4075326PRTHomo sapiensKDM3A Lysine-specific demethylase 3A;
Q9Y4C1_C251MOD_RES(20)..(20)Site of chemical conjugation 753Lys Ile Val
Asp Pro Ser Leu Ile His Val Glu Val Val His Asp Asn1 5
10 15Leu Val Thr Cys Gly Asn Ser Ala Arg
Ile 20 2575429PRTHomo sapiensDTWD1 DTW
domain-containing protein 1; Q8N5C7_C235MOD_RES(22)..(22)Site of
chemical conjugation 754Lys Ile Phe Thr Asp Glu Arg Leu Gln Gly Leu Leu
Gln Val Glu Leu1 5 10
15Lys Thr Arg Lys Thr Cys Phe Trp Arg His Gln Lys Gly 20
2575510PRTHomo sapiensGNL3 Guanine nucleotide-binding
protein-like 3; Q9BVP2_C131MOD_RES(5)..(5)Site of chemical
conjugation 755Lys Lys Leu Tyr Cys Gln Glu Leu Lys Lys1 5
1075628PRTHomo sapiensWDFY4 WD repeat- and FYVE
domain-containing protein 4; Q6ZS81_C1963MOD_RES(23)..(23)Site of
chemical conjugation 756Arg Asp Gly Lys Glu Pro Gln Pro Ser Ala Glu Ala
Ala Ala Ala Pro1 5 10
15Ser Leu Ala Asn Ile Ser Cys Phe Thr Gln Lys Leu 20
2575730PRTHomo sapiensIPO13 Importin-13;
O94829_C217MOD_RES(27)..(27)Site of chemical conjugation 757Arg Thr Ser
Leu Ala Val Glu Cys Gly Ala Val Phe Pro Leu Leu Glu1 5
10 15Gln Leu Leu Gln Gln Pro Ser Ser Pro
Ser Cys Val Arg Gln 20 25
3075825PRTHomo sapiensMIEN1 Migration and invasion enhancer 1;
Q9BRT3_C33MOD_RES(10)..(10)Site of chemical conjugation 758Arg Ile Val
Val Glu Tyr Cys Glu Pro Cys Gly Phe Glu Ala Thr Tyr1 5
10 15Leu Glu Leu Ala Ser Ala Val Lys Glu
20 2575910PRTHomo sapiensNUDT16L1 Protein
syndesmos; Q9BRJ7_C171MOD_RES(2)..(2)Site of chemical conjugation
759Lys Cys Gln Leu Leu Phe Ala Leu Lys Val1 5
1076013PRTHomo sapiensFRMD6 FERM domain-containing protein 6;
Q96NE9_C306MOD_RES(9)..(9)Site of chemical conjugation 760Arg Lys Leu Ile
Tyr Tyr Thr Gly Cys Pro Met Arg Ser1 5
1076128PRTHomo sapiensUSP34 Ubiquitin carboxyl-terminal hydrolase 34;
Q70CQ2_C1090MOD_RES(10)..(10)Site of chemical conjugation 761Arg Leu Ala
Thr Ser Ala Tyr Asp Gly Cys Ser Asn Ser Glu Leu Cys1 5
10 15Gly Met Asp Gln Phe Trp Gly Ile Ala
Leu Arg Ala 20 2576210PRTHomo sapiensCYR61
Protein CYR61; O00622_C70MOD_RES(5)..(5)Site of chemical conjugation
762Lys Thr Gln Pro Cys Asp His Thr Lys Gly1 5
1076313PRTHomo sapiensATF7IP Activating transcription factor
7-interacting prot; Q6VMQ6_C612MOD_RES(7)..(7)Site of chemical
conjugation 763Lys Leu Cys Ala Leu Gln Cys Ala Val Phe Asp Lys Thr1
5 1076441PRTHomo sapiensTUBB4B Tubulin beta-4B
chain; P68371_C213MOD_RES(28)..(28)Site of chemical
conjugationMOD_RES(38)..(38)Site of chemical conjugation 764Lys Val Ser
Asp Thr Val Val Glu Pro Tyr Asn Ala Thr Leu Ser Val1 5
10 15His Gln Leu Val Glu Asn Thr Asp Glu
Thr Tyr Cys Ile Asp Asn Glu 20 25
30Ala Leu Tyr Asp Ile Cys Phe Arg Thr 35
4076521PRTHomo sapiensNR3C1 Glucocorticoid receptor;
P04150_C622MOD_RES(9)..(9)Site of chemical conjugation 765Arg Gln Ser Ser
Ala Asn Leu Leu Cys Phe Ala Pro Asp Leu Ile Ile1 5
10 15Asn Glu Gln Arg Met
