SCREENING METHODS FOR PARP MODULATORS

Abstract

The present disclosure is related to methods of identifying Poly(ADP-ribose) polymerases (PARP) inhibitors, and the methods of using PARP probes.

Description

CLAIM OF PRIORITY

[0001] This application is a divisional application of U.S. patent application Ser. No. 16/397,410, filed on Apr. 29, 2019, which claims priority to U.S. Provisional Patent Application Ser. No. 62/664,595, filed on Apr. 30, 2018, the entire contents of which are hereby incorporated by reference.

SEQUENCE LISTING

[0002] This application contains a Sequence Listing that has been submitted electronically as an ASCII text file named SequenceListing.txt. The ASCII text file, created on Mar. 17, 2022, is 126 kilobytes in size. The material in the ASCII text file is hereby incorporated by reference in its entirety.

TECHNICAL FIELD

[0003] The present disclosure is related to methods of identifying Poly(ADP-ribose) polymerase (PARP) inhibitors.

BACKGROUND

[0004] Poly(ADP-ribose) polymerases (PARPs) are a family of 17 enzymes that can transfer ADP-ribose from nicotinamide adenine dinucleotide (NAD.sup.+) to protein and nucleic acid substrates.sup.1-2. The PARP enzymes family is comprised of two subfamilies, monoPARPs and polyPARPs. MonoPARP enzymes catalyze the transfer of a single ADP-ribose group to a target amino acid, while polyPARP enzymes catalyze the transfer of multiple ADP-ribose groups to form polymers. While approved drugs that target the polyPARPs exist, there are no potent and selective inhibitors of the monoPARPs. MonoPARPs are important regulators of the immune response.sup.3-4 and are implicated in human diseases such as inflammation.sup.5 and cancer.sup.6-7, therefore small molecules that modulate the enzymatic activity of monoPARPs can be useful therapeutics. Despite interest in the development of monoPARP inhibitors, the field is lacking effective high-throughput assays that can be used to screen for and characterize modulators of monoPARP function. Thus, there is an urgent need for high-throughput assays that can be used to screen monoPARP modulators.

SUMMARY

[0005] The present disclosure is related to methods of identifying PARP modulators.

[0006] The present invention is directed to a method of identifying an inhibitor for PARPs, the method comprising:

[0007] combining (i) a polypeptide comprising a PARP catalytic domain wherein the polypeptide is labeled with a donor fluorophore, (ii) a PARP probe, wherein the PARP probe is labeled with an acceptor fluorophore, and (iii) a test compound;

[0008] exposing the donor fluorophore to excitation light;

[0009] measuring a signal produced by the acceptor fluorophore; and

[0010] identifying the test compound as an inhibitor for PARP based on the signal produced by the acceptor fluorophore.

[0011] The present invention is further directed to a method of identifying an inhibitor for PARPs, the method comprising:

[0012] combining (i) a polypeptide comprising a PARP catalytic domain, wherein the polypeptide is labeled with an acceptor fluorophore; (ii) a PARP probe, wherein the PARP probe is labeled with a donor fluorophore, and (iii) a test compound;

[0013] exposing the donor fluorophore to excitation light;

[0014] measuring a signal produced by the acceptor fluorophore in the presence of a test compound; and

[0015] identifying the test compound as an inhibitor for PARP based on the signal produced by the acceptor fluorophore.

[0016] The present invention is further directed to a method of identifying an inhibitor for PARPs, the method comprising:

[0017] contacting a polypeptide comprising a PARP catalytic domain with a PARP probe in the presence of a test compound, wherein the PARP probe comprises a fluorophore;

[0018] exposing the probe to polarized excitation light, thereby generating fluorescence;

[0019] determining a fluorescence polarization value of the fluorescence; and

[0020] identifying the test compound as an inhibitor for PARP based on the fluorescence polarization value of the fluorescence.

[0021] The present invention is further directed to a method of identifying an inhibitor for PARPs, the method comprising:

[0022] contacting a fusion polypeptide with a PARP probe that comprises a fluorophore, wherein the fusion polypeptide comprises a PARP catalytic domain and a luciferase enzyme;

[0023] contacting the luciferase enzyme with a substrate to produce light, wherein the light can excite the fluorophore;

[0024] measuring a signal produced by the fluorophore in the presence of a test compound; and

[0025] identifying the test compound as an inhibitor for PARP based on the signal produced by the fluorophore.

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0027] Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

DESCRIPTION OF DRAWINGS

[0028] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0029] FIG. 1 is a schematic diagram illustrating an example of in vitro TR-FRET probe displacement assay. An inhibitor will outcompete the biotinylated probe and decrease TR-FRET signal.

[0030] FIG. 2 is a schematic diagram illustrating an example of in vitro fluorescence polarization probe displacement assay. An inhibitor will outcompete the fluorophore-labeled probe and decrease fluorescence polarization signal.

[0031] FIG. 3 is a schematic diagram illustrating an example of bioluminescence resonance energy transfer probe displacement assay. An inhibitor will outcompete the fluorophore-labeled probe and decrease NanoBRET signal.

[0032] FIGS. 4A-4F. Validation results of the in vitro probe displacement binding assays. Dose response curves for Compound A were generated using each assay to confirm that Probe C was outcompeted from the monoPARP enzyme. IC.sub.50 values for Compound A were 7 nM (TIPARP), 80 nM (PARP10), 200 nM (PARP12), 50 nM (PARP14), 60 nM (PARP15) and 100 nM (PARP16).

[0033] FIGS. 5A-5D. Validation results of the bioluminescence resonance energy transfer probe displacement binding assays. Dose response curves for control compounds were generated using each assay to confirm that Probe A was outcompeted from the monoPARP enzyme. IC.sub.50 values for catalytic TIPARP Compound B=7 nM, full-length TIPARP Compound B=4 nM, PARP10 Compound C=4 nM, PARP12 Compound B=170 nM, PARP14 Compound A=30 nM.

[0034] FIGS. 6A-6F show the amino acid sequences of PARPs.

DETAILED DESCRIPTION

[0035] PARPs refers to a family of proteins involved in a number of cellular processes such as DNA repair, genomic stability, and programmed cell death.

[0036] The primary function of PARPs is to post-translationally modify target proteins with ADP-ribose using NAD.sup.+ as substrate. The four best-studied family members, PARP1 and PARP5a along with their close functional homologs PARP2 and PARP5b respectively, all generate poly(ADP-ribose) (PAR). The main role of these PARPs is to detect and initiate an immediate cellular response to metabolic, chemical, or radiation-induced single-strand DNA breaks (SSB) by signaling the enzymatic machinery involved in the SSB repair. Once PARP detects a SSB, it binds to the DNA, undergoes a structural change, and begins the synthesis of a polymeric adenosine diphosphate ribose (poly (ADP-ribose)) chain, which acts as a signal for the other DNA-repairing enzymes.

[0037] However, many other PARPs do not generate PAR, and instead attach ADP-ribose as a monomer ADP-ribose (MAR) onto target proteins. Recent data has shown that many of these MAR-generating PARPs (monoPARPs) can have cancer relevant functions or inflammation related functions. MonoPARP enzymes have structurally related active sites which bind to nicotinamide adenine dinucleotide (NAD.sup.+) and catalyze the transfer of adenosine diphosphoribose to a substrate amino acid. Enzyme-linked immunosorbent assays (ELISA) measuring the incorporation of biotin-NAD.sup.+ to histones or to the monoPARP itself in an automodification reaction have been used to screen for monoPARP modulators.sup.8. These reactions are not catalytically efficient, and high concentrations of enzyme are needed to generate sufficient turnover to be detected. Since the lowest measurable IC.sub.50 in an enzyme assay is half of the total enzyme concentration.sup.9, these assays are usually unable to differentiate and rank order very potent compounds. Thermal shift assays (TSA) have also been used to screen inhibitors of monoPARPs.sup.10, however these assays consume large amounts of protein and are at best semi-quantitative since compounds with similar binding affinities can have different effects on protein stabilization.sup.11.

[0038] The present disclosure provides a more effective way to identify PARP modulators, and provides a series of high affinity active site probes that bind in the NAD.sup.+ pocket of monoPARPs which can be used to develop high-throughput biophysical assays.

Poly (ADP-Ribose) Polymerases

[0039] There are 17 PARPs. The enzymatic activity and cancer relevant functions of these PARPs are summarized in Table 1 below.

TABLE-US-00001 TABLE 1 Cancer Other NCBI Accession Related Cancers to PARP Names Activity No. Catalytic Domain Functions target PARP1 ARTD1 PAR NP_001609.2 Amino acids 788- DNA Repair, HR deficient (SEQ ID NO: 13) 1014 of accession ERK/ NF-kB Elevated no. NP_001609.2 signaling, Heat ERK/NF-kB shock signaling response PARP2 ARTD2 PAR NP_005475.2 Amino acids 356- DNA Repair HR deficient (SEQ ID NO: 14) 583 of accession no. NP_005475.2 PARP3 ARTD3 MAR NP_001003931.3 Amino acids 313- DNA Repair DNA repair (SEQ ID NO: 15) 533 of accession deficient no. NP_001003931.3 PARP4 vPARP MAR NP_006428.2 Amino acids 369- ARTD4 (SEQ ID NO: 16) 573 of accession no. NP_006428.2 PARP5a TNKS1 PAR NP_003738.2 Amino acids 1112- Telomere Elevated Wnt ARTD5 (SEQ ID NO: 17) 1317 of accession Maintenance, Signaling no. NP_003738.2 Wnt Signaling, Telomerase Proteasome Dependent Regulation, Stress Granule Stress Granule Positive Solid Assembly, Cell Tumors Division PARP5b TNKS2 PAR NP_079511.1 Amino acids 959- Telomere Elevated Wnt ARTD6 (SEQ ID NO: 18) 1164 of accession Maintenance, Signaling, no. NP_079511.1 Wnt Signaling Telomerase Dependent PARP6 ARTD17 MAR NP_001310451.1 Amino acids 394- Negative Potential (SEQ ID NO: 19) 620 of accession Regulator of Tumor no. Proliferation Suppressive NP_001310451.1 Functions TIPARP PARP7 MAR NP_056323.2 Amino acids 449- ARTD14 (SEQ ID NO: 1) 657 of accession no. NP_056323.2 PARP8 ARTD16 MAR AAH37386.1 Amino acids 328- (SEQ ID NO: 20) 494 of accession no. AAH37386.1 PARP9 BAL1 MAR AAH39580.1 Amino acids 628- Cell Migration Metastatic ARTD9 (SEQ ID NO: 21) 850 of accession Cancers no. AAH39580.1 PARP10 ARTD10 MAR NP_116178.2 Amino acids 806- Inhibits Potential (SEQ ID NO: 2) 1025 of accession Myc and NF- Tumor no. NP_116178.2 kB signaling Suppressive Pro-apoptotic Functions PARP11 ARTD11 MAR Q9NR21.2 Amino acids 123- (SEQ ID NO: 22) 338 of accession no. Q9NR21.2 PARP12 ARTD12 MAR NP_073587.1 Amino acids 484- Stress Granule Stress Granule (SEQ ID NO: 3) 698 of accession Assembly Positive Solid no. NP_073587.1 Tumors PARP13 ZAP MAR NP_064504.2 Amino acids 716- Stress Granule Stress Granule ZC3HAV1 (SEQ ID NO: 23) 902 of accession Assembly, Positive Solid ARTD13 no. NP_064504.2 miRNA-RISC Tumors regulation PARP14 BAL2 MAR NP_060024.2 Amino acids 1605- B cell survival, Hematopoeitic ARTD8 (SEQ ID NO: 4) 1801 of accession Cell Migration, malignancies, no. NP_060024.2 Stress Granule Metastatic Assembly Cancers PARP15 BAL3 MAR NP_689828.1 Amino acids 482- Stress Granule Stress Granule ARTD7 (SEQ ID NO: 5) 678 of accession Assembly Positive Solid no. NP_689828.1 Tumors PARP16 ARTD15 MAR NP_060321.3 Amino acids 94- ER Unfolded UPR (SEQ ID NO: 6) 279 of accession Protein dependent no. NP_060321.3 Response

[0040] Among these enzymes, PARP1, PARP2, PARP5a and PARP5b can generate poly (ADP-ribose) (PAR). PARP3, PARP4, PARP6, TIPARP, PARP8, PARP9, PARP10, PARP11, PARP12, PARP13, PARP14, PARP15, and PARP16 are monoPARPs.

[0041] PARPs have multiple diverse functions in physiological pathways including cell migration, transcriptional regulation, signal transduction, miRNA-mediated gene silencing, regulation of membrane organelles and telomere length regulation. Additionally, PARPs function in stress-responsive cellular pathways upon DNA damage, cytoplasmic stress, environmental stress and ER stress, activating DNA damage repair, stress granule assembly, the heat shock response and the ER unfolded protein response pathways in response. Many of these physiological and stress response pathways are misregulated in cancer or inflammation.

[0042] Thus, the inhibitors of PARPs (e.g., monoPARPs) can have various uses. For example, they can be used to modulate (e.g., inhibit or facilitate) cell migration, transcriptional regulation, signal transduction, and gene silencing. They can also be used to modulate (e.g., inhibit or facilitate) stress-responsive cellular pathways (e.g., upon DNA damage), or DNA damage repair pathways. In some embodiments, PARP inhibitors (e.g., monoPARP inhibitors) can be used to treat a disorder associated with PARP overexpression or overactivity. In some embodiments, PARP inhibitors (e.g., monoPARP inhibitors) can be used to treat cancers or inflammation.

[0043] A detailed description of PARPs and their functions can be found, e.g., in Vyas et al., "New PARP targets for cancer therapy," Nature Reviews Cancer 14.7 (2014): 502, which is incorporated herein by reference in its entirety.

[0044] The present disclosure provides methods of identifying PARP modulators, and also provides polypeptides (e.g., fusion polypeptides) comprising a catalytic domain of PARPs (e.g., PARP1, PARP2, PARP3, PARP4, PARP5a, PARP5b, PARP6, TIPARP, PARP8, PARP9, PARP10, PARP11, PARP12, PARP13, PARP14, PARP15, or PARP16). These polypeptides can be used in various assays for identifying the modulators of interest (e.g., PARP inhibitors). As used herein, the term "catalytic domain" refers to a portion of an enzyme that has a catalytic activity. The catalytic domain comprises the region of an enzyme that interacts with its substrate to cause the enzymatic reaction. In many cases, the active site is located in the catalytic domain, and the substrate binds to active site.

[0045] In some embodiments, the catalytic domain is the catalytic domain of TIPARP (e.g., residues 449-657 of NP_056323.2 (SEQ ID NO: 1)), the catalytic domain of PARP10 (e.g., residues 806-1025 of NP_116178.2 (SEQ ID NO: 2)), the catalytic domain of PARP12 (e.g., residues 484-698 of NP_073587.1 (SEQ ID NO: 3)), the catalytic domain of PARP14 (e.g., residues 1605-1801 of NP_060024.2 (SEQ ID NO: 4)), the catalytic domain of PARP15 (e.g., residues 482-678 of NP_689828.1 (SEQ ID NO: 5)), or the catalytic domain of PARP16 (e.g., residues 5-279 of NP_060321.3 (SEQ ID NO: 6)).

[0046] In some embodiments, the catalytic domain is the catalytic domain of PARP1 (e.g., residues 788-1014 of NP_001609.2 (SEQ ID NO: 13)), the catalytic domain of PARP2 (e.g., residues 356-583 of NP_005475.2 (SEQ ID NO: 14)), the catalytic domain of PARP3 (e.g., residues 313-533 of NP_001003931.3 (SEQ ID NO: 15)), the catalytic domain of PARP4 (e.g., residues 369-573 of NP_006428.2 (SEQ ID NO: 16)), the catalytic domain of PARP5a (e.g., residues 1112-1317 of NP_003738.2 (SEQ ID NO: 17)), the catalytic domain of PARP5b (e.g., residues 959-1164 of NP_079511.1 (SEQ ID NO: 18)), the catalytic domain of PARP6 (e.g., residues 394-620 of NP_001310451.1 (SEQ ID NO: 19)), the catalytic domain of PARP8 (e.g., residues 328-494 of AAH37386.1 (SEQ ID NO: 20)), the catalytic domain of PARP9 (e.g., residues 628-850 of AAH39580.1 (SEQ ID NO: 21)), the catalytic domain of PARP11 (e.g., residues 123-338 of Q9NR21.2 (SEQ ID NO: 22)), or the catalytic domain of PARP13 (e.g., residues 716-902 of NP_064504.2 (SEQ ID NO: 23)).

[0047] In some embodiments, the polypeptide comprises a catalytic domain that has a sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the sequence of a catalytic domain as described herein.

[0048] In some embodiments, the polypeptide (e.g., fusion polypeptide) comprises a region of a PARP, wherein the substrate binding pocket is located in this region of the PARP. In some embodiments, the polypeptide comprises residues 456-657 of NP_056323.2 (SEQ ID NO: 1), residues 808-1025 of NP_116178.2 (SEQ ID NO: 2), residues 489-684 of NP_073587.1 (SEQ ID NO: 3), residues 1611-1801 of NP_060024.2 (SEQ ID NO: 4), residues 481-678 of NP_689828.1 (SEQ ID NO: 5), or residues 5-279 of NP_060321.3 (SEQ ID NO: 6).

[0049] In some embodiments, the polypeptide can be linked with a label (e.g., a fluorophore). As used herein, the term "linked" refers to being covalently or non-covalently associated, e.g., by a chemical bond (e.g., a peptide bond, or a carbon-carbon bond), by hydrophobic interaction, by Van der Waals interaction, and/or by electrostatic interaction.

[0050] The label can be a chemical or composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. For example, useful labels include fluorescent dyes (fluorophores), luminescent agents, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA, or luciferase), biotin, enzymes acting on a substrate (e.g., horseradish peroxidase), digoxigenin, .sup.32P and other isotopes, haptens, and proteins which can be made detectable, e.g., by incorporating a radiolabel into the peptide or used to detect antibodies specifically reactive with the peptide. The term includes combinations of single labeling agents, e.g., a combination of fluorophores that provides a unique detectable signature, e.g., at a particular wavelength or combination of wavelengths. Any method known in the art for conjugating label to a desired agent may be employed.

[0051] In some embodiments, the polypeptide can have a fusion tag (e.g., SEQ ID NO: 7, 8, 9, or 10). These fusion tags can be used to purify polypeptides.

[0052] In some embodiments, the polypeptide can have an epitope (e.g., 6.times.His) that can be specifically recognized by an antibody (e.g., anti-6.times.His antibody). A label (e.g., fluorophore) can be conjugated to the antibody, thereby associating with the polypeptide.

[0053] In some embodiments, the polypeptide is a fusion polypeptide and can comprise a luciferase (e.g., SEQ ID NO: 11). The luciferase can be located at the N-terminus or the C-terminus of the polypeptide.

[0054] The disclosure also provides a nucleic acid sequence that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to any nucleotide sequence as described herein, and an amino acid sequence that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to any amino acid sequence as described herein. In some embodiments, the nucleic acid sequence is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to any nucleotide sequence as described herein. In some embodiments, the amino acid sequence is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to any amino acid sequence as described herein.

[0055] To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). The length of a reference sequence aligned for comparison purposes is at least 80% of the length of the reference sequence, and in some embodiments is at least 90%, 95%, or 100%. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology"). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. For purposes of the present disclosure, the comparison of sequences and determination of percent identity between two sequences can be accomplished using a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

[0056] In some embodiments, the disclosure relates to nucleotide sequences encoding any peptides that are described herein, or any amino acid sequences that are encoded by any nucleotide sequences as described herein. In some embodiments, the nucleic acid sequence is less than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 150, 200, 250, 300, 350, 400, 500, 600, 700, 800, 900, or 1000 nucleotides. In some embodiments, the amino acid sequence is less than 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 400, 500, 600, 700, 800, 900, or 1000 amino acid residues.

PARP Probes

[0057] The present disclosure provides high affinity active site probes that bind in the NAD.sup.+ pocket of PARPs (e.g., monoPARPs) which can be used to develop high-throughput biophysical assays. These probes are based on the structure-activity relationship (SAR) and binding mode of inhibitors that bind to the NAD.sup.+ pocket of monoPARP enzymes. Thus, as used herein, the term "PARP probe" refers to an agent (e.g., a small molecule) that can bind to the active site of a PARP.

[0058] Many enzyme inhibitors inhibit the functions of enzymes by preventing a substrate from entering the enzyme's active site. Thus, if a test compound can prevent an active site probe from binding to the enzyme's active site, the test compound can also prevent a substrate from entering the enzyme's active site, thus working as an inhibitor. Therefore, in some embodiments, these PARP inhibitors can compete with PARP probes, bind to or occupy enzyme's active site, and/or displace the PARP probes.

[0059] In some embodiments, the PARP probe has a structure according to Formula (I):

##STR00001##

or a salt thereof, wherein:

[0060] L is a linking group having 10-30 spacer atoms selected from C, N, O, and S connecting the N atom of the piperidinyl group of Formula (I) with group A; and

[0061] A is a fluorophore or an affinity tag.

[0062] In some embodiments,

L is:

##STR00002##

[0063] wherein: a is 0, 1, or 2; b is 1-26; and c is 0, 1, or 2; wherein the sum of a+b+c is 1 to 26, or L is a chain of 5-30 atoms in length comprising --(CH.sub.2CH.sub.2O).sub.d-- wherein d is 2-10.

[0064] In some embodiments, A is an organic dye.

[0065] In some embodiments, A is biotin.

[0066] In some embodiments, A is:

##STR00003##

or a salt thereof.

[0067] As used herein, the term "fluorophore" refers to a chemical compound that can re-emit light upon light excitation. The donor is the fluorophore that emits light of shorter wavelength which is used to excite the acceptor, causing it to emit light of longer wavelength. Fluorophores can be organic molecules like fluorescein and similar organic dye moieties. Fluorophores can also include inorganic components like transition metals.

[0068] An affinity tag is a chemical or polypeptide group that can bind to other chemical or polypeptide molecules covalently or non-covalently (e.g., preferably with high affinity). Examples of non-covalent affinity tags are biotin which binds to streptavidin and antibodies, hexahistidine which binds to nickel-nitrilotriacetic acid and antibodies, glutathione which binds to glutathione S-transferase and antibodies, etc. Examples of covalent affinity tags are primary amines such as lysine which react with N-hydroxysuccinamide, as well as free thiols such as cysteine which react with other free thiols to form disulfide bonds. In some embodiments, the affinity tag is biotin.

[0069] Where a PARP probe "is labeled" with a fluorophore, the PARP probe can contain an affinity tag that covalently or non-covalently binds with a molecule (e.g., streptavidin or an antibody) having a fluorophore. Where a PARP probe comprises a fluorophore or an affinity tag, the fluorophore or affinity tag is generally understood to be part of the probe molecule.

[0070] The PARP probes of the invention, including salts thereof, can be prepared using known organic synthesis techniques and can be synthesized according to any of numerous possible synthetic routes.

[0071] The reactions for preparing compounds as described herein can be carried out in suitable solvents which can be readily selected by one of skill in the art of organic synthesis. Suitable solvents can be substantially nonreactive with the starting materials (reactants), the intermediates, or products at the temperatures at which the reactions are carried out, e.g., temperatures which can range from the solvent's freezing temperature to the solvent's boiling temperature. A given reaction can be carried out in one solvent or a mixture of more than one solvent. Depending on the particular reaction step, suitable solvents for a particular reaction step can be selected by the skilled artisan.

[0072] Preparation of compounds as described herein can involve the protection and deprotection of various chemical groups. The need for protection and deprotection, and the selection of appropriate protecting groups, can be readily determined by one skilled in the art. The chemistry of protecting groups can be found, for example, in T. W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 3rd. Ed., Wiley & Sons, Inc., New York (1999), which is incorporated herein by reference in its entirety.

[0073] Reactions can be monitored according to any suitable method known in the art. For example, product formation can be monitored by spectroscopic means, such as nuclear magnetic resonance spectroscopy (e.g., .sup.1H or .sup.13C), infrared spectroscopy, spectrophotometry (e.g., UV-visible), or mass spectrometry, or by chromatography such as high performance liquid chromatography (HPLC) or thin layer chromatography.

[0074] The expressions, "ambient temperature," "room temperature," and "r.t.", as used herein, are understood in the art, and refer generally to a temperature, e.g. a reaction temperature, that is about the temperature of the room in which the reaction is carried out, for example, a temperature from about 20.degree. C. to about 30.degree. C.

Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Probe Displacement Assay

[0075] The PARP probes can be used in time-resolved fluorescence resonance energy transfer (TR-FRET) probe displacement assays. TR-FRET is the combination of time-resolved fluorometry (TRF) with Forster resonance energy transfer (FRET). It can offer a powerful tool for studying the interactions between biomolecules.

[0076] FRET occurs when a donor fluorophore in its excited state transfers energy by a non-radiative dipole-dipole coupling mechanism to an acceptor fluorophore in close proximity (e.g., <10 nm). As a result, the acceptor emission is predominantly observed because of the intermolecular FRET from the donor to the acceptor. FRET can be quantified in cuvette-based experiments or in microscopy images on a pixel-by-pixel basis. This quantification can be based directly on detecting two emission channels under two different excitation conditions (primarily donor and primarily acceptor). However, for robustness reasons, FRET quantification is most often based on measuring changes in fluorescence intensity.

[0077] The biological fluids or serum commonly used in these research applications contain many compounds and proteins which are naturally fluorescent. Therefore, the use of conventional, steady-state fluorescence measurement presents serious limitations in assay sensitivity.

[0078] To reduce assay interference and increase data quality, a time-resolved FRET (TR-FRET) assay can be used to identify PARP inhibitors. TR-FRET generally employs a long-lifetime donor fluorophore (e.g., terbium chelate, samarium, europium, terbium, and dysprosium) and a suitable acceptor fluorophore (fluorescein or allophycocyanin).

[0079] As shown in FIG. 1, TR-FRET can be used to identify an agent that can inhibit the binding between the PARP probe and the PARP polypeptide. The methods involve providing a polypeptide comprising a PARP catalytic domain. The polypeptide is labeled with a donor fluorophore. In the meantime, the PARP probe is labeled with an acceptor fluorophore. When the donor fluorophore is exposed to excitation light, the energy is transferred from the donor fluorophore to the acceptor fluorophore. The signal produced by the acceptor fluorophore is measured. In the presence of an agent (e.g., a PARP inhibitor) that can inhibit the binding between the PARP probe and the PARP polypeptide, the PARP probe is not in close proximity with the PARP polypeptide, thus the energy cannot be effectively transferred from the donor fluorophore to the acceptor fluorophore. Therefore, the signal produced by the acceptor fluorophore will decrease.

[0080] In certain embodiments, an agent (e.g., a test compound) is identified as a PARP inhibitor if it results in a decrease in the signal produced by the acceptor fluorophore by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% as compared to the signal of the acceptor fluorophore in the absence of the agent or any agent that can inhibit or interfere with the binding between the PARP probe and the PARP polypeptide.

[0081] In some embodiments, the signal produced by the acceptor fluorophore can be compared to a reference level. The reference level can be the signal produced by the acceptor fluorophore in the absence of any agent that can inhibit or interfere with the binding between the PARP probe and the polypeptide.

[0082] In certain embodiments, an agent (e.g., a test compound) is identified as a PARP inhibitor if it results in a decrease in the ratio of the signal produced by the acceptor to the signal produced by the donor by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% as compared to the ratio in the absence of the agent or any agent that can inhibit or interfere with the binding between the PARP probe and the PARP polypeptide.

[0083] In some embodiments, the ratio of the signal produced by the acceptor to the signal produced by the donor can be compared to a reference level. The reference level can be the ratio of the acceptor signal to the donor signal in the absence of any agent that can inhibit or interfere with the binding between the PARP probe and the polypeptide.

[0084] In some embodiments, the polypeptide can be labeled with a donor fluorophore, and the PARP probe can be labeled with an acceptor fluorophore.

[0085] A variety of fluorophore combinations can be used in TR-FRET. In some embodiments, lanthanide ion complexes (Ln(III) chelates or cryptates) are used. In some embodiments, the donor fluorophore is Europium.sup.3+, and the acceptor fluorophore is allophycocyanin. In some embodiments, the donor fluorophore is Terbium3+, and the acceptor fluorophore is phycoerythrin.

[0086] TR-FRET measurements can be also carried out using any suitable technique. For example, a microscope image of donor emission can be taken with the acceptor being present. The acceptor is then bleached, such that it is incapable of accepting energy transfer and another donor emission image is acquired. A pixel based quantification using the second equation in the theory section above is then possible. An alternative way of temporarily deactivating the acceptor is based on its fluorescence saturation.

[0087] In some embodiments, the ratio between the signal produced by the acceptor and the signal produced by the donor is calculated. The % inhibition can be calculated as described below:

% .times. .times. inhibition = 100 .times. TRF cmpd - TRF min TRF max - TRF min ##EQU00001##

wherein TRF.sub.cmpd is the TR-FRET ratio from the compound treated solution, TRF.sub.min is the TR-FRET ratio from a positive control and TRF.sub.max is the TR-FRET ratio from the negative control (e.g., DMSO-treated).

[0088] The % inhibition values can be plotted as a function of compound concentration and the following 4-parameter fit can be applied to derive the IC.sub.50 values:

Y = Bottom + ( Top - Bottom ) ( 1 + ( X IC 50 ) Hill .times. .times. Coefficient ##EQU00002##

wherein top and bottom can be normally allowed to float, but may be fixed at 100 or 0 respectively in a 3-parameter fit. The Hill Coefficient can be normally allowed to float but may also be fixed at 1 in a 3-parameter fit. Y is the % inhibition and X is the compound concentration. IC.sub.50 is the concentration of an inhibitor where the response (or binding) is reduced by half. It is a measure of the potency of a substance in inhibiting a specific biological or biochemical function. Based on the modeling, IC.sub.50 can be estimated.

[0089] FRET and TR-FRET methods, protocols, techniques, and assays are described generally and specifically in a number of patents and patent applications, including, e.g., U.S. Pat. Nos. 6,908,769; 6,824,990; 6,762,280; 6,689,574; 6,661,909; 6,642,001; 6,639,078; 6,472,156; 6,456,734; 6,376,257; 6,348,322; 6,323,039; 6,291,201; 6,280,981; 5,914,245; 5,661,035; and US 20080113444; US 2009021510; Du et al. "A time-resolved fluorescence resonance energy transfer assay for high-throughput screening of 14-3-3 protein-protein interaction inhibitors." Assay and drug development technologies 11.6 (2013): 367-381; each of which is incorporated herein by reference in its entirety.

Fluorescence Polarization Probe Displacement Assay

[0090] The PARP probes can also be used in a fluorescence polarization probe displacement assay. Fluorescence polarization (FP) is a homogeneous method that allows rapid and quantitative analysis of diverse molecular interactions and enzyme activities. This technique has been widely used in clinical and biomedical settings, and high-throughput screening (HTS).

[0091] In fluorescence polarization assays, a fluorophore is excited with polarized excitation light; the polarized fluorescence is then measured through an emission polarizer either parallel or perpendicular to the exciting light's plane of polarization. If a fluorescent molecule is stationary and exposed to plane-polarized light, it will become excited and consequently emit radiation back to the polarized-plane. However, if the excited fluorescent molecule is in motion (rotational or translational) during the fluorescent lifetime, it will emit light in a different direction than the excitation plane. The rate at which a molecule rotates is indicative of its size. When a fluorescent-labelled molecule binds to another molecule, the rotational motion will change, resulting in an altered intensity of plane-polarized light, which results in altered fluorescence polarization.

[0092] As shown in FIG. 2, when the PARP probe binds to the PARP polypeptide, the complex has a relatively larger molecular size. When an agent (e.g., PARP inhibitor) inhibits the binding between the PARP probe and the PARP polypeptide, the PARP probe with the fluorophore will not bind to the PARP polypeptide, thus the probe can rotate more quickly, resulting in a decrease of fluorescence polarization. This change can be detected, thereby determining whether an agent is a PARP inhibitor. In certain embodiments, an agent (e.g., a test compound) is identified as a PARP inhibitor if the fluorescence polarization decreases by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% as compared to the fluorescence polarization in the absence of the agent or any agent that can inhibit or interfere with the binding between the PARP probe and the PARP polypeptide.

[0093] A detailed description of fluorescence polarization and the method of implementing it is described, e.g., in Lea, Wendy A., and Anton Simeonov. "Fluorescence polarization assays in small molecule screening." Expert opinion on drug discovery 6.1 (2011): 17-32; U.S. Pat. No. 6,432,632; US20030082665; each of which is incorporated herein by reference in its entirety.

Bioluminescence Resonance Energy Transfer (BRET) Probe Displacement Assay

[0094] The PARP probes can also be used in bioluminescence resonance energy transfer (BRET) probe displacement assays. A limitation of FRET is the requirement for external illumination to initiate the fluorescence transfer, which can lead to background noise. Bioluminescence resonance energy transfer involves a bioluminescent luciferase (e.g., the luciferase from Renilla reniformis, or Oplophorus gracilirostris) to produce an initial photon emission.

[0095] As compared to FRET, in BRET, the donor fluorophore is replaced by a luciferase. As shown in FIG. 3, in the presence of a substrate, bioluminescence from the luciferase excites the acceptor fluorophore through Forster resonance energy transfer mechanisms. Thus, if the acceptor fluorophore is in close proximity with the luciferase, the acceptor fluorophore accepts the energy from the luciferase and emits a light with different length.

[0096] When an agent (e.g., PARP inhibitor) inhibits the binding between PARP probe and the PARP polypeptide, the PARP probe with the acceptor fluorophore will not be in close proximity with the PARP polypeptide, thus the light from the luciferase enzyme reaction cannot be transferred to the acceptor fluorophore, resulting a decrease of fluorescence emitted from the acceptor fluorophore. This change can be detected, thereby determining whether an agent is a PARP inhibitor. In certain embodiments, an agent (e.g., a test compound) is identified as a PARP inhibitor if it results in a decrease in the fluorescence emitted from the acceptor fluorophore by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% as compared to the fluorescence emitted from the acceptor fluorophore in the absence of the agent or any agent that can inhibit or interfere with the binding between the PARP probe and the PARP polypeptide.

[0097] In some embodiments, the luciferase is NanoLuc, and the acceptor fluorophore is NanoBRET.RTM. 590SE.

[0098] BRET ratio can be measured b the formula as shown below:

BRET .times. .times. ratio = Emission .times. .times. of .times. .times. the .times. .times. acceptor .times. .times. fluorophore Luminescence ##EQU00003##

[0099] Control wells containing a negative control or a positive control are used to calculate the % inhibition as described below:

% .times. .times. inhibition = 100 .times. BRET .times. .times. ratio cmpd - BRET .times. .times. ratio min BRET .times. .times. ratio max - BRET .times. .times. ratio min ##EQU00004##

wherein BRET ratio.sub.cmpd is the BRET ratio from the compound treated solution, BRET ratio.sub.min is the BRET ratio from the positive control and BRET ratio.sub.max is the BRET ratio from the negative control.

[0100] The % inhibition values are plotted as a function of compound concentration and the following 4-parameter fit is applied to derive the IC.sub.50 values:

Y = Bottom + ( Top - Bottom ) ( 1 + ( X IC 50 ) Hill .times. .times. Coefficient ##EQU00005##

wherein top and bottom can be normally allowed to float, but may be fixed at 100 or 0 respectively in a 3-parameter fit. The Hill Coefficient can be normally allowed to float but may also be fixed at 1 in a 3-parameter fit. Y is the % inhibition and X is the compound concentration. The IC.sub.50 value can be derived from the modeling.

High Throughput Screening and Compound Library

[0101] The assays as described herein can be used in high throughput screening for PARP modulators (e.g., monoPARP inhibitors). Such assays can be used to screen small molecule libraries available from various commercial sources. Screening of such libraries, including combinatorially generated libraries (e.g., peptide libraries), is a rapid and efficient way to screen a large number of related (and unrelated) compounds for activity.

[0102] This disclosure provides methods for screening test compounds, e.g., polypeptides (including, e.g., antibodies and antigen-binding fragments thereof), polynucleotides, inorganic or organic large or small molecule test compounds, to identify agents useful for modulating PARP enzymatic activity, and for the treatment of disorders associated with PARP overexpression or overactivity (e.g., cancer or inflammation).

[0103] As used herein, "small molecules" refers to small organic or inorganic molecules of molecular weight below about 3,000 Daltons. In general, small molecules useful for the methods as described herein can have a molecular weight of less than 3,000 Daltons (Da). The small molecules can be, e.g., from at least about 100 Da to about 3,000 Da (e.g., between about 100 to about 3,000 Da, about 100 to about 2500 Da, about 100 to about 2,000 Da, about 100 to about 1,750 Da, about 100 to about 1,500 Da, about 100 to about 1,250 Da, about 100 to about 1,000 Da, about 100 to about 750 Da, about 100 to about 500 Da, about 200 to about 1500, about 500 to about 1000, about 300 to about 1000 Da, or about 100 to about 250 Da).

[0104] The test compounds can be, e.g., natural products or members of a combinatorial chemistry library. A set of diverse molecules should be used to cover a variety of functions such as charge, aromaticity, hydrogen bonding, flexibility, size, length of side chain, hydrophobicity, and rigidity. Combinatorial techniques suitable for synthesizing small molecules are known in the art, e.g., as exemplified by Obrecht and Villalgordo, Solid-Supported Combinatorial and Parallel Synthesis of Small-Molecular-Weight Compound Libraries, Pergamon-Elsevier Science Limited (1998), and include those such as the "split and pool" or "parallel" synthesis techniques, solid-phase and solution-phase techniques, and encoding techniques (see, for example, Czarnik, Curr. Opin. Chem. Bio. 1:60-6 (1997)). In addition, a number of small molecule libraries are commercially available.

[0105] Libraries screened using the methods as described herein can comprise a variety of types of test compounds. A given library can comprise a set of structurally related or unrelated test compounds. In some embodiments, the test compounds are peptide or peptidomimetic molecules. In some embodiments, the test compounds are nucleic acids. In some embodiments, the test compounds are antibodies or antigen-binding fragments thereof.

[0106] In some embodiments, the test compounds and libraries thereof can be obtained by systematically altering the structure of a first test compound, e.g., a first test compound that is structurally similar to a known natural binding partner of the target polypeptide, or a first small molecule identified as capable of binding the target polypeptide, e.g., using methods known in the art or the methods described herein, and correlating that structure to a resulting biological activity, e.g., a structure-activity relationship study. As one of skill in the art will appreciate, there are a variety of standard methods for creating such a structure-activity relationship. Thus, in some instances, the work may be largely empirical, and in others, the three-dimensional structure of an endogenous polypeptide or portion thereof can be used as a starting point for the rational design of a small molecule compound or compounds. For example, in one embodiment, a general library of small molecules is screened, e.g., using the methods described herein.

[0107] In some embodiments, a test compound is applied to a test sample, e.g., a cell or living tissue, e.g., tumor tissue, and one or more effects of the test compound is evaluated. For example, the ability of the test compound to inhibit cell growth or tumor growth is evaluated.

[0108] In some embodiments, the test sample is, or is derived from (e.g., a sample taken from) an in vivo model. For example, an animal model, e.g., a rodent such as a rat, can be used.

[0109] Thus, test compounds identified as "hits" (e.g., test compounds that can inhibit PARP) in a first screen can be selected and systematically altered, e.g., using rational design, to optimize binding affinity, avidity, specificity, or other parameter. Such optimization can also be screened for using the methods described herein. Thus, in one embodiment, the disclosure includes screening a first library of compounds using a method known in the art and/or described herein, identifying one or more hits in that library, subjecting those hits to systematic structural alteration to create a second library of compounds structurally related to the hit, and screening the second library using the methods described herein.

[0110] Test compounds identified as hits can be considered candidate therapeutic compounds, useful in treating disorders associated with PARP overexpression or overactivity (e.g., cancer). A variety of techniques useful for determining the structures of "hits" can be used in the methods described herein, e.g., NMR, mass spectrometry, gas chromatography equipped with electron capture detectors, fluorescence and absorption spectroscopy. Thus, the disclosure also includes compounds identified as "hits" by the methods described herein, and methods for their administration and use in the treatment, prevention, or delay of development or progression of a disorder described herein.

[0111] Test compounds identified as candidate therapeutic compounds can be further screened by administration to an animal model of a disorder associated with PARP overexpression or overactivity (e.g., cancer). The animal can be monitored for a change in the disorder, e.g., for an improvement in a parameter of the disorder, e.g., a parameter related to clinical outcome. In some embodiments, the parameter is tumor growth, and an improvement would inhibit tumor growth.

[0112] In some embodiments, the PARP modulators obtained from the screening are inhibitors of PARP1, PARP2, PARP3, PARP4, PARP5a, PARP5b, PARP6, TIPARP, PARP8, PARP9, PARP10, PARP11, PARP12, PARP13, PARP14, PARP15, or PARP16. In some embodiments, the inhibitors have an IC.sub.50 for a PARP (e.g., PARP1, PARP2, PARP3, PARP4, PARP5a, PARP5b, PARP6, TIPARP, PARP8, PARP9, PARP10, PARP11, PARP12, PARP13, PARP14, PARP15, or PARP16) of less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nM.

[0113] In some embodiments, the binding affinity of the PARP inhibitors (K.sub.d) (between the compound and PARP) is less than 1.times.10.sup.-6 M, less than 1.times.10.sup.-7 M, less than 1.times.10.sup.-8 M, less than 1.times.10.sup.-9 M, or less than 1.times.10.sup.-10 M. In some embodiments, the K.sub.d is less than 50 nM, 30 nM, 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM. In some embodiments, K.sub.d is greater than 1.times.10.sup.-7 M, greater than 1.times.10.sup.-8 M, greater than 1.times.10.sup.-9 M, greater than 1.times.10.sup.-10 M, greater than 1.times.10.sup.-11 M, or greater than 1.times.10.sup.-12 M.

Methods of Treatment

[0114] The methods described herein include methods for the treatment of diseases or disorders associated with PARP overexpression or overactivity comprising administering to a patient in need thereof a therapeutically effective amount of a compound identified according to one or more of the assays described herein. In some embodiments, the disease or disorder is cancer. Generally, the methods include administering a therapeutically effective amount of PARP modulators (e.g., inhibitors) identified by the methods as described herein, to a subject who is in need of, or who has been determined to be in need of, such treatment. As used in this context, to "treat" means to ameliorate at least one symptom of the disorder associated with PARP overexpression or overactivity. Often, the treatment results in a decreased activity of PARP.

[0115] In some embodiments, the disease or disorder is cancer. In some embodiments, administration of a therapeutically effective amount of a compound described herein can result in a decrease of tumor size or tumor volume, a decrease of tumor growth, a reduction of the increase rate of tumor volume in a subject (e.g., as compared to the rate of increase in tumor volume in the same subject prior to treatment or in another subject without such treatment), a decrease in the risk of developing a metastasis or the risk of developing one or more additional metastasis, an increase of survival rate, and an increase of life expectancy, etc.