2076638PRTHomo sapiensCHTF18 Chromosome transmission fidelity
protein 18 homolo; Q8WVB6_C280MOD_RES(28)..(28)Site of chemical
conjugation 766Arg Ser Gly Glu Glu Glu Ala Ala Gln Pro Leu Gly Ala Pro
Glu Glu1 5 10 15Glu Pro
Thr Asp Gly Gln Asp Ala Ser Ser His Cys Leu Trp Val Asp 20
25 30Glu Phe Ala Pro Arg His
3576716PRTHomo sapiensDENND4A C-myc promoter-binding protein;
Q7Z401_C1289MOD_RES(12)..(12)Site of chemical conjugation 767Arg Leu Trp
Ser Ser Pro Ala Phe Ser Pro Thr Cys Pro Phe Arg Glu1 5
10 1576838PRTHomo sapiensHNRNPK
Heterogeneous nuclear ribonucleoprotein K;
P61978_C132MOD_RES(30)..(30)Site of chemical conjugation 768Lys Ile Ile
Pro Thr Leu Glu Glu Gly Leu Gln Leu Pro Ser Pro Thr1 5
10 15Ala Thr Ser Gln Leu Pro Leu Glu Ser
Asp Ala Val Glu Cys Leu Asn 20 25
30Tyr Gln His Tyr Lys Gly 3576915PRTHomo sapiensHNRNPH1
Heterogeneous nuclear; P31943_C267MOD_RES(6)..(6)Site of chemical
conjugation 769Arg Asp Leu Asn Tyr Cys Phe Ser Gly Met Ser Asp His Arg
Tyr1 5 10 1577011PRTHomo
sapiensCORO1C Coronin-1C; Q9ULV4_C420MOD_RES(3)..(3)Site of chemical
conjugation 770Lys Lys Cys Asp Leu Ile Ser Ile Pro Lys Lys1
5 1077125PRTHomo sapiensADD1 Alpha-adducin;
P35611_C68MOD_RES(12)..(12)Site of chemical conjugation 771Arg Val Ser
Met Ile Leu Gln Ser Pro Ala Phe Cys Glu Glu Leu Glu1 5
10 15Ser Met Ile Gln Glu Gln Phe Lys Lys
20 2577229PRTHomo sapiensCECR5 Cat eye syndrome
critical region protein 5; Q9BXW7_C392MOD_RES(4)..(4)Site of
chemical conjugation 772Arg Asp Leu Cys Phe Ser Pro Gly Leu Met Glu Ala
Ser His Val Val1 5 10
15Asn Asp Val Asn Glu Ala Val Gln Leu Val Phe Arg Lys 20
2577346PRTHomo sapiensPDIA3 Protein disulfide-isomerase A3;
P30101_C406MOD_RES(41)..(41)Site of chemical conjugation 773Lys Ser Glu
Pro Ile Pro Glu Ser Asn Asp Gly Pro Val Lys Val Val1 5
10 15Val Ala Glu Asn Phe Asp Glu Ile Val
Asn Asn Glu Asn Lys Asp Val 20 25
30Leu Ile Glu Phe Tyr Ala Pro Trp Cys Gly His Cys Lys Asn 35
40 4577418PRTHomo sapiensPDIA4 Protein
disulfide-isomerase A4; P13667_C555MOD_RES(13)..(13)Site of chemical
conjugation 774Lys Lys Asp Val Leu Ile Glu Phe Tyr Ala Pro Trp Cys Gly
His Cys1 5 10 15Lys
Gln77524PRTHomo sapiensSMARCC2 SWI/SNF complex subunit SMARCC2;
Q8TAQ2_C145MOD_RES(17)..(17)Site of chemical conjugation 775Lys Ser Leu
Val Gln Asn Asn Cys Leu Ser Arg Pro Asn Ile Phe Leu1 5
10 15Cys Pro Glu Ile Glu Pro Lys Leu
2077620PRTHomo sapiensZC3HAV1 Zinc finger CCCH-type antiviral
protein 1; Q7Z2W4_C645MOD_RES(17)..(17)Site of chemical conjugation
776Lys Asn Ser Asn Val Asp Ser Ser Tyr Leu Glu Ser Leu Tyr Gln Ser1
5 10 15Cys Pro Arg Gly
2077728PRTHomo sapiensMACROD1 O-acetyl-ADP-ribose deacetylase
MACROD1; Q9BQ69_C186MOD_RES(24)..(24)Site of chemical conjugation 777Lys
Leu Glu Val Asp Ala Ile Val Asn Ala Ala Asn Ser Ser Leu Leu1
5 10 15Gly Gly Gly Gly Val Asp Gly
Cys Ile His Arg Ala 20 2577813PRTHomo