[0116] As used herein, the term "cancer" refers to cells having the capacity for autonomous growth, i.e., an abnormal state or condition characterized by rapidly proliferating cell growth. The term is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness. The term "tumor" as used herein refers to cancerous cells, e.g., a mass of cancerous cells.

[0117] As used herein, the terms "subject" and "patient" are used interchangeably throughout the specification and describe an animal, human or non-human, to whom treatment according to the methods of the present invention is provided. Veterinary and non-veterinary applications are contemplated by the present invention. Human patients can be adult humans or juvenile humans (e.g., humans below the age of 18 years old). In addition to humans, patients include but are not limited to mice, rats, hamsters, guinea-pigs, rabbits, ferrets, cats, dogs, and primates. Included are, for example, non-human primates (e.g., monkey, chimpanzee, gorilla, and the like), rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits), lagomorphs, swine (e.g., pig, miniature pig), equine, canine, feline, bovine, and other domestic, farm, and zoo animals.

Kits and Compositions

[0118] The present disclosure also provides kits for PARP inhibitor screening. The kit can include a PARP probe comprising a fluorophore or an affinity tag. The kit can also include a polypeptide comprising a PARP catalytic domain and optionally a fusion/affinity tag (e.g., hexahistidine). In some embodiments, the polypeptide is labeled with a fluorophore.

[0119] The present disclosure also provides compositions for the PARP inhibitors. In some embodiments, the compositions are formulated with a pharmaceutically acceptable carrier. The pharmaceutical compositions and formulations can be administered parenterally, topically, orally or by local administration, such as by aerosol or transdermally. The pharmaceutical compositions can be formulated in any way and can be administered in a variety of unit dosage forms depending upon the condition or disease and the degree of illness, the general medical condition of each patient, the resulting preferred method of administration and the like. Details on techniques for formulation and administration of pharmaceuticals are well described in the scientific and patent literature, see, e.g., Remington: The Science and Practice of Pharmacy, 21st ed., 2005.

EXAMPLES

[0120] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

Materials and Methods

[0121] The following equipment and methods were used in the following examples.

[0122] .sup.1H NMR Spectra were recorded at 300 or 400 MHz using a Bruker AVANCE 400 MHz spectrometer. NMR interpretation was performed using MestReC or MestReNova software to assign chemical shift and multiplicity. In cases where two adjacent peaks of equal or unequal height were observed, these two peaks may be labeled as either a multiplet or as a doublet. In the case of a doublet, a coupling constant using this software may be assigned. In any given example, one or more protons may not be observed due to obscurity by water and/or solvent peaks.

[0123] Liquid chromatography-mass spectrometry (LCMS) equipment and conditions were as follows:

[0124] 1. Liquid chromatography (LC): Agilent Technologies 1290 series, Binary Pump, Diode Array Detector. Agilent Poroshell 120 EC-C18, 2.7 .mu.m, 4.6.times.50 mm column. Mobile phase: A: 0.05% Formic acid in water (v/v), B: 0.05% Formic acid in ACN (v/v). Flow Rate: 1 mL/min at 25.degree. C. Detector: 214 nm, 254 nm. Gradient stop time, 10 min. Timetable is shown below:

TABLE-US-00002

[0124] TABLE 2 T (min) A (%) B (%) 0.0 90 10 0.5 90 10 8.0 10 90 10.0 0 100



[0125] 2. Mass spectrometry (MS): G6120A, Quadrupole LC/MS, Ion Source: ES-API, TIC: 70.about.1000 m/z, Fragmentor: 60, Drying gas flow: 10 L/min, Nebulizer pressure: 35 psi, Drying gas temperature: 350.degree. C., Vcap: 3000V.

[0126] 3. Sample preparation: samples were dissolved in ACN or methanol at 1.about.10 mg/mL, then filtered through a 0.22 .mu.m filter membrane. Injection volume: 1.about.10.

[0127] The following abbreviations are used in the disclosure: ACN (acetonitrile); Boc (tert-butoxycarbonyl); Boc2O (di-tert-butyl dicarbonate); CuI (copper iodide); CDCl.sub.3 (deuterated chloroform); CD.sub.3OD (deuterated methanol); DCM (dichloromethane); DIPEA (N,N-diisopropylethylamine); DMF (N,N-dimethylformamide); DMSO (dimethylsulfoxide); DMSO-d.sub.6 (deuterated dimethylsulfoxide); eq (equivalent); EtOAc (ethyl acetate); g (gram); h (hour); HATU (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate); .sup.1H NMR (proton nuclear magnetic resonance); HCl (hydrochloric acid); Hz (hertz); L (litre); LiCl (lithium chloride); LCMS (liquid chromatography-mass spectrometry); M (molar); MeOH (methanol); mg (milligrams); MHz (megahertz); min (minutes); mL (millilitres), mmol (millimoles); NMP (N-methyl-2-pyrrolidone); prep-HPLC (preparative high-performance liquid chromatography); Pd(OAc)2 (palladium (II) acetate); ppm (parts per million); Pd(allyl)Cl.sub.2 (Bis(.eta.3-allyl)di(.mu.-chloro)dipalladium(II)); Rockphos (2-Di(tert-butyl)phosphino-2,4,6-triisopropyl-3-methoxy-6-methylbiphenyl)- ; RT (room temperature); SEM (2-(trimethylsilyl)ethoxymethyl); SEMCl (2-(trimethylsilyl)ethoxymethyl chloride); TEA (triethyl amine); tBuBrettphos (2-(Di-tert-butylphosphino)-2,4,6-triisopropyl-3,6-dimethoxy-1,1-biphenyl- ); THF (tetrahydrofuran); TLC (thin layer chromatography); v/v (volume/volume).

Example 1: Synthesis of Intermediates

Int-A1: 5-Chloro-4-(trifluoromethyl)-2-[[2-(trimethylsilyl)ethoxy]methyl]-- 2,3-dihydropyridazin-3-one

##STR00004##

[0128] Step 1: 4,5-Dibromo-2-[[2-(trimethylsilyl)ethoxy]methyl]-2,3-dihydropyridazin-3-o- ne

[0129] To a solution of 4,5-dibromo-2,3-dihydropyridazin-3-one (3500 g, 13.78 mol, 1.00 equiv) in DMF (30 L) was added sodium hydride (400 g, 16.56 mol, 1.20 equiv) in batches at 0.degree. C. under nitrogen. The resulting solution was stirred for 1 hour at room temperature, then [2-(chloromethoxy)ethyl]trimethylsilane (2500 g, 15.2 mol, 1.10 equiv) was added dropwise at 0.degree. C. and stirred for 2 hours at room temperature. The reaction was then quenched by the addition of 30 L of water. The resulting solution was extracted with 3.times.50 L of ethyl acetate and the organic layers combined. The organic layers were washed with 3.times.30 L of brine, dried over anhydrous sodium sulfate and concentrated under reduced pressure to afford 4.2 kg of title compound. LCMS: [M+H].sup.+ 384.70.

Step 2: 4-Bromo-5-chloro-2-[[2-(trimethylsilyl)ethoxy]methyl]-2,3-dihydrop- yridazin-3-one

[0130] To a solution of 4,5-dibromo-2-[[2-(trimethylsilyl)ethoxy]methyl]-2,3-dihydropyridazin-3-o- ne (2200 g, 5.73 mol, 1.00 equiv) in NMP (6 L) was added chlorolithium (231 g, 5.73 mol, 1.00 equiv) and stirred for 4 hours at 95.degree. C. The reaction was then diluted by the addition of 10 L of water, extracted with 3.times.20 L of ethyl acetate and the organic layers combined. The organic layers were washed with 3.times.20 L of brine, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography (EtOAc:petroleum ether, 1:50, v/v) to afford 4.2 kg of 4,5-dibromo-2-[[2-(trimethylsilyl)ethoxy]methyl]-2,3-dihydropyridazin-3-o- ne. This was repeated 2 times resulting in 2.2 kg of 4-bromo-5-chloro-2-[[2-(trimethylsilyl)ethoxy]methyl]-2,3-dihydropyridazi- n-3-one. LCMS: [M+H].sup.+ 340.90.

Step 3: 5-Chloro-4-(trifluoromethyl)-2-[[2-(trimethylsilyl)ethoxy]methyl]-- 2,3-dihydropyridazin-3-one

[0131] To a solution of 4-bromo-5-chloro-2-[[2-(trimethylsilyl)ethoxy]methyl]-2,3-dihydropyridazi- n-3-one (1100 g, 3.23 mol, 1.00 equiv) in NMP (6 L) at room temperature was added CuI (56 g, 0.64 mol, 0.20 equiv) followed by dropwise addition of methyl 2,2-difluoro-2-(fluorosulfonyl)acetate (1865 g, 9.7 mol, 3.00 equiv). The resulting solution was stirred for 2 hours at 80.degree. C. The reaction was then quenched by the addition of 10 L of water and extracted with 3.times.10 L of ethyl acetate. The organic layers were combined and washed with 3.times.10 L of brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by column chromatography (ethyl acetate/petroleum ether, 1/100, v/v) to afford 1030 g (76%) of the title compound. LCMS: [M+H].sup.+ 329.00.

[0132] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.82 (s, 1H), 5.50 (d, J=27.3 Hz, 2H), 3.74 (dt, J=12.9, 8.2 Hz, 2H), 0.97 (td, J=8.3, 5.0 Hz, 2H), 0.01 (d, J=2.1 Hz, 9H).

Int-A2: 2-[4-([2-[6-Oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-4-yl]-2,3- -dihydro-1H-isoindol-5-yl]oxy)piperidin-1-yl]acetic acid

##STR00005##

[0133] Step 1: 5-(5-Hydroxy-2,3-dihydro-1H-isoindol-2-yl)-4-(trifluoromethyl)-2-[[2-(tri- methylsilyl)ethoxy]methyl]-2,3-dihydropyridazin-3-one

[0134] A solution of 5-chloro-4-(trifluoromethyl)-2-[[2-(trimethylsilyl)ethoxy]methyl]-2,3-dih- ydropyridazin-3-one (2.8 g, 8.52 mmol, 1.00 equiv), 2,3-dihydro-1H-isoindol-5-ol hydrobromide (4.27 g, 19.76 mmol, 1.00 equiv), and TEA (10 mL) in ethanol (40 mL) was stirred for 1 h at 60.degree. C. The resulting solution was extracted with 2.times.100 mL of ethyl acetate and the organic layers combined and concentrated under reduced pressure to afford 4.5 g of the title compound as a yellow oil. LCMS: [M+H].sup.+ 428.23.

Step 2: tert-Butyl 4-([2-[6-oxo-5-(trifluoromethyl)-1-[[2-(trimethylsilyl)ethoxy]methyl]-1,6- -dihydropyridazin-4-yl]-2,3-dihydro-1H-isoindol-5-yl]oxy)piperidine-1-carb- oxylate

[0135] A solution of 5-(5-hydroxy-2,3-dihydro-1H-isoindol-2-yl)-4-(trifluoromethyl)-2-[[2-(tri- methylsilyl)ethoxy]methyl]-2,3-dihydropyridazin-3-one (4.5 g, 10.53 mmol, 1.00 equiv), tert-butyl 4-iodopiperidine-1-carboxylate (20 g, 64.28 mmol, 8.00 equiv), potassium carbonate (15 g, 108.53 mmol, 10.00 equiv), and DMF (50 mL) was stirred for 2 days at 80.degree. C. The resulting solution was extracted with 2.times.200 mL of ethyl acetate and the organic layers combined and concentrated under reduced pressure. The residue was applied onto a silica gel column eluting with ethyl acetate/petroleum ether to afford the title compound (2 g, 31%) as a yellow oil. LCMS: [M+H].sup.+ 611.15.

Step 3: 5-[5-(Piperidin-4-yloxy)-2,3-dihydro-1H-isoindol-2-yl]-4-(trifluor- omethyl)-2-[[2-(trimethylsilyl)ethoxy]methyl]-2,3-dihydropyridazin-3-one

[0136] A solution of tert-butyl 4-([2-[6-oxo-5-(trifluoromethyl)-1-[[2-(trimethylsilyl)ethoxy]methyl]-1,6- -dihydropyridazin-4-yl]-2,3-dihydro-1H-isoindol-5-yl]oxy)piperidine-1-carb- oxylate (2 g, 3.27 mmol, 1.00 equiv), dioxane/HCl (5 mL), and dioxane (45 mL) was stirred for 6 h at 25.degree. C. The resulting mixture was concentrated under reduced pressure. The residue was applied onto a silica gel column and eluted with ethyl acetate/petroleum ether to afford 1 g of title compound as a yellow oil. LCMS: [M+H].sup.+ 511.28.

Step 4: tert-Butyl 2-[4-([2-[6-oxo-5-(trifluoromethyl)-1-[[2-(trimethylsilyl)ethoxy]methyl]-- 1,6-dihydropyridazin-4-yl]-2,3-dihydro-1H-isoindol-5-yl]oxy)piperidin-1-yl- ]acetate

[0137] A solution of 5-[5-(piperidin-4-yloxy)-2,3-dihydro-1H-isoindol-2-yl]-4-(trifluoromethyl- )-2-[[2-(trimethylsilyl)ethoxy]methyl]-2,3-dihydropyridazin-3-one (1 g, 1.96 mmol, 1.00 equiv), tert-butyl 2-chloroacetate (450 mg, 2.99 mmol, 3.00 equiv), DIPEA (5 mL), and dichloromethane (10 mL) was stirred overnight at 25.degree. C. The residue was purified by C18 reverse phase chromatography eluting with H.sub.2O/CH.sub.3CN to afford the title compound (540 mg, 44%) as a yellow oil. LCMS: [M+H].sup.+ 625.20.

Step 5: 2-[4-([2-[6-Oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-4-yl]-2,3- -dihydro-1H-isoindol-5-yl]oxy)piperidin-1-yl]acetic acid

[0138] A solution of tert-butyl 2-[4-([2-[6-oxo-5-(trifluoromethyl)-1-[[2-(trimethylsilyl)ethoxy]methyl]-- 1,6-dihydropyridazin-4-yl]-2,3-dihydro-1H-isoindol-5-yl]oxy)piperidin-1-yl- ]acetate (540 mg, 0.86 mmol, 1.00 equiv) and dioxane/HCl (8 mL) was stirred overnight at 25.degree. C. The resulting mixture was concentrated under reduced pressure. The residue was purified by C18 reverse phase chromatography eluting with H.sub.2O/CH.sub.3CN to afford 200 mg (53%) of title compound as a white solid. LCMS: [M+H].sup.+ 439.31.

Example 2: Synthesis of Probe A

##STR00006##

[0139] Step 1: tert-Butyl N-(6-[2-[4-([2-[6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-4-yl]-2,3-- dihydro-1H-isoindol-5-yl]oxy)piperidin-1-yl]acetamido]hexyl)carbamate

[0140] A solution of 2-[4-([2-[6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-4-yl]-2,3-dihydr- o-1H-isoindol-5-yl]oxy)piperidin-1-yl]acetic acid (Int-A2) (44 mg, 0.10 mmol, 1.00 equiv), DIPEA (52 mg, 0.40 mmol, 4.00 equiv), HATU (46 mg, 0.12 mmol, 1.20 equiv), and tert-butyl N-(6-aminohexyl)carbamate (24 mg, 0.11 mmol, 1.10 equiv) in DMF (1 mL) was stirred overnight at 25.degree. C. The crude product was purified by C18 reverse phase chromatography eluting with H.sub.2O/CH.sub.3CN to afford 38 mg (59%) of title compound as an off-white solid. LCMS: [M+H].sup.+ 637.31.

Step 2: N-(6-Aminohexyl)-2-[4-([2-[6-oxo-5-(trifluoromethyl)-1,6-dihydropy- ridazin-4-yl]-2,3-dihydro-1H-isoindol-5-yl]oxy)piperidin-1-yl]acetamide hydrochloride

[0141] A solution of tert-butyl N-(6-[2-[4-([2-[6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-4-yl]-2,3-- dihydro-1H-isoindol-5-yl]oxy)piperidin-1-yl]acetamido]hexyl)carbamate (38 mg, 0.06 mmol, 1.00 equiv) in hydrogen chloride/dioxane (10 mL) was stirred for 3 hours at 25.degree. C. The resulting mixture was concentrated under reduced pressure to afford the title compound as a gray solid (30 mg, 88%). LCMS: [M-Cl].sup.+: 537.27.

Step 3: 2,2-Difluoro-4-[2-[(6-[2-[4-([2-[6-oxo-5-(trifluoromethyl)-1,6-dih- ydropyridazin-4-yl]-2,3-dihydro-1H-isoindol-5-yl]oxy)piperidin-1-yl]acetam- ido]hexyl)carbamoyl]ethyl]-12-(1H-pyrrol-2-yl)-1{circumflex over ( )}[5],3-diaza-2{circumflex over ( )}[4]-boratricyclo[7.3.0.0{circumflex over ( )}[3,7]]dodeca-1(12),4,6,8,10-pentaen-1-yliumc

[0142] A solution of NanoBRET.RTM. 590SE (N-(6-aminohexyl)-2-[4-([2-[6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazi- n-4-yl]-2,3-dihydro-1H-isoindol-5-yl]oxy)piperidin-1-yl]acetamide hydrochloride (23 mg, 0.04 mmol, 2.00 equiv), DIPEA (52 mg, 0.40 mmol, 5.00 equiv), 4-3-[(2,5-dioxopyrrolidin-1-yl)oxy]-3-oxopropyl-2,2-difluoro-12-(1H-pyrro- l-2-yl)-1{circumflex over ( )}5,3-diaza-2{circumflex over ( )}4-boratricyclo[7.3.0.0{circumflex over ( )}3,7]dodeca-1(12),4,6,8,10-pentaen-1-ylium) (10 mg, 0.02 mmol, 1.00 equiv) in dichloromethane (2 mL) and methanol (2 mL) was stirred for 2 h at 25.degree. C. The resulting mixture was concentrated under reduced pressure and the crude product was purified by C18 reverse phase chromatography eluting with H.sub.2O/CH.sub.3CN to afford 6.9 mg of a purple solid (35%). LCMS: [M+H].sup.+ 848.38.

[0143] .sup.1H NMR (CD.sub.3OD, 400 MHz) .delta.: 7.98 (s, 1H), 7.28-7.14 (m, 5H), 7.02-6.84 (m, 4H), 6.37-6.26 (m, 2H), 4.93 (d, J=12.0 Hz, 4H), 4.45-4.35 (m, 1H), 3.29-3.13 (m, 6H), 3.01 (s, 2H), 2.81-2.70 (m, 2H), 2.60 (t, J=7.7 Hz, 2H), 2.43 (td, J=8.7, 4.6 Hz, 2H), 2.01 (dd, J=11.8, 7.0 Hz, 2H), 1.81 (ddt, J=15.8, 11.5, 5.5 Hz, 2H), 1.51 (q, J=7.3, 6.8 Hz, 4H), 1.38-1.26 (m, 4H).

Example 3: Synthesis of Probe B

##STR00007##

[0144] Step 1: tert-Butyl N-(6-[2-[4-([2-[6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-4-yl]-2,3-- dihydro-1H-isoindol-5-yl]oxy)piperidin-1-yl]acetamido]hexyl)carbamate

[0145] A solution of 2-[4-([2-[6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-4-yl]-2,3-dihydr- o-1H-isoindol-5-yl]oxy)piperidin-1-yl]acetic acid (Int-A2) (250 mg, 0.57 mmol, 1.00 equiv), tert-butyl N-(6-aminohexyl)carbamate (120 mg, 0.55 mmol, 1.00 equiv), HATU (220 mg, 0.58 mmol, 1.10 equiv), DIPEA (2 mL), and DMF (4 mL) was stirred for 0.5 h at 0.degree. C. The residue was purified by C18 reverse phase chromatography eluting with H.sub.2O/CH.sub.3CN to afford 190 mg (52%) of the title compound as a white solid. LCMS: [M+H].sup.+ 637.32.

Step 2: N-(6-Aminohexyl)-2-[4-([2-[6-oxo-5-(trifluoromethyl)-1,6-dihydropy- ridazin-4-yl]-2,3-dihydro-1H-isoindol-5-yl]oxy)piperidin-1-yl]acetamide hydrochloride

[0146] A solution of tert-butyl N-(6-[2-[4-([2-[6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-4-yl]-2,3-- dihydro-1H-isoindol-5-yl]oxy)piperidin-1-yl]acetamido]hexyl)carbamate (190 mg, 0.30 mmol, 1.00 equiv) and dioxane/HCl (6 mL) was stirred for 1 hour at 25.degree. C. The resulting mixture was concentrated under reduced pressure. The residue was purified by C18 reverse phase chromatography eluting with H.sub.2O/CH.sub.3CN to afford 100 mg (58%) of title compound as a yellow oil. LCMS: [M+H].sup.+ 537.27.