sapiensCCNH Cyclin-H; P51946_C244MOD_RES(3)..(3)Site of chemical
conjugation 778Arg Thr Cys Leu Ser Gln Leu Leu Asp Ile Met Lys Ser1
5 1077925PRTHomo sapiensZWINT ZW10 interactor;
O95229_C54MOD_RES(4)..(4)Site of chemical conjugation 779Lys Leu Leu
Cys Ser Gln Leu Gln Val Ala Asp Phe Leu Gln Asn Ile1 5
10 15Leu Ala Gln Glu Asp Thr Ala Lys Gly
20 2578017PRTHomo sapiensDIAPH1 Protein
diaphanous homolog 1; O60610_C1227MOD_RES(4)..(4)Site of chemical
conjugation 780Lys Ala Gly Cys Ala Val Thr Ser Leu Leu Ala Ser Glu Leu
Thr Lys1 5 10
15Asp78121PRTHomo sapiensPRDX4 Peroxiredoxin-4;
Q13162_C51MOD_RES(5)..(5)Site of chemical conjugation 781Arg Glu Glu Glu
Cys His Phe Tyr Ala Gly Gly Gln Val Tyr Pro Gly1 5
10 15Glu Ala Ser Arg Val
2078224PRTHomo sapiensUSP7 Ubiquitin carboxyl-terminal hydrolase 7;
Q93009_C223MOD_RES(7)..(7)Site of chemical conjugation 782Lys Asn Gln Gly
Ala Thr Cys Tyr Met Asn Ser Leu Leu Gln Thr Leu1 5
10 15Phe Phe Thr Asn Gln Leu Arg Lys
2078318PRTHomo sapiensLIMCH1 LIM and calponin homology domains-
containing protein; Q9UPQ0_C140MOD_RES(6)..(6)Site of chemical
conjugation 783Lys Ala Ala Asn Ser Cys Thr Ser Tyr Ser Gly Thr Thr Leu
Asn Leu1 5 10 15Lys
Glu78415PRTHomo sapiensTK1 Thymidine kinase, cytosolic;
P04183_C230MOD_RES(11)..(11)Site of chemical conjugation 784Lys Leu Phe
Ala Pro Gln Gln Ile Leu Gln Cys Ser Pro Ala Asn1 5
10 1578540PRTHomo sapiensTCOF1 Treacle protein;
Q13428_C1298MOD_RES(20)..(20)Site of chemical conjugation 785Lys Gly
Ala Gly Asn Pro Gln Ala Ser Thr Leu Ala Leu Gln Ser Asn1 5
10 15Ile Thr Gln Cys Leu Leu Gly Gln
Pro Trp Pro Leu Asn Glu Ala Gln 20 25
30Val Gln Ala Ser Val Val Lys Val 35
4078611PRTHomo sapiensXPO1 Exportin-1;
O14980_C528MOD_RES(7)..(7)Site of chemical conjugation 786Lys Asp Leu Leu
Gly Leu Cys Glu Gln Lys Arg1 5
1078731PRTHomo sapiensSCP2 Non-specific lipid-transfer protein;
P22307_C71MOD_RES(27)..(27)Site of chemical conjugation 787Lys Ala Leu
Ala Asp Ala Gln Ile Pro Tyr Ser Ala Val Asp Gln Ala1 5
10 15Cys Val Gly Tyr Val Phe Gly Asp Ser
Thr Cys Gly Gln Arg Ala 20 25
3078826PRTHomo sapiensECI2 Enoyl-CoA delta isomerase 2,
mitochondrial; O75521_C312MOD_RES(9)..(9)Site of chemical conjugation
788Lys Lys Leu Thr Ala Gly Glu Ala Cys Ala Gln Gly Leu Val Thr Glu1
5 10 15Val Phe Pro Asp Ser Thr
Phe Gln Lys Glu 20 2578911PRTHomo
sapiensNT5DC3 5-nucleotidase domain-containing protein 3;
Q86UY8_C276MOD_RES(4)..(4)Site of chemical conjugation 789Lys Tyr Ile Cys
Tyr Ala Glu Gln Thr Arg Ala1 5
1079026PRTHomo sapiensNR2F2 COUP transcription factor 2;
P24468_C326MOD_RES(11)..(11)Site of chemical conjugation 790Lys Ala Ile
Val Leu Phe Thr Ser Asp Ala Cys Gly Leu Ser Asp Val1 5
10 15Ala His Val Glu Ser Leu Gln Glu Lys
Ser 20 2579138PRTHomo sapiensNPEPL1 Probable
aminopeptidase NPEPL1; Q8NDH3_C81MOD_RES(36)..(36)Site of chemical
conjugation 791Arg Val Thr Glu Glu Leu Trp Gln Ala Ala Leu Ser Thr Leu