Step 3: 17-[2-Carboxylato-5-[(6-[2-[4-([2-[6-oxo-5-(trifluoromethyl)-1,6-d- ihydropyridazin-4-yl]-2,3-dihydro-1H-isoindol-5-yl]oxy)piperidin-1-yl]acet- amido]hexyl)carbamoyl]phenyl]-3-oxa-9{circumflex over ( )}[5],25-diazaheptacyclo[18.8.1.{circumflex over ( )}[5,9].0{circumflex over ( )}[2,18].0{circumflex over ( )}[4,16].0{circumflex over ( )}[25,29].0{circumflex over ( )}[14,30]]triaconta-1(29),2(18),4,9(30),14,16,19-heptaen-9-ylium

[0147] A solution of N-(6-aminohexyl)-2-[4-([2-[6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin- -4-yl]-2,3-dihydro-1H-isoindol-5-yl]oxy)piperidin-1-yl]acetamide hydrochloride (8 mg, 0.01 mmol, 1.00 equiv), 17-[2-carboxylato-5-(2,3,5,6-tetrafluorophenoxycarbonyl)phenyl]-3-oxa-9{c- ircumflex over ( )}5,25-diazaheptacyclo[18.8.1.1{circumflex over ( )}5,9.0{circumflex over ( )}2,18.0{circumflex over ( )}4,16.0{circumflex over ( )}25,29.0{circumflex over ( )}14,30]triaconta-1(29),2(18),4,9(30),14,16,19-heptaen-9-ylium (NanoBRET.RTM. 618TFP Ester) (10 mg, 0.01 mmol, 1.00 equiv), DIPEA (0.8 mL), and DMF (6 mL) was stirred for 1 h at 25.degree. C. The resulting mixture was concentrated under reduced pressure. The crude product was purified by Prep-HPLC (XBridge Prep C18 OBD column, 5 .mu.m, 19.times.150 mm column, eluting with water:acetonitrile (50:50, v:v) with 0.1% NH.sub.4HCO.sub.3, at a flow rate of 1.2 mL/min) to afford the title compound as a blue solid (0.8 mg, 5%). LCMS: [M+H].sup.+ 1081.45.

[0148] .sup.1H NMR (CD.sub.3OD, 400 MHz) .delta.: 8.09-8.02 (m, 3H), 7.68 (d, J=1.6 Hz, 1H), 7.22 (d, J=8.4 Hz, 1H), 6.93-6.83 (m, 4H), 4.91 (s, 4H), 4.55-4.51 (m, 1H), 3.72-3.69 (m, 3H), 3.49-3.54 (m, 3H), 3.35-3.42 (m, 2H), 3.27-3.23 (m, 2H), 3.15-3.01 (m, 5H), 2.84-2.81 (m, 5H), 2.45-2.41 (m, 2H), 2.08-1.92 (m, 15H), 1.84-1.80 (m, 4H), 1.66-1.37 (m, 7H), 0.92-0.85 (m, 2H).

Example 4: Synthesis of Probe C

##STR00008##

[0149] Step 1: Tert-butyl N-(6-[5-[(3aS,4S,6aR)-2-oxo-hexahydro-1H-thieno[3,4-d]imidazolidin-4-yl]p- entanamido]hexyl)carbamate

[0150] A solution of 5-[(3aS,4S,6aR)-2-oxo-hexahydro-1H-thieno[3,4-d]imidazolidin-4-yl]pentano- ic acid ((reagent was purchased from Beijing Dragon Rui Trading Company, 976 mg, 3.99 mmol, 1.00 equiv), DIPEA (1.55 g, 11.99 mmol, 3.00 equiv), HATU (1.82 g, 4.79 mmol, 1.20 equiv), and tert-butyl N-(6-aminohexyl)carbamate (864 mg, 3.99 mmol, 1.00 equiv) in DMF (15 mL) was stirred overnight at 25.degree. C. The reaction was then quenched by the addition of 50 mL of water. The solids were collected by filtration to afford 1.5 g (85%) of the title compound as a white solid. LCMS: [M+H].sup.+ 443.26.

Step 2: 5-[(3aS,4S,6aR)-2-Oxo-hexahydro-1H-thieno[3,4-d]imidazolidin-4-yl]- -N-(6-aminohexyl)pentanamide hydrochloride

[0151] A solution of tert-butyl N-(6-[5-[(3aS,4S,6aR)-2-oxo-hexahydro-1H-thieno[3,4-d]imidazolidin-4-yl]p- entanamido]hexyl)carbamate (800 mg, 1.81 mmol, 1.00 equiv) in hydrogen chloride/dioxane (20 mL) was stirred overnight at 25.degree. C. The resulting mixture was concentrated under reduced pressure to afford 600 mg (88%) of the title compound as a gray crude oil. LCMS: [M+H].sup.+ 343.21.

Step 3: 5-[(3aS,4S,6aR)-2-Oxo-hexahydro-1H-thieno[3,4-d]imidazolidin-4-yl]- -N-(6-[2-[4-([2-[6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-4-yl]-2,3-- dihydro-1H-isoindol-5-yl]oxy)piperidin-1-yl]acetamido]hexyl)pentanamide

[0152] A solution of 2-[4-([2-[6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-4-yl]-2,3-dihydr- o-1H-isoindol-5-yl]oxy)piperidin-1-yl]acetic acid (175 mg, 0.40 mmol, 1.00 equiv), DIPEA (258 mg, 2.00 mmol, 5.00 equiv), HATU (228 mg, 0.60 mmol, 1.50 equiv), 5-[(3aS,4S,6aR)-2-oxo-hexahydro-1H-thieno[3,4-d]imidazolidin-4-yl]-N-(6-a- minohexyl)pentanamide hydrochloride (228 mg, 0.60 mmol, 1.50 equiv) in DMF (3 mL) was stirred for 4 h at 25.degree. C. The crude product was purified by C18 reverse phase chromatography eluting with H.sub.2O/CH.sub.3CN to afford the title compound as a white solid (118.3 mg, 39%). LCMS: [M+H].sup.+ 763.35.

[0153] .sup.1H NMR (DMSO-d.sub.6, 400 MHz) .delta.:12.52 (s, 1H), 7.98 (s, 1H), 7.81-7.68 (m, 2H), 7.26 (d, J=8.4 Hz, 1H), 7.00 (d, J=2.2 Hz, 1H), 6.91 (dd, J=8.4, 2.3 Hz, 1H), 6.45-6.39 (m, 1H), 6.36 (s, 1H), 4.91 (d, J=6.1 Hz, 4H), 4.45 (m, 1H), 4.26 (m, 1H), 4.17-4.08 (m, 1H), 3.14-2.96 (m, 5H), 2.91 (s, 2H), 2.82 (dd, J=12.4, 5.1 Hz, 1H), 2.73-2.63 (m, 2H), 2.58 (d, J=12.4 Hz, 1H), 2.33 (ddd, J=11.8, 9.4, 3.1 Hz, 2H), 2.11-1.90 (m, 4H), 1.76-1.54 (m, 3H), 1.57-1.20 (m, 13H).

Example 5: Synthesis of Probe D

##STR00009##

[0154] Step 1: Tert-butyl 2-(4-hydroxypiperidin-1-yl)acetate

[0155] A solution of piperidin-4-ol (10.1 g, 99.85 mmol, 1.00 equiv), DIPEA (14.2 g, 109.87 mmol, 1.10 equiv), tert-butyl 2-chloroacetate (14.5 g, 96.28 mmol, 1.00 equiv) in THF (500 mL) was stirred overnight at 25.degree. C. The solids were filtered and concentration under reduced pressure afforded the crude residue which was purified by silica gel chromatography eluting with EtOAC/petroleum ether (1/1) to afford 10.2 g (47%) of the title compound as a white solid. LCMS: [M+H].sup.+ 216.15.

Step 2: Tert-butyl 2-[4-(methanesulfonyloxy)piperidin-1-yl]acetate

[0156] A solution of tert-butyl 2-(4-hydroxypiperidin-1-yl)acetate (10.2 g, 47.38 mmol, 1.00 equiv), TEA (9.53 g, 94.18 mmol, 2.00 equiv), Ms.sub.2O (9.86 g, 1.20 equiv) in DCM (200 mL) was stirred for 3 hours at 25.degree. C. The reaction was then quenched by the addition of 300 mL of water. The resulting solution was extracted with 200 mL of dichloromethane and the organic layers were combined. The resulting mixture was concentrated under reduced pressure to afford 11 g (79%) of title compound as an off-white solid. LCMS: [M+H].sup.+ 294.13.

Step 3: Tert-butyl 2-[4-([2-[6-oxo-5-(trifluoromethyl)-1-[[2-(trimethylsilyl)ethoxy]methyl]-- 1,6-dihydropyridazin-4-yl]-2,3-dihydro-1H-isoindol-5-yl]oxy)piperidin-1-yl- ]acetate

[0157] A solution of 5-(5-hydroxy-2,3-dihydro-1H-isoindol-2-yl)-4-(trifluoromethyl)-2-[[2-(tri- methylsilyl)ethoxy]methyl]-2,3-dihydropyridazin-3-one (4.27 g, 9.99 mmol, 1.00 equiv), potassium carbonate (6.9 g, 49.92 mmol, 5.00 equiv), tert-butyl 2-[4-(methanesulfonyloxy)piperidin-1-yl]acetate (5.86 g, 19.97 mmol, 2.00 equiv) in DMF (50 mL) was stirred for 2 days at 80.degree. C. in an oil bath. The reaction was then quenched by the addition of 100 mL of water. The resulting solution was extracted with 4.times.100 mL of EtOAc and the organic layers were combined. After concentration, the residue was purified by C18 reverse phase chromatography eluting with H.sub.2O/CH.sub.3CN to afford 1.5 g (24%) of the title compound as a light yellow solid. LCMS: [M+H].sup.+ 625.30.

Step 4: 2-[4-([2-[6-Oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-4-yl]-2,3- -dihydro-1H-isoindol-5-yl]oxy)piperidin-1-yl]acetic acid

[0158] A solution of tert-butyl 2-[4-([2-[6-oxo-5-(trifluoromethyl)-1-[[2-(trimethylsilyl)ethoxy]methyl]-- 1,6-dihydropyridazin-4-yl]-2,3-dihydro-1H-isoindol-5-yl]oxy)piperidin-1-yl- ]acetate (1.5 g, 2.40 mmol, 1.00 equiv) in hydrogen chloride/dioxane (40 mL) was stirred overnight at 25.degree. C. The resulting mixture was concentrated under vacuum. The crude product was purified by C18 reverse phase chromatography eluting with H.sub.2O/CH.sub.3CN to afford 600 mg (57%) of the title compound as a gray solid. LCMS: [M+H].sup.+ 439.15.

Step 5: Tert-butyl N-(6-[3',6'-dihydroxy-3-oxo-3H-spiro[2-benzofuran-1,9'-xanthene]-6-ylform- amido]hexyl)carbamate

[0159] A solution of 3',6'-dihydroxy-3-oxo-3H-spiro[2-benzofuran-1,9'-xanthene]-6-carboxylic acid (752 mg, 2.00 mmol, 1.00 equiv), DIPEA (774 mg, 5.99 mmol, 3.00 equiv), HATU (912 mg, 2.40 mmol, 1.20 equiv), tert-butyl N-(6-aminohexyl)carbamate (475 mg, 2.20 mmol, 1.00 equiv) in DMF (10 mL) was stirred for 3 h at 25.degree. C. The reaction was then quenched by the addition of 50 mL of water. The solids were filtered and the crude product was purified by C18 reverse phase chromatography eluting with H.sub.2O/CH.sub.3CN to afford 400 mg (35%) of the title compound as a yellow solid. LCMS: [M+H].sup.+ 575.23.

Step 6: N-(6-Aminohexyl)-3',6'-dihydroxy-3-oxo-3H-spiro[2-benzofuran-1,9'-- xanthene]-6-carboxamide

[0160] A solution of tert-butyl N-(6-[3',6'-dihydroxy-3-oxo-3H-spiro[2-benzofuran-1,9'-xanthene]-6-ylform- amido]hexyl)carbamate (400 mg, 0.70 mmol, 1.00 equiv) in HCl/dioxane (20 mL) was stirred overnight at 25.degree. C. The resulting mixture was concentrated under reduced pressure and the crude product was purified by C18 reverse phase chromatography eluting with H.sub.2O/CH.sub.3CN to afford 200 mg of the title compound (61%) as a yellow solid. LCMS: [M+H].sup.+ 475.18.

Step 7. N-(6-[3-Dihydroxy-3-oxo-3H-spiro[2-benzofuran-1,9'-xanthene]]-6-yl- formamido]hexyl)-2-[4-([2-[6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-- 4-yl]-2,3-dihyro-H-isoindol-5-yl]oxy)piperidin-1-yl]acetamide

[0161] A solution of 2-[4-([2-[6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-4-yl]-2,3-dihydr- o-1H-isoindol-5-yl]oxy)piperidin-1-yl]acetic acid (Int-A2) (150 mg, 0.34 mmol, 1.00 equiv), DIPEA (176 mg, 1.36 mmol, 4.00 equiv), HATU (183 mg, 0.48 mmol, 1.40 equiv), N-(6-aminohexyl)-3-dihydroxy-3-oxo-3H-spiro[2-benzofuran-1,9'-xanthene]-6- -carboxamide (228 mg, 0.48 mmol, 1.40 equiv) in DMF (5 mL) was stirred for 3 h at 25.degree. C. After concentration, the residue was purified by C18 reverse phase chromatography eluting with H.sub.2O/CH.sub.3CN to afford the title compound as an orange solid (26.9 mg, 9%). LCMS: [M+H].sup.+ 895.32.

[0162] .sup.1H NMR (DMSO-d.sub.6, 400 MHz) .delta.: 8.61 (t, J=5.6 Hz, 1H), 8.14-8.02 (m, 2H), 7.96 (s, 1H), 7.72-7.61 (m, 2H), 7.22 (d, J=8.4 Hz, 1H), 6.96 (d, J=2.2 Hz, 1H), 6.86 (dd, J=8.4, 2.2 Hz, 1H), 6.58 (d, J=8.8 Hz, 4H), 6.49 (s, 2H), 4.88 (d, J=5.4 Hz, 4H), 4.34 (dp, J=8.2, 3.7 Hz, 1H), 3.17 (q, J=6.2 Hz, 2H), 3.03 (q, J=6.6 Hz, 2H), 2.86 (s, 2H), 2.70-2.58 (m, 2H), 2.33-2.22 (m, 2H), 1.91 (d, J=13.5 Hz, 2H), 1.65 (dtd, J=12.6, 8.8, 3.2 Hz, 2H), 1.39 (dq, J=27.4, 6.8, 6.2 Hz, 4H), 1.22 (d, J=6.8 Hz, 4H).

Example 6: Synthesis of Compound A

5-[5-(Piperidin-4-yloxy)-2,3-dihydro-1H-isoindol-2-yl]-4-(trifluoromethyl)- -2,3-dihydropyridazin-3-one

##STR00010##

[0163] Step 1: 5-(5-Hydroxy-2,3-dihydro-1H-isoindol-2-yl)-4-(trifluoromethyl)-2-[[2-(tri- methylsilyl)ethoxy]methyl]-2,3-dihydropyridazin-3-one

[0164] A solution of Int-A1 (2.8 g, 8.52 mmol, 1.00 equiv), 2,3-dihydro-1H-isoindol-5-ol hydrobromide (4.27 g, 19.76 mmol, 1.00 equiv), and TEA (10 mL) in ethanol (40 mL) was stirred for 1 h at 60.degree. C. The resulting solution was extracted with 2.times.100 mL of EtOAc and the organic layers combined and concentrated under reduced pressure to afford 4.5 g of the title compound as a yellow oil. LCMS: [M+H].sup.+ 428.23.

Step 2: tert-Butyl 4-([2-[6-oxo-5-(trifluoromethyl)-1-[[2-(trimethylsilyl)ethoxy]methyl]-1,6- -dihydropyridazin-4-yl]-2,3-dihydro-1H-isoindol-5-yl]oxy)piperidine-1-carb- oxylate

[0165] A solution of 5-(5-hydroxy-2,3-dihydro-1H-isoindol-2-yl)-4-(trifluoromethyl)-2-[[2-(tri- methylsilyl)ethoxy]methyl]-2,3-dihydropyridazin-3-one (4.5 g, 10.53 mmol, 1.00 equiv), tert-butyl 4-iodopiperidine-1-carboxylate (20 g, 64.28 mmol, 8.00 equiv), potassium carbonate (15 g, 108.53 mmol, 10.00 equiv), and DMF (50 mL) was stirred for 2 days at 80.degree. C. The resulting solution was extracted with 2.times.200 mL of EtOAc and the organic layers combined and concentrated under reduced pressure. The residue was applied onto a silica gel column eluting with EtOAc/petroleum ether to afford the title compound (2 g, 31%) as a yellow oil. LCMS: [M+H].sup.+ 611.15.

Step 3: 5-[5-(Piperidin-4-yloxy)-2,3-dihydro-1H-isoindol-2-yl]-4-(trifluor- omethyl)-2,3-dihydropyridazin-3-one

[0166] A solution of tert-butyl 4-([2-[6-oxo-5-(trifluoromethyl)-1-[[2-(trimethylsilyl)ethoxy]methyl]-1,6- -dihydropyridazin-4-yl]-2,3-dihydro-1H-isoindol-5-yl]oxy)piperidine-1-carb- oxylate (150 mg, 0.25 mmol, 1.00 equiv) in HCl/dioxane (5 mL) was stirred overnight at 45.degree. C. The resulting mixture was concentrated under reduced pressure and the crude product was purified by C18 reverse phase chromatography eluting with H.sub.2O/ACN to afford the title compound as a white solid LCMS: [M+H].sup.+ 381.28. .sup.1H NMR (400 MHz, Methanol-d.sub.4) .delta. 8.05 (s, 1H), 7.27 (d, J=8.4 Hz, 1H), 7.02-6.91 (m, 2H), 5.00 (d, J=10.6 Hz, 4H), 4.61-4.48 (m, 1H), 3.21-3.10 (m, 2H), 2.89-2.78 (m, 2H), 2.11-2.08 (m, 2H), 1.82-1.69 (m, 2H).

Example 7: Synthesis of Compound B

6-[4-[(3-[[(1S)-2-[6-Oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-4-yl]-2,- 3-dihydro-1H-isoindol-1-yl]methoxy]phenyl)carbonyl]piperazin-1-yl]pyridine- -3-carbonitrile

##STR00011##

[0167] Step 1: 5-[1-(Hydroxymethyl)-2,3-dihydro-1H-isoindol-2-yl]-4-(trifluoromethyl)-2-- [[2-(trimethylsilyl)ethoxy]methyl]-2,3-dihydropyridazin-3-one

[0168] A solution of 5-chloro-4-(trifluoromethyl)-2-[2-(trimethylsilyl)ethoxy]methyl-2,3-dihyd- ropyridazin-3-one (4.8 g, 14.60 mmol, 1.00 equiv), 2,3-dihydro-1H-isoindol-1-ylmethanol hydrochloride (2.7 g, 14.54 mmol, 1.00 equiv) and TEA (4.4 g, 43.48 mmol, 2.99 equiv) in ethanol (100 mL) was stirred for 1 h at 60.degree. C., and then the resulting solution was concentrated under vacuum and the residue was applied onto a silica gel column eluting with EtOAc/petroleum ether (45:55) to afford 2.9 g (45%) of the title compound as a brown solid. LCMS: [M+H].sup.+ 442.17.

Step 2: Methyl 3-([2-[6-oxo-5-(trifluoromethyl)-1-[[2-(trimethylsilyl)ethoxy]methyl]-1,6- -dihydropyridazin-4-yl]-2,3-dihydro-H-isoindol-1-yl]methoxy)benzoate

[0169] Under nitrogen, a solution of 5-[1-(hydroxymethyl)-2,3-dihydro-1H-isoindol-2-yl]-4-(trifluoromethyl)-2-- [[2-(trimethylsilyl)ethoxy]methyl]-2,3-dihydropyridazin-3-one (2.93 g, 6.64 mmol, 1.00 equiv), methyl 3-bromobenzoate (2.84 g, 13.21 mmol, 1.99 equiv), Pd(allyl)C12 (243 mg), Rockphos (311 mg) and Cs.sub.2CO.sub.3 (4.3 g, 13.20 mmol, 1.99 equiv) in Toluene (100 mL) was stirred for 18 h at 80.degree. C. The resulting solution was concentrated under vacuum and then the residue was applied onto a silica gel column eluting with EtOAc/petroleum ether (1:3) to afford 3 g (79%) of the title compound as a brown solid. LCMS: [M+H].sup.+ 576.21.

Step 3: 3-([2-[6-Oxo-5-(trifluoromethyl)-1-[[2-(trimethylsilyl)ethoxy]meth- yl]-1,6-dihydropyridazin-4-yl]-2,3-dihydro-1H-isoindol-1-yl]methoxy)benzoi- c acid

[0170] A solution of methyl 3-([2-[6-oxo-5-(trifluoromethyl)-1-[[2-(trimethylsilyl)ethoxy]methyl]-1,6- -dihydropyridazin-4-yl]-2,3-dihydro-1H-isoindol-1-yl]methoxy)benzoate (1.15 g, 2.00 mmol, 1.00 equiv) and LiOH (240 mg, 10.02 mmol, 5.02 equiv) in THF (12 mL) and water (3 mL) was stirred for 3 h at 60.degree. C. The resulting solution was concentrated under vacuum and the residue was diluted with 10 mL of H.sub.2O, and then the pH value of the solution was adjusted to 5 with HCl (36.5%). The solid was collected by filtration to afford 1.1 g (98%) of the title compound as a light yellow solid. LCMS: [M+H].sup.+ 562.19.

Step 4: 3-([2-[6-Oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-4-yl]-2,3-di- hydro-1H-isoindol-1-yl]methoxy)benzoic acid

[0171] A solution of 3-([2-[6-oxo-5-(trifluoromethyl)-1-[[2-(trimethylsilyl)ethoxy]methyl]-1,6- -dihydropyridazin-4-yl]-2,3-dihydro-1H-isoindol-1-yl]methoxy)benzoic acid (1.1 g, 1.96 mmol, 1.00 equiv) in HCl/dioxane (20 mL, 4M) was stirred for 3 h at RT, and then the resulting solution was concentrated under vacuum to afford 1 g of the title compound as a crude brown solid. LCMS: [M+H].sup.+ 432.11.