Asn Pro1 5 10 15Asn Pro
Thr Asp Ser Cys Pro Leu Tyr Leu Asn Tyr Ala Thr Val Ala 20
25 30Ala Leu Pro Cys Arg Val
3579216PRTHomo sapiensMCMBP Mini-chromosome maintenance complex-
binding protein; Q9BTE3_C325MOD_RES(12)..(12)Site of chemical conjugation
792Lys Leu Gln His Ile Asn Pro Leu Leu Pro Ala Cys Leu Asn Lys Glu1
5 10 1579319PRTHomo
sapiensTMPO Lamina-associated polypeptide 2, isoform alpha;
P42166_C629MOD_RES(10)..(10)Site of chemical conjugation 793Lys Thr Tyr
Asp Ala Ala Ser Tyr Ile Cys Glu Ala Ala Phe Asp Glu1 5
10 15Val Lys Met79423PRTHomo
sapiensMAD2L1BP MAD2L1-binding protein;
Q15013_C124MOD_RES(2)..(2)Site of chemical conjugation 794Lys Cys Gln Gln
Ala Leu Ala Glu Leu Glu Ser Val Leu Ser His Leu1 5
10 15Glu Asp Phe Phe Ala Arg Thr
207959PRTHomo sapiensNUBP2 Cytosolic Fe-S cluster assembly factor
NUBP2; Q9Y5Y2_C72MOD_RES(6)..(6)Site of chemical conjugation 795Arg Ala
Val His Gln Cys Asp Arg Gly1 579637PRTHomo sapiensCD3EAP
DNA-directed RNA polymerase I subunit RPA34;
O15446_C86MOD_RES(6)..(6)Site of chemical conjugation 796Arg Val Leu Ser
Ser Cys Pro Gln Ala Gly Glu Ala Thr Leu Leu Ala1 5
10 15Pro Ser Thr Glu Ala Gly Gly Gly Leu Thr
Cys Ala Ser Ala Pro Gln 20 25
30Gly Thr Leu Arg Ile 3579716PRTHomo sapiensTSTA3 GDP-L-fucose
synthase; Q13630_C116MOD_RES(10)..(10)Site of chemical conjugation
797Arg Lys Val Val Ser Cys Leu Ser Thr Cys Ile Phe Pro Asp Lys Thr1
5 10 1579822PRTHomo
sapiensCTSC Dipeptidyl peptidase 1; P53634_C258MOD_RES(9)..(9)Site
of chemical conjugation 798Arg Asn Gln Ala Ser Cys Gly Ser Cys Tyr Ser
Phe Ala Ser Met Gly1 5 10
15Met Leu Glu Ala Arg Ile 2079934PRTHomo sapiensFADD Protein
FADD; Q13158_C98MOD_RES(21)..(21)Site of chemical conjugation 799Arg
Val Asp Asp Phe Glu Ala Gly Ala Ala Ala Gly Ala Ala Pro Gly1
5 10 15Glu Glu Asp Leu Cys Ala Ala
Phe Asn Val Ile Cys Asp Asn Val Gly 20 25
30Lys Asp80015PRTHomo sapiensWAPAL Wings apart-like protein
homolog; Q7Z5K2_C160MOD_RES(12)..(12)Site of chemical conjugation
800Arg Ile Val Glu Asp Asp Ala Ser Ile Ser Ser Cys Asn Lys Leu1
5 10 1580110PRTHomo sapiensTMPO
Lamina-associated polypeptide 2, isoform alpha;
P42166_C518MOD_RES(8)..(8)Site of chemical conjugation 801Arg Gln Leu Pro
Ser Leu Ala Cys Lys Tyr1 5 1080212PRTHomo
sapiensUSP22 Ubiquitin carboxyl-terminal hydrolase 22;
Q9UPT9_C171MOD_RES(6)..(6)Site of chemical conjugation 802Lys Ile Thr Ser
Asn Cys Thr Ile Gly Leu Arg Gly1 5
1080331PRTHomo sapiensIRF2BP1 Interferon regulatory factor 2-binding
protein 1; Q8IU81_C363MOD_RES(25)..(25)Site of chemical conjugation
803Arg Ser Phe Arg Glu Pro Ala Pro Ala Glu Ala Leu Pro Gln Gln Tyr1
5 10 15Pro Glu Pro Ala Pro Ala
Ala Leu Cys Gly Pro Pro Pro Arg Ala 20 25
3080426PRTHomo sapiensDENND1C DENN domain-containing protein
1C; Q8IV53_C174MOD_RES(9)..(9)Site of chemical conjugation 804Arg
Gly Asn Ser Lys Pro Leu Ser Cys Phe Val Ala Pro Asp Ser Gly1
5 10 15Arg Leu Pro Ser Ile Pro Glu
Asn Arg Asn 20 2580516PRTHomo sapiensAPOBEC3C
Probable DNA dC- dU-editing enzyme APOBEC-3C;
Q9NRW3_C130MOD_RES(9)..(9)Site of chemical conjugation 805Arg Leu Tyr Tyr
Phe Gln Tyr Pro Cys Tyr Gln Glu Gly Leu Arg Ser1 5
10 1580610PRTHomo sapiensIRAK4 Interleukin-1
receptor-associated kinase 4; Q9NWZ3_C13MOD_RES(2)..(2)Site of
chemical conjugation 806Arg Cys Leu Asn Val Gly Leu Ile Arg Lys1
5 1080715PRTHomo sapiensTDRKH Tudor and KH
domain-containing protein; Q9Y2W6_C109MOD_RES(13)..(13)Site of
chemical conjugation 807Arg Val Leu Leu Ile Ser Gly Phe Pro Val Gln Val
Cys Lys Ala1 5 10
1580811PRTHomo sapiensNADSYN1 Glutamine-dependent NAD(+) synthetase;
Q6IA69_C428MOD_RES(8)..(8)Site of chemical conjugation 808Lys Asn Ser Ser
Gln Glu Thr Cys Thr Arg Ala1 5
108099PRTHomo sapiensIKBKB Inhibitor of nuclear factor kappa-B
kinase subunit; O14920_C464MOD_RES(5)..(5)Site of chemical conjugation
809Arg Asn Asn Ser Cys Leu Ser Lys Met1 581014PRTHomo
sapiensADA Adenosine deaminase; P00813_C75MOD_RES(12)..(12)Site of
chemical conjugation 810Lys Phe Asp Tyr Tyr Met Pro Ala Ile Ala Gly Cys
Arg Glu1 5 1081113PRTHomo sapiensVDAC3
Voltage-dependent anion-selective channel protein;
Q9Y277_C65MOD_RES(3)..(3)Site of chemical conjugation 811Lys Val Cys Asn
Tyr Gly Leu Thr Phe Thr Gln Lys Trp1 5
1081210PRTHomo sapiensAARS Alanine--tRNA ligase, cytoplasmic;
P49588_C773MOD_RES(2)..(2)Site of chemical conjugation 812Lys Cys Leu Ser
Val Met Glu Ala Lys Val1 5 1081329PRTHomo
sapiensUBE2L6 Ubiquitin/ISG15-conjugating enzyme E2 L6;
O14933_C98MOD_RES(26)..(26)Site of chemical conjugation 813Lys Ile Tyr
His Pro Asn Val Asp Glu Asn Gly Gln Ile Cys Leu Pro1 5
10 15Ile Ile Ser Ser Glu Asn Trp Lys Pro
Cys Thr Lys Thr 20 2581411PRTHomo sapiensPGLS
6-phosphogluconolactonase; O95336_C32MOD_RES(4)..(4)Site of chemical
conjugation 814Arg Ala Ala Cys Cys Leu Ala Gly Ala Arg Ala1
5 1081512PRTHomo sapiensPGP Phosphoglycolate
phosphatase; A6NDG6_C297MOD_RES(8)..(8)Site of chemical conjugation
815Lys Asn Asn Gln Glu Ser Asp Cys Val Ser Lys Lys1 5
1081626PRTHomo sapiensHUWE1 E3 ubiquitin-protein ligase
HUWE1; Q7Z6Z7_C3372MOD_RES(3)..(3)Site of chemical conjugation
816Lys Ala Cys Ser Pro Cys Ser Ser Gln Ser Ser Ser Ser Gly Ile Cys1
5 10 15Thr Asp Phe Trp Asp Leu
Leu Val Lys Leu 20 2581717PRTHomo
sapiensBCL2A1 Bcl-2-related protein A1;
Q16548_C55MOD_RES(3)..(3)Site of chemical conjugation 817Lys Ser Cys Leu
Asp Asn Val Asn Val Val Ser Val Asp Thr Ala Arg1 5
10 15Thr81811PRTHomo sapiensPYGB Glycogen
phosphorylase, brain form; P11216_C326MOD_RES(3)..(3)Site of
chemical conjugation 818Arg Thr Cys Phe Glu Thr Phe Pro Asp Lys Val1
5 1081913PRTHomo sapiensAGFG2 Arf-GAP domain
and FG repeat-containing protein 2; O95081_C39MOD_RES(6)..(6)Site of
chemical conjugation 819Arg Glu Leu Gly Gly Cys Ser Gln Ala Gly Asn Arg