Step 5: 6-[4-[(3-[[(1R)-2-[6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-- 4-yl]-2,3-dihydro-1H-isoindol-1-yl]methoxy]phenyl)carbonyl]piperazin-1-yl]- pyridine-3-carbonitrile and 6-[4-[(3-[[(S)-2-[6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-4-yl]-2,- 3-dihydro-1H-isoindol-1-yl]methoxy]phenyl)carbonyl]piperazin-1-yl]pyridine- -3-carbonitrile

[0172] A solution of 3-([2-[6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-4-yl]-2,3-dihydro-1- H-isoindol-1-yl]methoxy)benzoic acid (500 mg, 1.16 mmol, 1.00 equiv), HATU (528 mg, 1.39 mmol, 1.20 equiv), DIPEA (449 mg, 3.47 mmol, 3.00 equiv) and Int-A4 (240 mg, 1.27 mmol, 1.1 equiv) in DMF (5 mL) was stirred for 2 h at RT. After concentration by reduced pressure, the resulting solution was purified by C18 reverse phase chromatography eluting with H.sub.2O/ACN. The residue was further purified by Prep-HPLC and Chiral-Prep-HPLC (CHIRAL Repaired IA, 5 .mu.m, 0.46.times.10 cm column, eluting with a gradient of (Hexanes:DCM=3:1)(0.1% DEA):EtOH=50:50, at a flow rate of 1 mL/min) yielding the title compound as a white solid. The absolute stereochemistry was assigned based on an X-ray crystal structure which confirmed (S)-absolute stereochemistry.

[0173] LCMS: [M+H].sup.+ 602.05, .sup.1H NMR (300 MHz, Methanol-d.sub.4) .delta. 8.43 (d, J=1.8 Hz, 1H), 8.42 (s, 1H), 7.79 (dd, J=9.0, 2.4 Hz, 1H), 7.53-7.50 (m, 1H), 7.41-7.35 (m, 4H), 7.05-6.99 (m, 2H), 6.94-6.87 (m, 2H), 6.20 (s, 1H), 5.33 (d, J=14.8 Hz, 1H), 4.68 (d, J=14.7 Hz, 1H), 4.53 (dd, J=10.2, 3.3 Hz, 1H), 4.29 (dd, J=10.2, 6.6 Hz, 1H), 3.91-3.44 (m, 8H). tR=5.955 min.

Example 8: Synthesis of Compound C

5-[2-[(1-Acetylpiperidin-4-yl)oxy]-5H,6H,7H-pyrrolo[3,4-b]pyridin-6-yl]-4-- (trifluoromethyl)-2,3-dihydropyridazin-3-one

##STR00012##

[0174] Step 1: 5-[2-Chloro-5H,6H,7H-pyrrolo[3,4-b]pyridin-6-yl]-4-(trifluoromethyl)-2-[[- 2-(trimethylsilyl)ethoxy]methyl]-2,3-dihydropyridazin-3-one

[0175] A solution of 2-chloro-5H,6H,7H-pyrrolo[3,4-b]pyridin-6-yl hydrochloride (5 g, 26.31 mmol, 1.00 equiv), TEA (8 g, 79.06 mmol, 3.00 equiv), and Int-A1 (14.3 g, 43.49 mmol, 1.00 equiv) in EtOH (30 mL) was stirred for 2 h at 80.degree. C. After concentration under reduced pressure, the residue was applied onto a silica gel column with EtOAc/petroleum ether (1:4) to afford 9.3 g (79%) of title compound as a yellow oil. LCMS: [M+H].sup.+ 447.15.

Step 2: 5-[2-Hydroxy-5H,6H,7H-pyrrolo[3,4-b]pyridin-6-yl]-4-(trifluorometh- yl)-2-[[2-(trimethylsilyl)ethoxy]methyl]-2,3-dihydropyridazin-3-one

[0176] A solution of 5-[2-chloro-5H,6H,7H-pyrrolo[3,4-b]pyridin-6-yl]-4-(trifluoromethyl)-2-[[- 2-(trimethylsilyl)ethoxy]methyl]-2,3-dihydropyridazin-3-one (300 mg, 0.67 mmol, 1.00 equiv), tBuBrettphos (49 mg, 0.15 equiv), K.sub.3PO.sub.4 (427 mg, 2.01 mmol, 3.00 equiv), and Pd(OAc).sub.2 (15 mg, 0.07 mmol, 0.10 equiv) in dioxane (5 mL) and water (0.5 mL) was stirred for 2 h at 80.degree. C. in an oil bath under N.sub.2 atmosphere. After concentration, the residue was applied onto a silica gel column with DCM/methanol (85:15) to afford 200 mg (70%) of title compound as a yellow oil. LCMS: [M+H].sup.+ 429.15

Step 3: Tert-butyl 4-([6-[6-oxo-5-(trifluoromethyl)-1-[[2-(trimethylsilyl)ethoxy]methyl]-1,6- -dihydropyridazin-4-yl]-5H,6H,7H-pyrrolo[3,4-b]pyridin-2-yl]oxy)piperidine- -1-carboxylate

[0177] A solution of 5-[2-hydroxy-5H,6H,7H-pyrrolo[3,4-b]pyridin-6-yl]-4-(trifluoromethyl)-2-[- [2-(trimethylsilyl)ethoxy]methyl]-2,3-dihydropyridazin-3-one (200 mg, 0.47 mmol, 1.00 equiv), Ag.sub.2CO.sub.3 (247 mg, 2.00 equiv), and tert-butyl 4-iodopiperidine-1-carboxylate (416 mg, 1.34 mmol, 3.00 equiv) in DMF (15 mL) was stirred for 4 h at 80.degree. C. The resulting solution was extracted with 3.times.10 mL of EtOAc and the organic layers combined. After concentration, the residue was applied onto a silica gel column with EtOAc/petroleum ether (1:9) to afford 150 mg (53%) of title compound as a yellow oil. LCMS: [M+H].sup.+ 612.30.

Step 4: 5-[2-(Piperidin-4-yloxy)-5H,6H,7H-pyrrolo[3,4-b]pyridin-6-yl]-4-(t- rifluoromethyl)-2,3-dihydropyridazin-3-one

[0178] A solution of tert-butyl 4-([6-[6-oxo-5-(trifluoromethyl)-1-[[2-(trimethylsilyl)ethoxy]methyl]-1,6- -dihydropyridazin-4-yl]-5H,6H,7H-pyrrolo[3,4-b]pyridin-2-yl]oxy)piperidine- -1-carboxylate (150 mg, 0.25 mmol, 1.00 equiv) in HCl/dioxane (15 mL, 4M) was stirred overnight at 25.degree. C. The pH value of the solution was adjusted to 8 with ammonia (100%). The crude product was purified by Prep-HPLC to afford 64.8 mg (69%) of title compound as a white solid. LCMS: [M+H].sup.+ 382.15 [M+H].

Step 5: 5-[2-[(1-Acetylpiperidin-4-yl)oxy]-5H,6H,7H-pyrrolo[3,4-b]pyridin-- 6-yl]-4-(trifluoromethyl)-2,3-dihydropyridazin-3-one

[0179] A solution of 5-[2-(piperidin-4-yloxy)-5H,6H,7H-pyrrolo[3,4-b]pyridin-6-yl]-4-(trifluor- omethyl)-2,3-dihydropyridazin-3-one (300 mg, 0.79 mmol, 1.00 equiv), TEA (239 mg, 2.36 mmol, 3.00 equiv), and EtOAc (160 mg, 1.57 mmol, 2.00 equiv) in DCM (15 mL) was stirred for 1 h at 25.degree. C. The resulting solution was quenched by 20 mL of water and extracted with 3.times.15 mL of DCM and the organic layers combined. After concentration, the crude product was purified by Flash-Prep-HPLC to afford 70.2 mg (21%) of title compound as a white solid. LCMS: [M+H].sup.+ 424.15. .sup.1H NMR (400 MHz, Methanol-d.sub.4) .delta. 8.07 (s, 1H), 7.69 (d, J=8.4 Hz, 1H), 6.76 (d, J=8.4 Hz, 1H), 5.39-5.28 (m, 1H), 5.03 (s, 2H), 4.92 (s, 2H), 3.97-3.89 (m, 1H), 3.86-3.75 (m, 1H), 3.57-3.44 (m, 2H), 2.15 (s, 3H), 2.14-1.97 (m, 2H), 1.89-1.70 (m, 2H).

Example 9: In Vitro Assays

Recombinant PARP Enzymes

[0180] A portion of TIPARP (residues 456 to 657 of NP_056323.2 (SEQ ID NO: 1), GenBank Accession No. NM_015508.4) was overexpressed in E. coli cells. An N-terminal fusion tag, MHHHHHHSSGVDLGTENLYFQSNAGLNDIFEAQKIEWHE (SEQ ID NO: 7), was used to purify the protein from cell lysates. The fusion tag was left on the protein for use in the probe displacement assay.

[0181] A portion of PARP10 (residues 808 to 1025 of NP_116178.2 (SEQ ID NO: 2), GenBank Accession No. NM_032789.4) was overexpressed in E. coli cells. An N-terminal fusion tag, MAHHHHHHENLYFQSM (SEQ ID NO: 8), was used to purify the protein from cell lysates. The fusion tag was left on the protein for use in the probe displacement assay.

[0182] A portion of PARP12 (residues 489 to 684 of NP_073587.1 (SEQ ID NO: 3), GenBank Accession No. NM_022750.3) was overexpressed in Sf9 cells. An N-terminal fusion tag, MAHHHHHHENLYFQSM (SEQ ID NO: 8), was used to purify the protein from cell lysates. The fusion tag was left on the protein for use in the probe displacement assay.

[0183] A portion of PARP14 (residues 1611 to 1801 of NP_060024.2 (SEQ ID NO: 4), GenBank Accession No. NM_017554) was overexpressed in E. coli cells. An N-terminal fusion tag, MHHHHHHSSGVDLGTENLYFQSNA (SEQ ID NO: 9), was used to purify the protein from cell lysates. The fusion tag was left on the protein for use in the probe displacement assay.

[0184] A portion of PARP15 (residues 481 to 678 of NP_689828.1 (SEQ ID NO: 5), GenBank Accession No. NM_152615) was overexpressed in f9 cells. An N-terminal fusion tag, MAHHHHHHSSGVDLGTENLYFQSM (SEQ ID NO: 10), was used to purify the protein from cell lysates. The fusion tag was left on the protein for use in the probe displacement assay.

[0185] A portion of PARP16 (residues 5 to 279 of NP_060321.3 (SEQ ID NO: 6), GenBank Accession No. NM_017851) was overexpressed in E. coli cells. An N-terminal fusion tag, MHHHHHHSSGVDLGTENLYFQSNA (SEQ ID NO: 9), was used to purify the protein from cell lysates. The fusion tag was left on the protein for use in the probe displacement assay.

In Vitro Probe Displacement Assay for Assessing Binding of Inhibitors to TIPARP, PARP10, PARP14 and PARP16

[0186] Displacement of a Probe C binding to monoPARP active sites was measured using a time-resolved fluorescence resonance energy transfer (TR-FRET) assay. 20 nL of a dose response curve of each test compound was spotted in black 384-well polystyrene proxiplates (Perkin Elmer) using a Mosquito (TTP Labtech). Reactions were performed in an 8 .mu.L volume by adding 6 .mu.L of the monoPARP and Probe C in assay buffer (20 mM HEPES pH=8, 100 mM NaCl, 0.1% bovine serum albumin, 2 mM DTT and 0.002% Tween20), incubating with test compound at 25.degree. C. for 30 min, then adding 2 .mu.L of ULight-anti 6.times.His and LANCE Eu-W1024 labeled streptavidin (Perkin Elmer). The final concentrations of monoPARP, Probe C, ULight-anti 6.times.His and LANCE Eu-W1024 labeled streptavidin are listed in Table 3. Binding reactions were equilibrated at 25.degree. C. for an additional 30 min, then read on an Envision platereader equipped with a LANCE/DELFIA top mirror (Perkin Elmer) using excitation of 320 nm and emission of 615 nm and 665 nM with a 90 .mu.s delay. The ratio of the 665/615 nm emission were calculated for each well to determine the amount of complex of monoPARP and Probe C in each well.

TABLE-US-00003 TABLE 3 Assay conditions for monoPARP probe displacement assays where the streptavidin is labeled with TR-FRET donor LANCE Eu- Enzyme Probe C ULight-anti W1024 labeled Target (nM) (nM) 6xHis (nM) streptavidin (nM) TIPARP 6 2 4 0.25 PARP10 6 0.5 2 0.25 PARP14 6 2 10 0.25 PARP16 3 1 6 0.25

In Vitro Probe Displacement Assay for Assessing Binding of Inhibitors to PARP12 and PARP15

[0187] Displacement of a Probe C binding to monoPARP active sites was measured using a time-resolved fluorescence resonance energy transfer (TR-FRET) assay. 20 nL of a dose response curve of each test compound was spotted in black 384-well polystyrene proxiplates (Perkin Elmer) using a Mosquito (TTP Labtech). Reactions were performed in a 8 .mu.L volume by adding 6 .mu.L of the monoPARP and Probe C in assay buffer (20 mM HEPES pH=8, 100 mM NaCl, 0.1% bovine serum albumin, 2 mM DTT and 0.002% Tween20), incubating with test compound at 25.degree. C. for 30 min, then adding 2 .mu.L of ULight-streptavidin and LANCE Eu-W1024 Anti-6.times.His (Perkin Elmer). The final concentrations of monoPARP, Probe C, ULight-streptavidin and LANCE Eu-W1024 Anti-6.times.His are listed in Table 4. Binding reactions were equilibrated at 25.degree. C. for an additional 30 min, then read on an Envision platereader equipped with a LANCE/DELFIA top mirror (Perkin Elmer) using excitation of 320 nm and emission of 615 nm and 665 nM with a 90 .mu.s delay. The ratio of the 665/615 nm emission were calculated for each well to determine the amount of complex of monoPARP and Probe C in each well.

TABLE-US-00004 TABLE 4 Assay conditions for monoPARP probe displacement assays where the anti-His antibody is labeled with TR-FRET donor LANCE Eu- Enzyme Probe C ULight- labeled W1024 anti Target (nM) (nM) streptavidin (nM) 6xHis (nM) PARP12 6 32 10 1.25 PARP15 1.5 8 2 0.5

Data Analysis for all In Vitro Assays

[0188] Control wells containing a negative control of 0.25% DMSO vehicle or a positive control of 100 .mu.M Compound A were used to calculate the % inhibition as described below:

% .times. .times. inhibition = 100 .times. TRF cmpd - TRF min TRF max - TRF min ##EQU00006##

[0189] where TRF.sub.cmpd is the TR-FRET ratio from the compound treated well, TRF.sub.min is the TR-FRET ratio from the Compound A-treated positive control well and TRF.sub.max is the TR-FRET ratio from the DMSO-treated negative control well.

[0190] The % inhibition values were plotted as a function of compound concentration and the following 4-parameter fit was applied to derive the IC.sub.50 values:

Y = Bottom + ( Top - Bottom ) ( 1 + ( X IC 50 ) Hill .times. .times. Coefficient ##EQU00007##

[0191] where top and bottom are normally allowed to float, but may be fixed at 100 or 0 respectively in a 3-parameter fit. The Hill Coefficient is normally allowed to float but may also be fixed at 1 in a 3-parameter fit. Y is the % inhibition and X is the compound concentration.

Validation of In Vitro Probe Displacement Assays

[0192] The probe displacement assays were validated by outcompeting Probe C with Compound A, an analog that does not contain a linker or biotin group. The assays for TIPARP, PARP10, PARP12, PARP14, PARP15 and PARP16 were set up as described above, and the results are shown in FIGS. 4A-4F.

[0193] FIGS. 4A-4F are validation results of the in vitro probe displacement binding assays. Dose response curves for Compound A were generated using each assay to confirm that Probe C was able to be outcompeted from the monoPARP enzyme. IC.sub.50 values were TIPARP=7 nM, PARP10=80 nM, PARP12=200 nM, PARP14=50 nM, PARP15=60 nM and PARP16=100 nM.

Example 10: Live Cell Assays

NanoLuc Plasmids

[0194] MonoPARP genes from Table 5 were cloned into the pcDNA3.1-mammalian expression vector as a NanoLuc fusion with NanoLuc on the N- or C-terminus as indicated. The sequence of the NanoLuc tag is as follows:

TABLE-US-00005 NanoLuc amino acid sequence (SEQ ID NO: 11): MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSG ENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLV IDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGS LLFRVTINGVTGWRLCERILA NanoLuc nucleic acid sequence (SEQ ID NO: 12): atggtcttcacactcgaagatttcgttggggactggcgacagacagccgg ctacaacctggaccaagtccttgaacagggaggtgtgtccagtttgtttc agaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggt gaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggtct gagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtacc ctgtggatgatcatcactttaaggtgatcctgcactatggcacactggta atcgacggggttacgccgaacatgatcgactatttcggacggccgtatga aggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgt ggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctcc ctgctgttccgagtaaccatcaacggagtgaccggctggcggctgtgcga acgcattctggcgtaa

TABLE-US-00006 TABLE 5 Details for the monoPARP-NanoLuc fusion genes used Genbank Amino Acid NanoLuc Accession Plasmid Name Number Residues Position Full-length TIPARP NM_015508 1-657 of C-terminus NP_056323.2 (SEQ ID NO: 1) Catalytic domain NM_015508 456-657 of C-terminus TIPARP NP_056323.2 (SEQ ID NO: 1) PARP10 NM_032789 808-1025 of C-terminus NP_116178.2 (SEQ ID NO: 2) PARP12 NM_022750.3 489-684 of C-terminus NP_073587.1 (SEQ ID NO: 3) PARP14 NM_017554 1611-1801 of N-terminus NP_060024.2 (SEQ ID NO: 4)

NanoBRET Probe Displacement Assay for Assessing Binding of Inhibitors to TIPARP, PARP10, PARP12 and PARP14

[0195] Displacement of a fluorescently-labeled compound Probe A binding to NanoLuc-tagged monoPARP enzymes was measured in live cells using a bioluminescence resonance energy transfer (NanoBRET) assay. TIPARP, PARP10, PARP12 or PARP14 fused to a NanoLuc tag were overexpressed in 293T cells (ATCC) using the plasmids described herein. Plasmid DNA and empty vector DNA were added to phenol red free OptiMEM (Thermo Fisher) as shown in Table 6 in a total volume of 2.456 mL. 157 .mu.L of Fugene HD (Promega) was added to the DNA mixture and allowed to incubate 5 min at 25.degree. C.

TABLE-US-00007 TABLE 6 Concentration of Concentration of Plasmid Empty Vector Probe A Plasmid Name (.mu.g/mL) (.mu.g/mL) (nM) Full-length TIPARP 0.2 19.8 9 Catalytic domain 0.2 19.8 80 TIPARP PARP10 2 18 7 PARP12 2 18 300 PARP14 0.2 19.8 100

[0196] Next, 2.375 mL of the plasmid-Fugene mixture were added to 20 million 293T cells in DMEM (Thermo Fisher) supplemented with 10% FBS (VWR). The transfection was incubated for 24 h at 37.degree. C. in an incubator containing air supplemented with 5% CO.sub.2. The cells were resuspended in phenol red free OptiMEM media. Transfected 293T cells were diluted to 500,000 cells per mL and Probe A was added to a final concentration as shown in Table 6. 40 .mu.L of cells were then added to white polystyrene 384-well non-binding surface microplate (Corning). 40 nL of a dose response curve diluted in DMSO of each test compound was added to the cell plate using a Mosquito (TTP Labtech) and the plate was incubated at 37.degree. C. in an incubator containing air supplemented with 5% CO.sub.2 for 2 h. The assay plate was allowed to equilibrate to room temperature (25.degree. C.), then 20 .mu.L per well of NanoBRET substrate (Promega) was added to the plate (1:166 dilution of NanoBRET substrate, 1:500 dilution of NanoLuc extracellular inhibitor in OptiMEM without phenol red). Filtered luminescence was measured on an Envision (Perkin Elmer) equipped with a dual 585 nm mirror, 460.+-.40 nm bandpass filter (donor) and 610.+-.50 nm longpass filter (acceptor).

TABLE-US-00008 TABLE 7 Positive control Final Concentration Assay compound (.mu.M) Full-length TIPARP Compound B 0.1 Catalytic domain TIPARP Compound B 0.2 PARP10 Compound C 2 PARP12 Compound B 1 PARP14 Compound D 5

Data Analysis for NanoBRET Assays

[0197] BRET ratio was measured as shown below:

BRET .times. .times. ratio = Emission .times. .times. at .times. .times. 610 .times. .times. nm Luminescence ##EQU00008##

[0198] Control wells containing a negative control of 0.2% DMSO vehicle or a positive control were used to calculate the % inhibition as described below:

% .times. .times. inhibition = 100 .times. BRET .times. .times. ratio cmpd - BRET .times. .times. ratio min BRET .times. .times. ratio max - BRET .times. .times. ratio min ##EQU00009##

[0199] where BRET ratio.sub.cmpd is the BRET ratio from the compound treated well, BRET ratio.sub.min is the BRET ratio from the positive control wells and BRET ratio.sub.max is the BRET ratio from the DMSO treated negative control well.

[0200] The % inhibition values were plotted as a function of compound concentration and the following 4-parameter fit was applied to derive the IC.sub.50 values:

Y = Bottom + ( Top - Bottom ) ( 1 + ( X IC 50 ) Hill .times. .times. Coefficient ##EQU00010##

[0201] where top and bottom are normally allowed to float, but may be fixed at 100 or 0 respectively in a 3-parameter fit. The Hill Coefficient is normally allowed to float but may also be fixed at 1 in a 3-parameter fit. Y is the % inhibition and X is the compound concentration.