His1 5 1082020PRTHomo sapiensZC3HAV1 Zinc
finger CCCH-type antiviral protein 1;
Q7Z2W4_C645MOD_RES(17)..(17)Site of chemical conjugation 820Lys Asn Ser
Asn Val Asp Ser Ser Tyr Leu Glu Ser Leu Tyr Gln Ser1 5
10 15Cys Pro Arg Gly
2082111PRTHomo sapiensAIP AH receptor-interacting protein;
O00170_C122MOD_RES(4)..(4)Site of chemical conjugation 821Arg His Cys Cys
Gly Val Ala Gln Met Arg Glu1 5
1082211PRTHomo sapiensTRNT1 CCA tRNA nucleotidyltransferase 1,
mitochondrial; Q96Q11_C373MOD_RES(7)..(7)Site of chemical conjugation
822Lys Tyr Gln Gly Glu His Cys Leu Leu Lys Glu1 5
1082316PRTHomo sapiensRIN3 Ras and Rab interactor 3;
Q8TB24_C942MOD_RES(13)..(13)Site of chemical conjugation 823Arg Cys Phe
Gln Leu Ala Asp Asp Ala Leu Pro His Cys Ile Lys Gly1 5
10 1582414PRTHomo sapiensLAS1L Ribosomal
biogenesis protein LAS1L; Q9Y4W2_C456MOD_RES(5)..(5)Site of chemical
conjugation 824Arg Leu Phe Asn Cys Ser Ala Ser Leu Asp Trp Pro Arg Met1
5 1082513PRTHomo sapiensIRF8 Interferon
regulatory factor 8; Q02556_C306MOD_RES(11)..(11)Site of chemical
conjugation 825Arg Val Phe Cys Ser Gly Asn Ala Val Val Cys Lys Gly1
5 1082629PRTHomo sapiensMARS2 Methionine--tRNA
ligase, mitochondrial; Q96GW9_C425MOD_RES(12)..(12)Site of chemical
conjugation 826Arg Ile Asn Pro Ser Glu Thr Tyr Pro Ala Phe Cys Thr Thr
Cys Phe1 5 10 15Pro Ser
Glu Pro Gly Leu Val Gly Pro Ser Val Arg Ala 20
2582724PRTHomo sapiensIRF4 Interferon regulatory factor 4;
Q15306_C194MOD_RES(16)..(16)Site of chemical conjugation 827Arg Asp Tyr
Val Pro Asp Gln Pro His Pro Glu Ile Pro Tyr Gln Cys1 5
10 15Pro Met Thr Phe Gly Pro Arg Gly
2082824PRTHomo sapiensSPCS2 Signal peptidase complex subunit 2;
Q15005_C17MOD_RES(8)..(8)Site of chemical conjugation 828Arg Ser Gly Gly
Ser Gly Gly Cys Ser Gly Ala Gly Gly Ala Ser Asn1 5
10 15Cys Gly Thr Gly Ser Gly Arg Ser
2082922PRTHomo sapiensRARS Arginine--tRNA ligase, cytoplasmic;
P54136_C32MOD_RES(3)..(3)Site of chemical conjugation 829Lys Asn Cys Gly
Cys Leu Gly Ala Ser Pro Asn Leu Glu Gln Leu Gln1 5
10 15Glu Glu Asn Leu Lys Leu
2083052PRTHomo sapiensTUBGCP3 Gamma-tubulin complex component 3;
Q96CW5_C194MOD_RES(49)..(49)Site of chemical conjugation 830Arg Ser Ala
Gln Ser Ala Gln Ser Ser Gly Ser Val Gly Ser Ser Gly1 5
10 15Ile Ser Ser Ile Gly Leu Cys Ala Leu
Ser Gly Pro Ala Pro Ala Pro 20 25
30Gln Ser Leu Leu Pro Gly Gln Ser Asn Gln Ala Pro Gly Val Gly Asp
35 40 45Cys Leu Arg Gln
5083110PRTHomo sapiensCRKL Crk-like protein;
P46109_C249MOD_RES(5)..(5)Site of chemical conjugation 831Lys Arg Val Pro
Cys Ala Tyr Asp Lys Thr1 5 1083224PRTHomo
sapiensPUSL1 tRNA pseudouridine synthase-like 1;
Q8N0Z8_C292MOD_RES(13)..(13)Site of chemical conjugation 832Lys Ser Val
Leu Tyr Gly Asn Leu Gly Ala Ala Ser Cys Thr Leu Gln1 5
10 15Gly Pro Gln Phe Gly Ser His Gly
2083312PRTHomo sapiensUBR4 E3 ubiquitin-protein ligase UBR4;
Q5T4S7_C2554MOD_RES(5)..(5)Site of chemical conjugation 833Lys Ala Val
Gln Cys Leu Asn Thr Ser Ser Lys Glu1 5
1083413PRTHomo sapiensZNF346 Zinc finger protein 346;
Q9UL40_C68MOD_RES(4)..(4)Site of chemical conjugation 834Lys Asn Gln Cys
Leu Phe Thr Asn Thr Gln Cys Lys Val1 5
1083517PRTHomo sapiensFLII Protein flightless-1 homolog;
Q13045_C46MOD_RES(5)..(5)Site of chemical conjugation 835Arg Thr Gly Leu
Cys Tyr Leu Pro Glu Glu Leu Ala Ala Leu Gln Lys1 5
10 15Leu83628PRTHomo sapiensKIAA0528
Uncharacterized protein KIAA0528; Q86YS7_C993MOD_RES(21)..(21)Site
of chemical conjugation 836Arg Glu Ser Asp Leu Glu Val Val Ser Ser Gln
Gln Pro Thr Thr Asn1 5 10
15Cys Gln Ser Ser Cys Thr Glu Gly Glu Val Thr Thr 20
258379PRTHomo sapiensMTCH2 Mitochondrial carrier homolog 2;
Q9Y6C9_C296MOD_RES(4)..(4)Site of chemical conjugation 837Lys Thr Tyr Cys
Cys Asp Leu Lys Met1 583819PRTHomo sapiensDCXR L-xylulose
reductase; Q7Z4W1_C244MOD_RES(19)..(19)Site of chemical conjugation
838Arg Ser Gly Met Thr Thr Gly Ser Thr Leu Pro Val Glu Gly Gly Phe1
5 10 15Trp Ala Cys83930PRTHomo
sapiensPRKCQ Protein kinase C theta type;
Q04759_C14MOD_RES(9)..(9)Site of chemical conjugation 839Arg Ile Gly Leu
Ser Asn Phe Asp Cys Gly Ser Cys Gln Ser Cys Gln1 5
10 15Gly Glu Ala Val Asn Pro Tyr Cys Ala Val
Leu Val Lys Glu 20 25
3084018PRTHomo sapiensSON Protein SON; P18583_C92MOD_RES(2)..(2)Site
of chemical conjugation 840Arg Cys Val Ser Val Gln Thr Asp Pro Thr Asp
Glu Ile Pro Thr Lys1 5 10
15Lys Ser84110PRTHomo sapiensMAT2A S-adenosylmethionine synthase isoform
type-2; P31153_C56MOD_RES(4)..(4)Site of chemical conjugation 841Lys
Val Ala Cys Glu Thr Val Ala Lys Thr1 5
1084223PRTHomo sapiensBCL2A1 Bcl-2-related protein A1;
Q16548_C19MOD_RES(9)..(9)Site of chemical conjugation 842Arg Leu Ala Gln
Asp Tyr Leu Gln Cys Val Leu Gln Ile Pro Gln Pro1 5
10 15Gly Ser Gly Pro Ser Lys Thr
2084321PRTHomo sapiensIL16 Pro-interleukin-16;
Q14005_C1004MOD_RES(5)..(5)Site of chemical conjugation 843Lys Ser Leu
Leu Cys Leu Pro Ser Ser Ile Ser Cys Ala Gln Thr Pro1 5
10 15Cys Ile Pro Lys Glu
2084425PRTHomo sapiensMAT2A S-adenosylmethionine synthase isoform
type-2; P31153_C104MOD_RES(3)..(3)Site of chemical conjugation 844Lys Thr
Cys Asn Val Leu Val Ala Leu Glu Gln Gln Ser Pro Asp Ile1 5
10 15Ala Gln Gly Val His Leu Asp Arg
Asn 20 2584521PRTHomo sapiensVDAC3
Voltage-dependent anion-selective channel protein;
Q9Y277_C36MOD_RES(3)..(3)Site of chemical conjugation 845Lys Ser Cys Ser
Gly Val Glu Phe Ser Thr Ser Gly His Ala Tyr Thr1 5
10 15Asp Thr Gly Lys Ala
2084613PRTHomo sapiensSYNE2 Nesprin-2;
Q8WXH0_C553MOD_RES(7)..(7)Site of chemical conjugation 846Lys Asn Leu Ala
Gly Glu Cys Gln Asn Ile Asn Lys Gln1 5
1084717PRTHomo sapiensSCLY Selenocysteine lyase;
Q96I15_C22MOD_RES(14)..(14)Site of chemical conjugation 847Arg Asp Ala
Pro Ala Pro Ala Ala Ser Gln Pro Ser Gly Cys Gly Lys1 5