Validation of NanoBRET Probe Displacement Assays

[0202] The probe displacement assays were validated by outcompeting Probe A with Compound A in the PARP14 NanoBRET assay, Compound B in the TIPARP NanoBRET assay, Compound C in the PARP10 NanoBRET assay and Compound B in the PARP12 NanoBRET assay. The compounds used to test the probe displacement of Probe A are analogs that do not contain a linker or fluorescent tag. The assays for TIPARP full-length and catalytic domain, PARP10, PARP12 and PARP14 were set up as described above, and the results are shown in FIG. 5.

[0203] FIGS. 5A-5D are validation results of the NanoBRET probe displacement binding assays. Dose response curves for control compounds were generated using each assay to confirm that Probe A was able to be outcompeted from the monoPARP enzyme. IC.sub.50 values were full-length TIPARP Compound B=4 nM, catalytic domain TIPARP Compound B=7 nM, PARP10 Compound C=4 nM, PARP12 Compound B=170 nM, PARP14 compound A=30 nM.

OTHER EMBODIMENTS

[0204] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

REFERENCES

[0205] 1. Leidecker, O.; Bonfiglio, J. J.; Colby, T.; Zhang, Q.; Atanassov, I.; Zaja, R.; Palazzo, L.; Stockum, A.; Ahel, I.; Matic, I., Serine is a new target residue for endogenous ADP-ribosylation on histones. Nat Chem Biol 2016, 12 (12), 998-1000.

[0206] 2. Belousova, E. A.; Ishchenko capital A, C.; Lavrik, O. I., Dna is a New Target of Parp3. Sci Rep 2018, 8 (1), 4176.

[0207] 3. Yamada, T.; Horimoto, H.; Kameyama, T.; Hayakawa, S.; Yamato, H.; Dazai, M.; Takada, A.; Kida, H.; Bott, D.; Zhou, A. C.; Hutin, D.; Watts, T. H.; Asaka, M.; Matthews, J.; Takaoka, A., Constitutive aryl hydrocarbon receptor signaling constrains type I interferon-mediated antiviral innate defense. Nat Immunol 2016, 17(6), 687-94.

[0208] 4. Caprara, G.; Prosperini, E.; Piccolo, V.; Sigismondo, G.; Melacarne, A.; Cuomo, A.; Boothby, M.; Rescigno, M.; Bonaldi, T.; Natoli, G., PARP14 Controls the Nuclear Accumulation of a Subset of Type I IFN-Inducible Proteins. J Immunol 2018, 200 (7), 2439-2454.

[0209] 5. Zaffini, R.; Gotte, G.; Menegazzi, M., Asthma and poly(ADP-ribose) polymerase inhibition: a new therapeutic approach. Drug Des Devel Ther 2018, 12, 281-293.

[0210] 6. Jwa, M.; Chang, P., PARP16 is a tail-anchored endoplasmic reticulum protein required for the PERK- and IRE1alpha-mediated unfolded protein response. Nat Cell Biol 2012, 14 (11), 1223-30.

[0211] 7. Barbarulo, A.; Iansante, V.; Chaidos, A.; Naresh, K.; Rahemtulla, A.; Franzoso, G.; Karadimitris, A.; Haskard, D. O.; Papa, S.; Bubici, C., Poly(ADP-ribose) polymerase family member 14 (PARP14) is a novel effector of the JNK2-dependent pro-survival signal in multiple myeloma. Oncogene 2013, 32 (36), 4231-42.

[0212] 8. Thorsell, A. G.; Ekblad, T.; Karlberg, T.; Low, M.; Pinto, A. F.; Tresaugues, L.; Moche, M.; Cohen, M. S.; Schuler, H., Structural Basis for Potency and Promiscuity in Poly(ADP-ribose) Polymerase (PARP) and Tankyrase Inhibitors. J Med Chem 2017, 60 (4), 1262-1271.

[0213] 9. Copeland, R. A., Evaluation of enzyme inhibitors in drug discovery. A guide for medicinal chemists and pharmacologists. Methods Biochem Anal 2005, 46, 1-265.

[0214] 10. Wahlberg, E.; Karlberg, T.; Kouznetsova, E.; Markova, N.; Macchiarulo, A.; Thorsell, A. G.; Pol, E.; Frostell, A.; Ekblad, T.; Oncu, D.; Kull, B.; Robertson, G. M.; Pellicciari, R.; Schuler, H.; Weigelt, J., Family-wide chemical profiling and structural analysis of PARP and tankyrase inhibitors. Nat Biotechnol 2012, 30 (3), 283-8.

[0215] 11. Davis, B. J.; Erlanson, D. A., Learning from our mistakes: the `unknown knowns` in fragment screening. BioorgMed Chem Lett 2013, 23 (10), 2844-52.

Sequence CWU 1

1

231657PRTHomo sapiens 1Met Glu Met Glu Thr Thr Glu Pro Glu Pro Asp Cys Val Val Gln Pro1 5 10 15Pro Ser Pro Pro Asp Asp Phe Ser Cys Gln Met Arg Leu Ser Glu Lys 20 25 30Ile Thr Pro Leu Lys Thr Cys Phe Lys Lys Lys Asp Gln Lys Arg Leu 35 40 45Gly Thr Gly Thr Leu Arg Ser Leu Arg Pro Ile Leu Asn Thr Leu Leu 50 55 60Glu Ser Gly Ser Leu Asp Gly Val Phe Arg Ser Arg Asn Gln Ser Thr65 70 75 80Asp Glu Asn Ser Leu His Glu Pro Met Met Lys Lys Ala Met Glu Ile 85 90 95Asn Ser Ser Cys Pro Pro Ala Glu Asn Asn Met Ser Val Leu Ile Pro 100 105 110Asp Arg Thr Asn Val Gly Asp Gln Ile Pro Glu Ala His Pro Ser Thr 115 120 125Glu Ala Pro Glu Arg Val Val Pro Ile Gln Asp His Ser Phe Pro Ser 130 135 140Glu Thr Leu Ser Gly Thr Val Ala Asp Ser Thr Pro Ala His Phe Gln145 150 155 160Thr Asp Leu Leu His Pro Val Ser Ser Asp Val Pro Thr Ser Pro Asp 165 170 175Cys Leu Asp Lys Val Ile Asp Tyr Val Pro Gly Ile Phe Gln Glu Asn 180 185 190Ser Phe Thr Ile Gln Tyr Ile Leu Asp Thr Ser Asp Lys Leu Ser Thr 195 200 205Glu Leu Phe Gln Asp Lys Ser Glu Glu Ala Ser Leu Asp Leu Val Phe 210 215 220Glu Leu Val Asn Gln Leu Gln Tyr His Thr His Gln Glu Asn Gly Ile225 230 235 240Glu Ile Cys Met Asp Phe Leu Gln Gly Thr Cys Ile Tyr Gly Arg Asp 245 250 255Cys Leu Lys His His Thr Val Leu Pro Tyr His Trp Gln Ile Lys Arg 260 265 270Thr Thr Thr Gln Lys Trp Gln Ser Val Phe Asn Asp Ser Gln Glu His 275 280 285Leu Glu Arg Phe Tyr Cys Asn Pro Glu Asn Asp Arg Met Arg Met Lys 290 295 300Tyr Gly Gly Gln Glu Phe Trp Ala Asp Leu Asn Ala Met Asn Val Tyr305 310 315 320Glu Thr Thr Glu Phe Asp Gln Leu Arg Arg Leu Ser Thr Pro Pro Ser 325 330 335Ser Asn Val Asn Ser Ile Tyr His Thr Val Trp Lys Phe Phe Cys Arg 340 345 350Asp His Phe Gly Trp Arg Glu Tyr Pro Glu Ser Val Ile Arg Leu Ile 355 360 365Glu Glu Ala Asn Ser Arg Gly Leu Lys Glu Val Arg Phe Met Met Trp 370 375 380Asn Asn His Tyr Ile Leu His Asn Ser Phe Phe Arg Arg Glu Ile Lys385 390 395 400Arg Arg Pro Leu Phe Arg Ser Cys Phe Ile Leu Leu Pro Tyr Leu Gln 405 410 415Thr Leu Gly Gly Val Pro Thr Gln Ala Pro Pro Pro Leu Glu Ala Thr 420 425 430Ser Ser Ser Gln Ile Ile Cys Pro Asp Gly Val Thr Ser Ala Asn Phe 435 440 445Tyr Pro Glu Thr Trp Val Tyr Met His Pro Ser Gln Asp Phe Ile Gln 450 455 460Val Pro Val Ser Ala Glu Asp Lys Ser Tyr Arg Ile Ile Tyr Asn Leu465 470 475 480Phe His Lys Thr Val Pro Glu Phe Lys Tyr Arg Ile Leu Gln Ile Leu 485 490 495Arg Val Gln Asn Gln Phe Leu Trp Glu Lys Tyr Lys Arg Lys Lys Glu 500 505 510Tyr Met Asn Arg Lys Met Phe Gly Arg Asp Arg Ile Ile Asn Glu Arg 515 520 525His Leu Phe His Gly Thr Ser Gln Asp Val Val Asp Gly Ile Cys Lys 530 535 540His Asn Phe Asp Pro Arg Val Cys Gly Lys His Ala Thr Met Phe Gly545 550 555 560Gln Gly Ser Tyr Phe Ala Lys Lys Ala Ser Tyr Ser His Asn Phe Ser 565 570 575Lys Lys Ser Ser Lys Gly Val His Phe Met Phe Leu Ala Lys Val Leu 580 585 590Thr Gly Arg Tyr Thr Met Gly Ser His Gly Met Arg Arg Pro Pro Pro 595 600 605Val Asn Pro Gly Ser Val Thr Ser Asp Leu Tyr Asp Ser Cys Val Asp 610 615 620Asn Phe Phe Glu Pro Gln Ile Phe Val Ile Phe Asn Asp Asp Gln Ser625 630 635 640Tyr Pro Tyr Phe Val Ile Gln Tyr Glu Glu Val Ser Asn Thr Val Ser 645 650 655Ile21025PRTHomo sapiens 2Met Val Ala Met Ala Glu Ala Glu Ala Gly Val Ala Val Glu Val Arg1 5 10 15Gly Leu Pro Pro Ala Val Pro Asp Glu Leu Leu Thr Leu Tyr Phe Glu 20 25 30Asn Arg Arg Arg Ser Gly Gly Gly Pro Val Leu Ser Trp Gln Arg Leu 35 40 45Gly Cys Gly Gly Val Leu Thr Phe Arg Glu Pro Ala Asp Ala Glu Arg 50 55 60Val Leu Ala Gln Ala Asp His Glu Leu His Gly Ala Gln Leu Ser Leu65 70 75 80Arg Pro Ala Pro Pro Arg Ala Pro Ala Arg Leu Leu Leu Gln Gly Leu 85 90 95Pro Pro Gly Thr Thr Pro Gln Arg Leu Glu Gln His Val Gln Ala Leu 100 105 110Leu Arg Ala Ser Gly Leu Pro Val Gln Pro Cys Cys Ala Leu Ala Ser 115 120 125Pro Arg Pro Asp Arg Ala Leu Val Gln Leu Pro Lys Pro Leu Ser Glu 130 135 140Ala Asp Val Arg Val Leu Glu Glu Gln Ala Gln Asn Leu Gly Leu Glu145 150 155 160Gly Thr Leu Val Ser Leu Ala Arg Val Pro Gln Ala Arg Ala Val Arg 165 170 175Val Val Gly Asp Gly Ala Ser Val Asp Leu Leu Leu Leu Glu Leu Tyr 180 185 190Leu Glu Asn Glu Arg Arg Ser Gly Gly Gly Pro Leu Glu Asp Leu Gln 195 200 205Arg Leu Pro Gly Pro Leu Gly Thr Val Ala Ser Phe Gln Gln Trp Gln 210 215 220Val Ala Glu Arg Val Leu Gln Gln Glu His Arg Leu Gln Gly Ser Glu225 230 235 240Leu Ser Leu Val Pro His Tyr Asp Ile Leu Glu Pro Glu Glu Leu Ala 245 250 255Glu Asn Thr Ser Gly Gly Asp His Pro Ser Thr Gln Gly Pro Arg Ala 260 265 270Thr Lys His Ala Leu Leu Arg Thr Gly Gly Leu Val Thr Ala Leu Gln 275 280 285Gly Ala Gly Thr Val Thr Met Gly Ser Gly Glu Glu Pro Gly Gln Ser 290 295 300Gly Ala Ser Leu Arg Thr Gly Pro Met Val Gln Gly Arg Gly Ile Met305 310 315 320Thr Thr Gly Ser Gly Gln Glu Pro Gly Gln Ser Gly Thr Ser Leu Arg 325 330 335Thr Gly Pro Met Gly Ser Leu Gly Gln Ala Glu Gln Val Ser Ser Met 340 345 350Pro Met Gly Ser Leu Glu His Glu Gly Leu Val Ser Leu Arg Pro Val 355 360 365Gly Leu Gln Glu Gln Glu Gly Pro Met Ser Leu Gly Pro Val Gly Ser 370 375 380Ala Gly Pro Val Glu Thr Ser Lys Gly Leu Leu Gly Gln Glu Gly Leu385 390 395 400Val Glu Ile Ala Met Asp Ser Pro Glu Gln Glu Gly Leu Val Gly Pro 405 410 415Met Glu Ile Thr Met Gly Ser Leu Glu Lys Ala Gly Pro Val Ser Pro 420 425 430Gly Cys Val Lys Leu Ala Gly Gln Glu Gly Leu Val Glu Met Val Leu 435 440 445Leu Met Glu Pro Gly Ala Met Arg Phe Leu Gln Leu Tyr His Glu Asp 450 455 460Leu Leu Ala Gly Leu Gly Asp Val Ala Leu Leu Pro Leu Glu Gly Pro465 470 475 480Asp Met Thr Gly Phe Arg Leu Cys Gly Ala Gln Ala Ser Cys Gln Ala 485 490 495Ala Glu Glu Phe Leu Arg Ser Leu Leu Gly Ser Ile Ser Cys His Val 500 505 510Leu Cys Leu Glu His Pro Gly Ser Ala Arg Phe Leu Leu Gly Pro Glu 515 520 525Gly Gln His Leu Leu Gln Gly Leu Glu Ala Gln Phe Gln Cys Val Phe 530 535 540Gly Thr Glu Arg Leu Ala Thr Ala Thr Leu Asp Thr Gly Leu Glu Glu545 550 555 560Val Asp Pro Thr Glu Ala Leu Pro Val Leu Pro Gly Asn Ala His Thr 565 570 575Leu Trp Thr Pro Asp Ser Thr Gly Gly Asp Gln Glu Asp Val Ser Leu 580 585 590Glu Glu Val Arg Glu Leu Leu Ala Thr Leu Glu Gly Leu Asp Leu Asp 595 600 605Gly Glu Asp Trp Leu Pro Arg Glu Leu Glu Glu Glu Gly Pro Gln Glu 610 615 620Gln Pro Glu Glu Glu Val Thr Pro Gly His Glu Glu Glu Glu Pro Val625 630 635 640Ala Pro Ser Thr Val Ala Pro Arg Trp Leu Glu Glu Glu Ala Ala Leu 645 650 655Gln Leu Ala Leu His Arg Ser Leu Glu Pro Gln Gly Gln Val Ala Glu 660 665 670Gln Glu Glu Ala Ala Ala Leu Arg Gln Ala Leu Thr Leu Ser Leu Leu 675 680 685Glu Gln Pro Pro Leu Glu Ala Glu Glu Pro Pro Asp Gly Gly Thr Asp 690 695 700Gly Lys Ala Gln Leu Val Val His Ser Ala Phe Glu Gln Asp Val Glu705 710 715 720Glu Leu Asp Arg Ala Leu Arg Ala Ala Leu Glu Val His Val Gln Glu 725 730 735Glu Thr Val Gly Pro Trp Arg Arg Thr Leu Pro Ala Glu Leu Arg Ala 740 745 750Arg Leu Glu Arg Cys His Gly Val Ser Val Ala Leu Arg Gly Asp Cys 755 760 765Thr Ile Leu Arg Gly Phe Gly Ala His Pro Ala Arg Ala Ala Arg His 770 775 780Leu Val Ala Leu Leu Ala Gly Pro Trp Asp Gln Ser Leu Ala Phe Pro785 790 795 800Leu Ala Ala Ser Gly Pro Thr Leu Ala Gly Gln Thr Leu Lys Gly Pro 805 810 815Trp Asn Asn Leu Glu Arg Leu Ala Glu Asn Thr Gly Glu Phe Gln Glu 820 825 830Val Val Arg Ala Phe Tyr Asp Thr Leu Asp Ala Ala Arg Ser Ser Ile 835 840 845Arg Val Val Arg Val Glu Arg Val Ser His Pro Leu Leu Gln Gln Gln 850 855 860Tyr Glu Leu Tyr Arg Glu Arg Leu Leu Gln Arg Cys Glu Arg Arg Pro865 870 875 880Val Glu Gln Val Leu Tyr His Gly Thr Thr Ala Pro Ala Val Pro Asp 885 890 895Ile Cys Ala His Gly Phe Asn Arg Ser Phe Cys Gly Arg Asn Ala Thr 900 905 910Val Tyr Gly Lys Gly Val Tyr Phe Ala Arg Arg Ala Ser Leu Ser Val 915 920 925Gln Asp Arg Tyr Ser Pro Pro Asn Ala Asp Gly His Lys Ala Val Phe 930 935 940Val Ala Arg Val Leu Thr Gly Asp Tyr Gly Gln Gly Arg Arg Gly Leu945 950 955 960Arg Ala Pro Pro Leu Arg Gly Pro Gly His Val Leu Leu Arg Tyr Asp 965 970 975Ser Ala Val Asp Cys Ile Cys Gln Pro Ser Ile Phe Val Ile Phe His 980 985 990Asp Thr Gln Ala Leu Pro Thr His Leu Ile Thr Cys Glu His Val Pro 995 1000 1005Arg Ala Ser Pro Asp Asp Pro Ser Gly Leu Pro Gly Arg Ser Pro 1010 1015 1020Asp Thr 10253701PRTHomo sapiens 3Met Ala Gln Ala Gly Val Val Gly Glu Val Thr Gln Val Leu Cys Ala1 5 10 15Ala Gly Gly Ala Leu Glu Leu Pro Glu Leu Arg Arg Arg Leu Arg Met 20 25 30Gly Leu Ser Ala Asp Ala Leu Glu Arg Leu Leu Arg Gln Arg Gly Arg 35 40 45Phe Val Val Ala Val Arg Ala Gly Gly Ala Ala Ala Ala Pro Glu Arg 50 55 60Val Val Leu Ala Ala Ser Pro Leu Arg Leu Cys Arg Ala His Gln Gly65 70 75 80Ser Lys Pro Gly Cys Val Gly Leu Cys Ala Gln Leu His Leu Cys Arg 85 90 95Phe Met Val Tyr Gly Ala Cys Lys Phe Leu Arg Ala Gly Lys Asn Cys 100 105 110Arg Asn Ser His Ser Leu Thr Thr Glu His Asn Leu Ser Val Leu Arg 115 120 125Thr His Gly Val Asp His Leu Ser Tyr Asn Glu Leu Cys Gln Leu Leu 130 135 140Phe Gln Asn Asp Pro Trp Leu Leu Pro Glu Ile Cys Gln His Tyr Asn145 150 155 160Lys Gly Asp Gly Pro His Gly Ser Cys Ala Phe Gln Lys Gln Cys Ile 165 170 175Lys Leu His Ile Cys Gln Tyr Phe Leu Gln Gly Glu Cys Lys Phe Gly 180 185 190Thr Ser Cys Lys Arg Ser His Asp Phe Ser Asn Ser Glu Asn Leu Glu 195 200 205Lys Leu Glu Lys Leu Gly Met Ser Ser Asp Leu Val Ser Arg Leu Pro 210 215 220Thr Ile Tyr Arg Asn Ala His Asp Ile Lys Asn Lys Ser Ser Ala Pro225 230 235 240Ser Arg Val Pro Pro Leu Phe Val Pro Gln Gly Thr Ser Glu Arg Lys 245 250 255Asp Ser Ser Gly Ser Val Ser Pro Asn Thr Leu Ser Gln Glu Glu Gly 260 265 270Asp Gln Ile Cys Leu Tyr His Ile Arg Lys Ser Cys Ser Phe Gln Asp 275 280 285Lys Cys His Arg Val His Phe His Leu Pro Tyr Arg Trp Gln Phe Leu 290 295 300Asp Arg Gly Lys Trp Glu Asp Leu Asp Asn Met Glu Leu Ile Glu Glu305 310 315 320Ala Tyr Cys Asn Pro Lys Ile Glu Arg Ile Leu Cys Ser Glu Ser Ala 325 330 335Ser Thr Phe His Ser His Cys Leu Asn Phe Asn Ala Met Thr Tyr Gly 340 345 350Ala Thr Gln Ala Arg Arg Leu Ser Thr Ala Ser Ser Val Thr Lys Pro 355 360 365Pro His Phe Ile Leu Thr Thr Asp Trp Ile Trp Tyr Trp Ser Asp Glu 370 375 380Phe Gly Ser Trp Gln Glu Tyr Gly Arg Gln Gly Thr Val His Pro Val385 390 395 400Thr Thr Val Ser Ser Ser Asp Val Glu Lys Ala Tyr Leu Ala Tyr Cys 405 410 415Thr Pro Gly Ser Asp Gly Gln Ala Ala Thr Leu Lys Phe Gln Ala Gly 420 425 430Lys His Asn Tyr Glu Leu Asp Phe Lys Ala Phe Val Gln Lys Asn Leu 435 440 445Val Tyr Gly Thr Thr Lys Lys Val Cys Arg Arg Pro Lys Tyr Val Ser 450 455 460Pro Gln Asp Val Thr Thr Met Gln Thr Cys Asn Thr Lys Phe Pro Gly465 470 475 480Pro Lys Ser Ile Pro Asp Tyr Trp Asp Ser Ser Ala Leu Pro Asp Pro 485 490 495Gly Phe Gln Lys Ile Thr Leu Ser Ser Ser Ser Glu Glu Tyr Gln Lys 500 505 510Val Trp Asn Leu Phe Asn Arg Thr Leu Pro Phe Tyr Phe Val Gln Lys 515 520 525Ile Glu Arg Val Gln Asn Leu Ala Leu Trp Glu Val Tyr Gln Trp Gln 530 535 540Lys Gly Gln Met Gln Lys Gln Asn Gly Gly Lys Ala Val Asp Glu Arg545 550 555 560Gln Leu Phe His Gly Thr Ser Ala Ile Phe Val Asp Ala Ile Cys Gln 565 570 575Gln Asn Phe Asp Trp Arg Val Cys Gly Val His Gly Thr Ser Tyr Gly 580 585 590Lys Gly Ser Tyr Phe Ala Arg Asp Ala Ala Tyr Ser His His Tyr Ser 595 600 605Lys Ser Asp Thr Gln Thr His Thr Met Phe Leu Ala Arg Val Leu Val 610 615 620Gly Glu Phe Val Arg Gly Asn Ala Ser Phe Val Arg Pro Pro Ala Lys625 630 635 640Glu Gly Trp Ser Asn Ala Phe Tyr Asp Ser Cys Val Asn Ser Val Ser 645 650 655Asp Pro Ser Ile Phe Val Ile Phe Glu Lys His Gln Val Tyr Pro Glu 660 665 670Tyr Val Ile Gln Tyr Thr Thr Ser Ser Lys Pro Ser Val Thr Pro Ser 675 680 685Ile Leu Leu Ala Leu Gly Ser Leu Phe Ser Ser Arg Gln 690 695 70041801PRTHomo sapiens 4Met Ala Val Pro Gly Ser Phe Pro Leu Leu Val Glu Gly Ser Trp Gly1 5 10 15Pro Asp Pro Pro Lys Asn Leu Asn Thr Lys Leu Gln Met Tyr Phe Gln 20 25 30Ser Pro Lys Arg Ser Gly Gly Gly Glu Cys Glu Val Arg Gln Asp Pro 35 40 45Arg Ser Pro Ser Arg Phe Leu Val Phe Phe Tyr Pro Glu Asp Val Arg 50 55 60Gln Lys Val Leu Glu Arg Lys Asn His Glu Leu Val Trp Gln Gly Lys65 70 75 80Gly

Thr Phe Lys Leu Thr Val Gln Leu Pro Ala Thr Pro Asp Glu Ile 85 90 95Asp His Val Phe Glu Glu Glu Leu Leu Thr Lys Glu Ser Lys Thr Lys 100 105 110Glu Asp Val Lys Glu Pro Asp Val Ser Glu Glu Leu Asp Thr Lys Leu 115 120 125Pro Leu Asp Gly Gly Leu Asp Lys Met Glu Asp Ile Pro Glu Glu Cys 130 135 140Glu Asn Ile Ser Ser Leu Val Ala Phe Glu Asn Leu Lys Ala Asn Val145 150 155 160Thr Asp Ile Met Leu Ile Leu Leu Val Glu Asn Ile Ser Gly Leu Ser 165 170 175Asn Asp Asp Phe Gln Val Glu Ile Ile Arg Asp Phe Asp Val Ala Val 180 185 190Val Thr Phe Gln Lys His Ile Asp Thr Ile Arg Phe Val Asp Asp Cys 195 200 205Thr Lys His His Ser Ile Lys Gln Leu Gln Leu Ser Pro Arg Leu Leu 210 215 220Glu Val Thr Asn Thr Ile Arg Val Glu Asn Leu Pro Pro Gly Ala Asp225 230 235 240Asp Tyr Ser Leu Lys Leu Phe Phe Glu Asn Pro Tyr Asn Gly Gly Gly 245 250 255Arg Val Ala Asn Val Glu Tyr Phe Pro Glu Glu Ser Ser Ala Leu Ile 260 265 270Glu Phe Phe Asp Arg Lys Val Leu Asp Thr Ile Met Ala Thr Lys Leu 275 280 285Asp Phe Asn Lys Met Pro Leu Ser Val Phe Pro Tyr Tyr Ala Ser Leu 290 295 300Gly Thr Ala Leu Tyr Gly Lys Glu Lys Pro Leu Ile Lys Leu Pro Ala305 310 315 320Pro Phe Glu Glu Ser Leu Asp Leu Pro Leu Trp Lys Phe Leu Gln Lys 325 330 335Lys Asn His Leu Ile Glu Glu Ile Asn Asp Glu Met Arg Arg Cys His 340 345 350Cys Glu Leu Thr Trp Ser Gln Leu Ser Gly Lys Val Thr Ile Arg Pro 355 360 365Ala Ala Thr Leu Val Asn Glu Gly Arg Pro Arg Ile Lys Thr Trp Gln 370 375 380Ala Asp Thr Ser Thr Thr Leu Ser Ser Ile Arg Ser Lys Tyr Lys Val385 390 395 400Asn Pro Ile Lys Val Asp Pro Thr Met Trp Asp Thr Ile Lys Asn Asp 405 410 415Val Lys Asp Asp Arg Ile Leu Ile Glu Phe Asp Thr Leu Lys Glu Met 420 425 430Val Ile Leu Ala Gly Lys Ser Glu Asp Val Gln Ser Ile Glu Val Gln 435 440 445Val Arg Glu Leu Ile Glu Ser Thr Thr Gln Lys Ile Lys Arg Glu Glu 450 455 460Gln Ser Leu Lys Glu Lys Met Ile Ile Ser Pro Gly Arg Tyr Phe Leu465 470 475 480Leu Cys His Ser Ser Leu Leu Asp His Leu Leu Thr Glu Cys Pro Glu 485 490 495Ile Glu Ile Cys Tyr Asp Arg Val Thr Gln His Leu Cys Leu Lys Gly 500 505 510Pro Ser Ala Asp Val Tyr Lys Ala Lys Cys Glu Ile Gln Glu Lys Val 515 520 525Tyr Thr Met Ala Gln Lys Asn Ile Gln Val Ser Pro Glu Ile Phe Gln 530 535 540Phe Leu Gln Gln Val Asn Trp Lys Glu Phe Ser Lys Cys Leu Phe Ile545 550 555 560Ala Gln Lys Ile Leu Ala Leu Tyr Glu Leu Glu Gly Thr Thr Val Leu 565 570 575Leu Thr Ser Cys Ser Ser Glu Ala Leu Leu Glu Ala Glu Lys Gln Met 580 585 590Leu Ser Ala Leu Asn Tyr Lys Arg Ile Glu Val Glu Asn Lys Glu Val 595 600 605Leu His Gly Lys Lys Trp Lys Gly Leu Thr His Asn Leu Leu Lys Lys 610 615 620Gln Asn Ser Ser Pro Asn Thr Val Ile Ile Asn Glu Leu Thr Ser Glu625 630 635 640Thr Thr Ala Glu Val Ile Ile Thr Gly Cys Val Lys Glu Val Asn Glu 645 650 655Thr Tyr Lys Leu Leu Phe Asn Phe Val Glu Gln Asn Met Lys Ile Glu 660 665 670Arg Leu Val Glu Val Lys Pro Ser Leu Val Ile Asp Tyr Leu Lys Thr 675 680 685Glu Lys Lys Leu Phe Trp Pro Lys Ile Lys Lys Val Asn Val Gln Val 690 695 700Ser Phe Asn Pro Glu Asn Lys Gln Lys Gly Ile Leu Leu Thr Gly Ser705 710 715 720Lys Thr Glu Val Leu Lys Ala Val Asp Ile Val Lys Gln Val Trp Asp 725 730 735Ser Val Cys Val Lys Ser Val His Thr Asp Lys Pro Gly Ala Lys Gln 740 745 750Phe Phe Gln Asp Lys Ala Arg Phe Tyr Gln Ser Glu Ile Lys Arg Leu 755 760 765Phe Gly Cys Tyr Ile Glu Leu Gln Glu Asn Glu Val Met Lys Glu Gly 770 775 780Gly Ser Pro Ala Gly Gln Lys Cys Phe Ser Arg Thr Val Leu Ala Pro785 790 795 800Gly Val Val Leu Ile Val Gln Gln Gly Asp Leu Ala Arg Leu Pro Val 805 810 815Asp Val Val Val Asn Ala Ser Asn Glu Asp Leu Lys His Tyr Gly Gly 820 825 830Leu Ala Ala Ala Leu Ser Lys Ala Ala Gly Pro Glu Leu Gln Ala Asp 835 840 845Cys Asp Gln Ile Val Lys Arg Glu Gly Arg Leu Leu Pro Gly Asn Ala 850 855 860Thr Ile Ser Lys Ala Gly Lys Leu Pro Tyr His His Val Ile His Ala865 870 875 880Val Gly Pro Arg Trp Ser Gly Tyr Glu Ala Pro Arg Cys Val Tyr Leu 885 890 895Leu Arg Arg Ala Val Gln Leu Ser Leu Cys Leu Ala Glu Lys Tyr Lys 900 905 910Tyr Arg Ser Ile Ala Ile Pro Ala Ile Ser Ser Gly Val Phe Gly Phe 915 920 925Pro Leu Gly Arg Cys Val Glu Thr Ile Val Ser Ala Ile Lys Glu Asn 930 935 940Phe Gln Phe Lys Lys Asp Gly His Cys Leu Lys Glu Ile Tyr Leu Val945 950 955 960Asp Val Ser Glu Lys Thr Val Glu Ala Phe Ala Glu Ala Val Lys Thr 965 970 975Val Phe Lys Ala Thr Leu Pro Asp Thr Ala Ala Pro Pro Gly Leu Pro 980 985 990Pro Ala Ala Ala Gly Pro Gly Lys Thr Ser Trp Glu Lys Gly Ser Leu 995 1000 1005Val Ser Pro Gly Gly Leu Gln Met Leu Leu Val Lys Glu Gly Val 1010 1015 1020Gln Asn Ala Lys Thr Asp Val Val Val Asn Ser Val Pro Leu Asp 1025 1030 1035Leu Val Leu Ser Arg Gly Pro Leu Ser Lys Ser Leu Leu Glu Lys 1040 1045 1050Ala Gly Pro Glu Leu Gln Glu Glu Leu Asp Thr Val Gly Gln Gly 1055 1060 1065Val Ala Val Ser Met Gly Thr Val Leu Lys Thr Ser Ser Trp Asn 1070 1075 1080Leu Asp Cys Arg Tyr Val Leu His Val Val Ala Pro Glu Trp Arg 1085 1090 1095Asn Gly Ser Thr Ser Ser Leu Lys Ile Met Glu Asp Ile Ile Arg 1100 1105 1110Glu Cys Met Glu Ile Thr Glu Ser Leu Ser Leu Lys Ser Ile Ala 1115 1120 1125Phe Pro Ala Ile Gly Thr Gly Asn Leu Gly Phe Pro Lys Asn Ile 1130 1135 1140Phe Ala Glu Leu Ile Ile Ser Glu Val Phe Lys Phe Ser Ser Lys 1145 1150 1155Asn Gln Leu Lys Thr Leu Gln Glu Val His Phe Leu Leu His Pro 1160 1165 1170Ser Asp His Glu Asn Ile Gln Ala Phe Ser Asp Glu Phe Ala Arg 1175 1180 1185Arg Ala Asn Gly Asn Leu Val Ser Asp Lys Ile Pro Lys Ala Lys 1190 1195 1200Asp Thr Gln Gly Phe Tyr Gly Thr Val Ser Ser Pro Asp Ser Gly 1205 1210 1215Val Tyr Glu Met Lys Ile Gly Ser Ile Ile Phe Gln Val Ala Ser 1220 1225 1230Gly Asp Ile Thr Lys Glu Glu Ala Asp Val Ile Val Asn Ser Thr 1235 1240 1245Ser Asn Ser Phe Asn Leu Lys Ala Gly Val Ser Lys Ala Ile Leu 1250 1255 1260Glu Cys Ala Gly Gln Asn Val Glu Arg Glu Cys Ser Gln Gln Ala 1265 1270 1275Gln Gln Arg Lys Asn Asp Tyr Ile Ile Thr Gly Gly Gly Phe Leu 1280 1285 1290Arg Cys Lys Asn Ile Ile His Val Ile Gly Gly Asn Asp Val Lys 1295 1300 1305Ser Ser Val Ser Ser Val Leu Gln Glu Cys Glu Lys Lys Asn Tyr 1310 1315 1320Ser Ser Ile Cys Leu Pro Ala Ile Gly Thr Gly Asn Ala Lys Gln 1325 1330 1335His Pro Asp Lys Val Ala Glu Ala Ile Ile Asp Ala Ile Glu Asp 1340 1345 1350Phe Val Gln Lys Gly Ser Ala Gln Ser Val Lys Lys Val Lys Val 1355 1360 1365Val Ile Phe Leu Pro Gln Val Leu Asp Val Phe Tyr Ala Asn Met 1370 1375 1380Lys Lys Arg Glu Gly Thr Gln Leu Ser Ser Gln Gln Ser Val Met 1385 1390 1395Ser Lys Leu Ala Ser Phe Leu Gly Phe Ser Lys Gln Ser Pro Gln 1400 1405 1410Lys Lys Asn His Leu Val Leu Glu Lys Lys Thr Glu Ser Ala Thr 1415 1420 1425Phe Arg Val Cys Gly Glu Asn Val Thr Cys Val Glu Tyr Ala Ile 1430 1435 1440Ser Trp Leu Gln Asp Leu Ile Glu Lys Glu Gln Cys Pro Tyr Thr 1445 1450 1455Ser Glu Asp Glu Cys Ile Lys Asp Phe Asp Glu Lys Glu Tyr Gln 1460 1465 1470Glu Leu Asn Glu Leu Gln Lys Lys Leu Asn Ile Asn Ile Ser Leu 1475 1480 1485Asp His Lys Arg Pro Leu Ile Lys Val Leu Gly Ile Ser Arg Asp 1490 1495 1500Val Met Gln Ala Arg Asp Glu Ile Glu Ala Met Ile Lys Arg Val 1505 1510 1515Arg Leu Ala Lys Glu Gln Glu Ser Arg Ala Asp Cys Ile Ser Glu 1520 1525 1530Phe Ile Glu Trp Gln Tyr Asn Asp Asn Asn Thr Ser His Cys Phe 1535 1540 1545Asn Lys Met Thr Asn Leu Lys Leu Glu Asp Ala Arg Arg Glu Lys 1550 1555 1560Lys Lys Thr Val Asp Val Lys Ile Asn His Arg His Tyr Thr Val 1565 1570 1575Asn Leu Asn Thr Tyr Thr Ala Thr Asp Thr Lys Gly His Ser Leu 1580 1585 1590Ser Val Gln Arg Leu Thr Lys Ser Lys Val Asp Ile Pro Ala His 1595 1600 1605Trp Ser Asp Met Lys Gln Gln Asn Phe Cys Val Val Glu Leu Leu 1610 1615 1620Pro Ser Asp Pro Glu Tyr Asn Thr Val Ala Ser Lys Phe Asn Gln 1625 1630 1635Thr Cys Ser His Phe Arg Ile Glu Lys Ile Glu Arg Ile Gln Asn 1640 1645 1650Pro Asp Leu Trp Asn Ser Tyr Gln Ala Lys Lys Lys Thr Met Asp 1655 1660 1665Ala Lys Asn Gly Gln Thr Met Asn Glu Lys Gln Leu Phe His Gly 1670 1675 1680Thr Asp Ala Gly Ser Val Pro His Val Asn Arg Asn Gly Phe Asn 1685 1690 1695Arg Ser Tyr Ala Gly Lys Asn Ala Val Ala Tyr Gly Lys Gly Thr 1700 1705 1710Tyr Phe Ala Val Asn Ala Asn Tyr Ser Ala Asn Asp Thr Tyr Ser 1715 1720 1725Arg Pro Asp Ala Asn Gly Arg Lys His Val Tyr Tyr Val Arg Val 1730 1735 1740Leu Thr Gly Ile Tyr Thr His Gly Asn His Ser Leu Ile Val Pro 1745 1750 1755Pro Ser Lys Asn Pro Gln Asn Pro Thr Asp Leu Tyr Asp Thr Val 1760 1765 1770Thr Asp Asn Val His His Pro Ser Leu Phe Val Ala Phe Tyr Asp 1775 1780 1785Tyr Gln Ala Tyr Pro Glu Tyr Leu Ile Thr Phe Arg Lys 1790 1795 18005444PRTHomo sapiens 5Met Leu Gln Arg Ile Gly Leu Ile Phe Leu His Asn Ile Val Val Val1 5 10 15Ser Asn Cys Phe Tyr Phe Gln Ala Phe Leu Asp Glu Phe Thr Asn Trp 20 25 30Ser Arg Ile Asn Pro Asn Lys Ala Arg Ile Pro Met Ala Gly Asp Thr 35 40 45Gln Gly Val Val Gly Thr Val Ser Lys Pro Cys Phe Thr Ala Tyr Glu 50 55 60Met Lys Ile Gly Ala Ile Thr Phe Gln Val Ala Thr Gly Asp Ile Ala65 70 75 80Thr Glu Gln Val Asp Val Ile Val Asn Ser Thr Ala Arg Thr Phe Asn 85 90 95Arg Lys Ser Gly Val Ser Arg Ala Ile Leu Glu Gly Ala Gly Gln Ala 100 105 110Val Glu Ser Glu Cys Ala Val Leu Ala Ala Gln Pro His Arg Asp Phe 115 120 125Ile Ile Thr Pro Gly Gly Cys Leu Lys Cys Lys Ile Ile Ile His Val 130 135 140Pro Gly Gly Lys Asp Val Arg Lys Thr Val Thr Ser Val Leu Glu Glu145 150 155 160Cys Glu Gln Arg Lys Tyr Thr Ser Val Ser Leu Pro Ala Ile Gly Thr 165 170 175Gly Asn Ala Gly Lys Asn Pro Ile Thr Val Ala Asp Asn Ile Ile Asp 180 185 190Ala Ile Val Asp Phe Ser Ser Gln His Ser Thr Pro Ser Leu Lys Thr 195 200 205Val Lys Val Val Ile Phe Gln Pro Glu Leu Leu Asn Ile Phe Tyr Asp 210 215 220Ser Met Lys Lys Arg Asp Leu Ser Ala Ser Leu Asn Phe Gln Ser Thr225 230 235 240Phe Ser Met Thr Thr Cys Asn Leu Pro Glu His Trp Thr Asp Met Asn 245 250 255His Gln Leu Phe Cys Met Val Gln Leu Glu Pro Gly Gln Ser Glu Tyr 260 265 270Asn Thr Ile Lys Asp Lys Phe Thr Arg Thr Cys Ser Ser Tyr Ala Ile 275 280 285Glu Lys Ile Glu Arg Ile Gln Asn Ala Phe Leu Trp Gln Ser Tyr Gln 290 295 300Val Lys Lys Arg Gln Met Asp Ile Lys Asn Asp His Lys Asn Asn Glu305 310 315 320Arg Leu Leu Phe His Gly Thr Asp Ala Asp Ser Val Pro Tyr Val Asn 325 330 335Gln His Gly Phe Asn Arg Ser Cys Ala Gly Lys Asn Ala Val Ser Tyr 340 345 350Gly Lys Gly Thr Tyr Phe Ala Val Asp Ala Ser Tyr Ser Ala Lys Asp 355 360 365Thr Tyr Ser Lys Pro Asp Ser Asn Gly Arg Lys His Met Tyr Val Val 370 375 380Arg Val Leu Thr Gly Val Phe Thr Lys Gly Arg Ala Gly Leu Val Thr385 390 395 400Pro Pro Pro Lys Asn Pro His Asn Pro Thr Asp Leu Phe Asp Ser Val 405 410 415Thr Asn Asn Thr Arg Ser Pro Lys Leu Phe Val Val Phe Phe Asp Asn 420 425 430Gln Ala Tyr Pro Glu Tyr Leu Ile Thr Phe Thr Ala 435 4406323PRTHomo sapiens 6Met Gln Pro Ser Gly Trp Ala Ala Ala Arg Glu Ala Ala Gly Arg Asp1 5 10 15Met Leu Ala Ala Asp Leu Arg Cys Ser Leu Phe Ala Ser Ala Leu Gln 20 25 30Ser Tyr Lys Arg Asp Ser Val Leu Arg Pro Phe Pro Ala Ser Tyr Ala 35 40 45Arg Gly Asp Cys Lys Asp Phe Glu Ala Leu Leu Ala Asp Ala Ser Lys 50 55 60Leu Pro Asn Leu Lys Glu Leu Leu Gln Ser Ser Gly Asp Asn His Lys65 70 75 80Arg Ala Trp Asp Leu Val Ser Trp Ile Leu Ser Ser Lys Val Leu Thr 85 90 95Ile His Ser Ala Gly Lys Ala Glu Phe Glu Lys Ile Gln Lys Leu Thr 100 105 110Gly Ala Pro His Thr Pro Val Pro Ala Pro Asp Phe Leu Phe Glu Ile 115 120 125Glu Tyr Phe Asp Pro Ala Asn Ala Lys Phe Tyr Glu Thr Lys Gly Glu 130 135 140Arg Asp Leu Ile Tyr Ala Phe His Gly Ser Arg Leu Glu Asn Phe His145 150 155 160Ser Ile Ile His Asn Gly Leu His Cys His Leu Asn Lys Thr Ser Leu 165 170 175Phe Gly Glu Gly Thr Tyr Leu Thr Ser Asp Leu Ser Leu Ala Leu Ile 180 185 190Tyr Ser Pro His Gly His Gly Trp Gln His Ser Leu Leu Gly Pro Ile 195 200 205Leu Ser Cys Val Ala Val Cys Glu Val Ile Asp His Pro Asp Val Lys 210 215 220Cys Gln Thr Lys Lys Lys Asp Ser Lys Glu Ile Asp Arg Arg Arg Ala225 230 235 240Arg Ile Lys His Ser Glu Gly Gly Asp Ile Pro Pro Lys Tyr Phe Val 245 250 255Val Thr Asn Asn Gln Leu Leu Arg Val Lys Tyr Leu Leu Val Tyr Ser 260 265 270Gln Lys Pro Pro Lys Ser Arg Ala Ser Ser Gln Leu Ser Trp Phe Ser 275 280 285Ser His Trp Phe Thr Val Met Ile Ser