10 15His84810PRTHomo sapiensPML Protein
PML; P29590_C479MOD_RES(2)..(2)Site of chemical conjugation 848Lys
Cys Ser Gln Thr Gln Cys Pro Arg Lys1 5
1084919PRTHomo sapiensTHNSL1 Threonine synthase-like 1;
Q8IYQ7_C324MOD_RES(16)..(16)Site of chemical conjugation 849Arg Leu Gly
Glu Met Ile Glu Thr Ala Tyr Gly Glu Asn Phe Ala Cys1 5
10 15Ser Lys Ile85012PRTHomo sapiensUSP7
Ubiquitin carboxyl-terminal hydrolase 7;
Q93009_C315MOD_RES(4)..(4)Site of chemical conjugation 850Lys Gly Thr Cys
Val Glu Gly Thr Ile Pro Lys Leu1 5
1085111PRTHomo sapiensTNFAIP3 Tumor necrosis factor alpha-induced
protein 3; P21580_C54MOD_RES(3)..(3)Site of chemical conjugation 851Arg
Thr Cys Gln Phe Cys Pro Gln Phe Arg Glu1 5
1085212PRTHomo sapiensGAK Cyclin-G-associated kinase;
O14976_C87MOD_RES(8)..(8)Site of chemical conjugation 852Arg Ala Ile Ile
Gln Glu Val Cys Phe Met Lys Lys1 5
1085310PRTHomo sapiensTHOC1 THO complex subunit 1;
Q96FV9_C49MOD_RES(2)..(2)Site of chemical conjugation 853Lys Cys Thr Leu
Asp Gln Ala Phe Arg Gly1 5 108549PRTHomo
sapiensPRKDC DNA-dependent protein kinase catalytic subunit;
P78527_C4045MOD_RES(5)..(5)Site of chemical conjugation 854Arg Gln Lys
Ile Cys Tyr Ala Lys Arg1 585517PRTHomo sapiensCHRAC1
Chromatin accessibility complex protein 1;
Q9NRG0_C55MOD_RES(9)..(9)Site of chemical conjugation 855Lys Ala Thr Glu
Leu Phe Val Gln Cys Leu Ala Thr Tyr Ser Tyr Arg1 5
10 15His85619PRTHomo sapiensGHDC GH3
domain-containing protein; Q8N2G8_C502MOD_RES(7)..(7)Site of
chemical conjugation 856Arg Ala Ala Leu Ala Ala Cys Pro Ser Ser Pro Phe
Pro Pro Ala Met1 5 10
15Pro Arg Val8574PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 857Asp Glu Val Asp18584PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 858Ile
Glu Thr Asp18594PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 859Ala Glu Val Asp18607PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 860Gly
Gln Phe Tyr Leu Asn Glu1 58616PRTArtificial
SequenceDescription of Artificial Sequence Synthetic 6xHis tag
861His His His His His His1 58626PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 862Arg
Arg Arg Arg Arg Arg1 58634PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 863Trp Glu His
Asp186414PRTHomo sapiensCASP8 C360MOD_RES(7)..(7)Site of chemical
conjugation 864Val Phe Phe Ile Gln Ala Cys Gln Gly Asp Asn Tyr Gln Lys1
5 1086518PRTHomo sapiens 865Ser Asn Phe Asp
Cys Gly Ser Cys Gln Ser Cys Gln Gly Glu Ala Val1 5
10 15Asn Pro86618PRTMus sp. 866Ser Asn Phe Asp
Cys Gly Thr Cys Gln Ala Cys Gln Gly Glu Ala Val1 5
10 15Asn Pro86717PRTHomo sapiens 867Asn Ser Tyr
Glu Leu Gly Ser Leu Gln Ala Glu Asp Glu Ala Asn Gln1 5
10 15Pro86826PRTHomo sapiens 868Ala Val Ser
Leu Lys Pro Thr Ala Trp Ser Leu Arg His Ala Val Gly1 5
10 15Pro Arg Pro Gln Thr Phe Leu Leu Asp
Pro 20 25
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