Leu Tyr Leu Leu Leu Leu Leu 290 295 300Ile Val Ser Val Ile Asn Ser Ser Ala Phe Gln His Phe Trp Asn Arg305 310 315 320Ala Lys Arg739PRTArtificial sequenceFusion tag 7Met His His His His His His Ser Ser Gly Val Asp Leu Gly Thr Glu1 5 10 15Asn Leu Tyr Phe Gln Ser Asn Ala Gly Leu Asn Asp Ile Phe Glu Ala 20 25 30Gln Lys Ile Glu Trp His Glu 35816PRTArtificial sequenceFusion tag 8Met Ala His His His His His His Glu Asn Leu Tyr Phe Gln Ser Met1 5 10 15924PRTArtificial sequenceFusion tag 9Met His His His His His His Ser Ser Gly Val Asp Leu Gly Thr Glu1 5 10 15Asn Leu Tyr Phe Gln Ser Asn Ala 201024PRTArtificial sequenceFusion tag 10Met Ala His His His His His His Ser Ser Gly Val Asp Leu Gly Thr1 5 10 15Glu Asn Leu Tyr Phe Gln Ser Met 2011171PRTArtificial sequenceNanoLuc 11Met Val Phe Thr Leu Glu Asp Phe Val Gly Asp Trp Arg Gln Thr Ala1 5 10 15Gly Tyr Asn Leu Asp Gln Val Leu Glu Gln Gly Gly Val Ser Ser Leu 20 25 30Phe Gln Asn Leu Gly Val Ser Val Thr Pro Ile Gln Arg Ile Val Leu 35 40 45Ser Gly Glu Asn Gly Leu Lys Ile Asp Ile His Val Ile Ile Pro Tyr 50 55 60Glu Gly Leu Ser Gly Asp Gln Met Gly Gln Ile Glu Lys Ile Phe Lys65 70 75 80Val Val Tyr Pro Val Asp Asp His His Phe Lys Val Ile Leu His Tyr 85 90 95Gly Thr Leu Val Ile Asp Gly Val Thr Pro Asn Met Ile Asp Tyr Phe 100 105 110Gly Arg Pro Tyr Glu Gly Ile Ala Val Phe Asp Gly Lys Lys Ile Thr 115 120 125Val Thr Gly Thr Leu Trp Asn Gly Asn Lys Ile Ile Asp Glu Arg Leu 130 135 140Ile Asn Pro Asp Gly Ser Leu Leu Phe Arg Val Thr Ile Asn Gly Val145 150 155 160Thr Gly Trp Arg Leu Cys Glu Arg Ile Leu Ala 165 17012516DNAArtificial sequenceNanoLuc 12atggtcttca cactcgaaga tttcgttggg gactggcgac agacagccgg ctacaacctg 60gaccaagtcc ttgaacaggg aggtgtgtcc agtttgtttc agaatctcgg ggtgtccgta 120actccgatcc aaaggattgt cctgagcggt gaaaatgggc tgaagatcga catccatgtc 180atcatcccgt atgaaggtct gagcggcgac caaatgggcc agatcgaaaa aatttttaag 240gtggtgtacc ctgtggatga tcatcacttt aaggtgatcc tgcactatgg cacactggta 300atcgacgggg ttacgccgaa catgatcgac tatttcggac ggccgtatga aggcatcgcc 360gtgttcgacg gcaaaaagat cactgtaaca gggaccctgt ggaacggcaa caaaattatc 420gacgagcgcc tgatcaaccc cgacggctcc ctgctgttcc gagtaaccat caacggagtg 480accggctggc ggctgtgcga acgcattctg gcgtaa 516131014PRTHomo sapiens 13Met Ala Glu Ser Ser Asp Lys Leu Tyr Arg Val Glu Tyr Ala Lys Ser1 5 10 15Gly Arg Ala Ser Cys Lys Lys Cys Ser Glu Ser Ile Pro Lys Asp Ser 20 25 30Leu Arg Met Ala Ile Met Val Gln Ser Pro Met Phe Asp Gly Lys Val 35 40 45Pro His Trp Tyr His Phe Ser Cys Phe Trp Lys Val Gly His Ser Ile 50 55 60Arg His Pro Asp Val Glu Val Asp Gly Phe Ser Glu Leu Arg Trp Asp65 70 75 80Asp Gln Gln Lys Val Lys Lys Thr Ala Glu Ala Gly Gly Val Thr Gly 85 90 95Lys Gly Gln Asp Gly Ile Gly Ser Lys Ala Glu Lys Thr Leu Gly Asp 100 105 110Phe Ala Ala Glu Tyr Ala Lys Ser Asn Arg Ser Thr Cys Lys Gly Cys 115 120 125Met Glu Lys Ile Glu Lys Gly Gln Val Arg Leu Ser Lys Lys Met Val 130 135 140Asp Pro Glu Lys Pro Gln Leu Gly Met Ile Asp Arg Trp Tyr His Pro145 150 155 160Gly Cys Phe Val Lys Asn Arg Glu Glu Leu Gly Phe Arg Pro Glu Tyr 165 170 175Ser Ala Ser Gln Leu Lys Gly Phe Ser Leu Leu Ala Thr Glu Asp Lys 180 185 190Glu Ala Leu Lys Lys Gln Leu Pro Gly Val Lys Ser Glu Gly Lys Arg 195 200 205Lys Gly Asp Glu Val Asp Gly Val Asp Glu Val Ala Lys Lys Lys Ser 210 215 220Lys Lys Glu Lys Asp Lys Asp Ser Lys Leu Glu Lys Ala Leu Lys Ala225 230 235 240Gln Asn Asp Leu Ile Trp Asn Ile Lys Asp Glu Leu Lys Lys Val Cys 245 250 255Ser Thr Asn Asp Leu Lys Glu Leu Leu Ile Phe Asn Lys Gln Gln Val 260 265 270Pro Ser Gly Glu Ser Ala Ile Leu Asp Arg Val Ala Asp Gly Met Val 275 280 285Phe Gly Ala Leu Leu Pro Cys Glu Glu Cys Ser Gly Gln Leu Val Phe 290 295 300Lys Ser Asp Ala Tyr Tyr Cys Thr Gly Asp Val Thr Ala Trp Thr Lys305 310 315 320Cys Met Val Lys Thr Gln Thr Pro Asn Arg Lys Glu Trp Val Thr Pro 325 330 335Lys Glu Phe Arg Glu Ile Ser Tyr Leu Lys Lys Leu Lys Val Lys Lys 340 345 350Gln Asp Arg Ile Phe Pro Pro Glu Thr Ser Ala Ser Val Ala Ala Thr 355 360 365Pro Pro Pro Ser Thr Ala Ser Ala Pro Ala Ala Val Asn Ser Ser Ala 370 375 380Ser Ala Asp Lys Pro Leu Ser Asn Met Lys Ile Leu Thr Leu Gly Lys385 390 395 400Leu Ser Arg Asn Lys Asp Glu Val Lys Ala Met Ile Glu Lys Leu Gly 405 410 415Gly Lys Leu Thr Gly Thr Ala Asn Lys Ala Ser Leu Cys Ile Ser Thr 420 425 430Lys Lys Glu Val Glu Lys Met Asn Lys Lys Met Glu Glu Val Lys Glu 435 440 445Ala Asn Ile Arg Val Val Ser Glu Asp Phe Leu Gln Asp Val Ser Ala 450 455 460Ser Thr Lys Ser Leu Gln Glu Leu Phe Leu Ala His Ile Leu Ser Pro465 470 475 480Trp Gly Ala Glu Val Lys Ala Glu Pro Val Glu Val Val Ala Pro Arg 485 490 495Gly Lys Ser Gly Ala Ala Leu Ser Lys Lys Ser Lys Gly Gln Val Lys 500 505 510Glu Glu Gly Ile Asn Lys Ser Glu Lys Arg Met Lys Leu Thr Leu Lys 515 520 525Gly Gly Ala Ala Val Asp Pro Asp Ser Gly Leu Glu His Ser Ala His 530 535 540Val Leu Glu Lys Gly Gly Lys Val Phe Ser Ala Thr Leu Gly Leu Val545 550 555 560Asp Ile Val Lys Gly Thr Asn Ser Tyr Tyr Lys Leu Gln Leu Leu Glu 565 570 575Asp Asp Lys Glu Asn Arg Tyr Trp Ile Phe Arg Ser Trp Gly Arg Val 580 585 590Gly Thr Val Ile Gly Ser Asn Lys Leu Glu Gln Met Pro Ser Lys Glu 595 600 605Asp Ala Ile Glu His Phe Met Lys Leu Tyr Glu Glu Lys Thr Gly Asn 610 615 620Ala Trp His Ser Lys Asn Phe Thr Lys Tyr Pro Lys Lys Phe Tyr Pro625 630 635 640Leu Glu Ile Asp Tyr Gly Gln Asp Glu Glu Ala Val Lys Lys Leu Thr 645 650 655Val Asn Pro Gly Thr Lys Ser Lys Leu Pro Lys Pro Val Gln Asp Leu 660 665 670Ile Lys Met Ile Phe Asp Val Glu Ser Met Lys Lys Ala Met Val Glu 675 680 685Tyr Glu Ile Asp Leu Gln Lys Met Pro Leu Gly Lys Leu Ser Lys Arg 690 695 700Gln Ile Gln Ala Ala Tyr Ser Ile Leu Ser Glu Val Gln Gln Ala Val705 710 715 720Ser Gln Gly Ser Ser Asp Ser Gln Ile Leu Asp Leu Ser Asn Arg Phe 725 730 735Tyr Thr Leu Ile Pro His Asp Phe Gly Met Lys Lys Pro Pro Leu Leu 740 745 750Asn Asn Ala Asp Ser Val Gln Ala Lys Val Glu Met Leu Asp Asn Leu 755 760 765Leu Asp Ile Glu Val Ala Tyr Ser Leu Leu Arg Gly Gly Ser Asp Asp 770 775 780Ser Ser Lys Asp Pro Ile Asp Val Asn Tyr Glu Lys Leu Lys Thr Asp785 790 795 800Ile Lys Val Val Asp Arg Asp Ser Glu Glu Ala Glu Ile Ile Arg Lys 805 810 815Tyr Val Lys Asn Thr His Ala Thr Thr His Asn Ala Tyr Asp Leu Glu 820 825 830Val Ile Asp Ile Phe Lys Ile Glu Arg Glu Gly Glu Cys Gln Arg Tyr 835 840 845Lys Pro Phe Lys Gln Leu His Asn Arg Arg Leu Leu Trp His Gly Ser 850 855 860Arg Thr Thr Asn Phe Ala Gly Ile Leu Ser Gln Gly Leu Arg Ile Ala865 870 875 880Pro Pro Glu Ala Pro Val Thr Gly Tyr Met Phe Gly Lys Gly Ile Tyr 885 890 895Phe Ala Asp Met Val Ser Lys Ser Ala Asn Tyr Cys His Thr Ser Gln 900 905 910Gly Asp Pro Ile Gly Leu Ile Leu Leu Gly Glu Val Ala Leu Gly Asn 915 920 925Met Tyr Glu Leu Lys His Ala Ser His Ile Ser Lys Leu Pro Lys Gly 930 935 940Lys His Ser Val Lys Gly Leu Gly Lys Thr Thr Pro Asp Pro Ser Ala945 950 955 960Asn Ile Ser Leu Asp Gly Val Asp Val Pro Leu Gly Thr Gly Ile Ser 965 970 975Ser Gly Val Asn Asp Thr Ser Leu Leu Tyr Asn Glu Tyr Ile Val Tyr 980 985 990Asp Ile Ala Gln Val Asn Leu Lys Tyr Leu Leu Lys Leu Lys Phe Asn 995 1000 1005Phe Lys Thr Ser Leu Trp 101014583PRTHomo sapiens 14Met Ala Ala Arg Arg Arg Arg Ser Thr Gly Gly Gly Arg Ala Arg Ala1 5 10 15Leu Asn Glu Ser Lys Arg Val Asn Asn Gly Asn Thr Ala Pro Glu Asp 20 25 30Ser Ser Pro Ala Lys Lys Thr Arg Arg Cys Gln Arg Gln Glu Ser Lys 35 40 45Lys Met Pro Val Ala Gly Gly Lys Ala Asn Lys Asp Arg Thr Glu Asp 50 55 60Lys Gln Asp Gly Met Pro Gly Arg Ser Trp Ala Ser Lys Arg Val Ser65 70 75 80Glu Ser Val Lys Ala Leu Leu Leu Lys Gly Lys Ala Pro Val Asp Pro 85 90 95Glu Cys Thr Ala Lys Val Gly Lys Ala His Val Tyr Cys Glu Gly Asn 100 105 110Asp Val Tyr Asp Val Met Leu Asn Gln Thr Asn Leu Gln Phe Asn Asn 115 120 125Asn Lys Tyr Tyr Leu Ile Gln Leu Leu Glu Asp Asp Ala Gln Arg Asn 130 135 140Phe Ser Val Trp Met Arg Trp Gly Arg Val Gly Lys Met Gly Gln His145 150 155 160Ser Leu Val Ala Cys Ser Gly Asn Leu Asn Lys Ala Lys Glu Ile Phe 165 170 175Gln Lys Lys Phe Leu Asp Lys Thr Lys Asn Asn Trp Glu Asp Arg Glu 180 185 190Lys Phe Glu Lys Val Pro Gly Lys Tyr Asp Met Leu Gln Met Asp Tyr 195 200 205Ala Thr Asn Thr Gln Asp Glu Glu Glu Thr Lys Lys Glu Glu Ser Leu 210 215 220Lys Ser Pro Leu Lys Pro Glu Ser Gln Leu Asp Leu Arg Val Gln Glu225 230 235 240Leu Ile Lys Leu Ile Cys Asn Val Gln Ala Met Glu Glu Met Met Met 245 250 255Glu Met Lys Tyr Asn Thr Lys Lys Ala Pro Leu Gly Lys Leu Thr Val 260 265 270Ala Gln Ile Lys Ala Gly Tyr Gln Ser Leu Lys Lys Ile Glu Asp Cys 275 280 285Ile Arg Ala Gly Gln His Gly Arg Ala Leu Met Glu Ala Cys Asn Glu 290 295 300Phe Tyr Thr Arg Ile Pro His Asp Phe Gly Leu Arg Thr Pro Pro Leu305 310 315 320Ile Arg Thr Gln Lys Glu Leu Ser Glu Lys Ile Gln Leu Leu Glu Ala 325 330 335Leu Gly Asp Ile Glu Ile Ala Ile Lys Leu Val Lys Thr Glu Leu Gln 340 345 350Ser Pro Glu His Pro Leu Asp Gln His Tyr Arg Asn Leu His Cys Ala 355 360 365Leu Arg Pro Leu Asp His Glu Ser Tyr Glu Phe Lys Val Ile Ser Gln 370 375 380Tyr Leu Gln Ser Thr His Ala Pro Thr His Ser Asp Tyr Thr Met Thr385 390 395 400Leu Leu Asp Leu Phe Glu Val Glu Lys Asp Gly Glu Lys Glu Ala Phe 405 410 415Arg Glu Asp Leu His Asn Arg Met Leu Leu Trp His Gly Ser Arg Met 420 425 430Ser Asn Trp Val Gly Ile Leu Ser His Gly Leu Arg Ile Ala Pro Pro 435 440 445Glu Ala Pro Ile Thr Gly Tyr Met Phe Gly Lys Gly Ile Tyr Phe Ala 450 455 460Asp Met Ser Ser Lys Ser Ala Asn Tyr Cys Phe Ala Ser Arg Leu Lys465 470 475 480Asn Thr Gly Leu Leu Leu Leu Ser Glu Val Ala Leu Gly Gln Cys Asn 485 490 495Glu Leu Leu Glu Ala Asn Pro Lys Ala Glu Gly Leu Leu Gln Gly Lys 500 505 510His Ser Thr Lys Gly Leu Gly Lys Met Ala Pro Ser Ser Ala His Phe 515 520 525Val Thr Leu Asn Gly Ser Thr Val Pro Leu Gly Pro Ala Ser Asp Thr 530 535 540Gly Ile Leu Asn Pro Asp Gly Tyr Thr Leu Asn Tyr Asn Glu Tyr Ile545 550 555 560Val Tyr Asn Pro Asn Gln Val Arg Met Arg Tyr Leu Leu Lys Val Gln 565 570 575Phe Asn Phe Leu Gln Leu Trp 58015540PRTHomo sapiens 15Met Ser Leu Leu Phe Leu Ala Met Ala Pro Lys Pro Lys Pro Trp Val1 5 10 15Gln Thr Glu Gly Pro Glu Lys Lys Lys Gly Arg Gln Ala Gly Arg Glu 20 25 30Glu Asp Pro Phe Arg Ser Thr Ala Glu Ala Leu Lys Ala Ile Pro Ala 35 40 45Glu Lys Arg Ile Ile Arg Val Asp Pro Thr Cys Pro Leu Ser Ser Asn 50 55 60Pro Gly Thr Gln Val Tyr Glu Asp Tyr Asn Cys Thr Leu Asn Gln Thr65 70 75 80Asn Ile Glu Asn Asn Asn Asn Lys Phe Tyr Ile Ile Gln Leu Leu Gln 85 90 95Asp Ser Asn Arg Phe Phe Thr Cys Trp Asn Arg Trp Gly Arg Val Gly 100 105 110Glu Val Gly Gln Ser Lys Ile Asn His Phe Thr Arg Leu Glu Asp Ala 115 120 125Lys Lys Asp Phe Glu Lys Lys Phe Arg Glu Lys Thr Lys Asn Asn Trp 130 135 140Ala Glu Arg Asp His Phe Val Ser His Pro Gly Lys Tyr Thr Leu Ile145 150 155 160Glu Val Gln Ala Glu Asp Glu Ala Gln Glu Ala Val Val Lys Val Asp 165 170 175Arg Gly Pro Val Arg Thr Val Thr Lys Arg Val Gln Pro Cys Ser Leu 180 185 190Asp Pro Ala Thr Gln Lys Leu Ile Thr Asn Ile Phe Ser Lys Glu Met 195 200 205Phe Lys Asn Thr Met Ala Leu Met Asp Leu Asp Val Lys Lys Met Pro 210 215 220Leu Gly Lys Leu Ser Lys Gln Gln Ile Ala Arg Gly Phe Glu Ala Leu225 230 235 240Glu Ala Leu Glu Glu Ala Leu Lys Gly Pro Thr Asp Gly Gly Gln Ser 245 250 255Leu Glu Glu Leu Ser Ser His Phe Tyr Thr Val Ile Pro His Asn Phe 260 265 270Gly His Ser Gln Pro Pro Pro Ile Asn Ser Pro Glu Leu Leu Gln Ala 275 280 285Lys Lys Asp Met Leu Leu Val Leu Ala Asp Ile Glu Leu Ala Gln Ala 290 295 300Leu Gln Ala Val Ser Glu Gln Glu Lys Thr Val Glu Glu Val Pro His305 310 315 320Pro Leu Asp Arg Asp Tyr Gln Leu Leu Lys Cys Gln Leu Gln Leu Leu 325 330 335Asp Ser Gly Ala Pro Glu Tyr Lys Val Ile Gln Thr Tyr Leu Glu Gln 340 345 350Thr Gly Ser Asn His Arg Cys Pro Thr Leu Gln His Ile Trp Lys Val 355 360 365Asn Gln Glu Gly Glu Glu Asp Arg Phe Gln Ala His Ser Lys Leu Gly 370 375 380Asn Arg Lys Leu Leu Trp His Gly Thr Asn Met Ala Val Val Ala Ala385 390 395 400Ile Leu Thr Ser Gly Leu Arg Ile Met Pro His Ser Gly Gly Arg Val 405 410 415Gly Lys Gly Ile Tyr Phe Ala Ser Glu Asn Ser Lys Ser Ala Gly Tyr 420 425 430Val Ile Gly Met Lys Cys Gly Ala His His Val Gly Tyr Met Phe Leu 435 440 445Gly Glu Val Ala Leu Gly Arg Glu His His Ile Asn

Thr Asp Asn Pro 450 455 460Ser Leu Lys Ser Pro Pro Pro Gly Phe Asp Ser Val Ile Ala Arg Gly465 470 475 480His Thr Glu Pro Asp Pro Thr Gln Asp Thr Glu Leu Glu Leu Asp Gly 485 490 495Gln Gln Val Val Val Pro Gln Gly Gln Pro Val Pro Cys Pro Glu Phe 500 505 510Ser Ser Ser Thr Phe Ser Gln Ser Glu Tyr Leu Ile Tyr Gln Glu Ser 515 520 525Gln Cys Arg Leu Arg Tyr Leu Leu Glu Val His Leu 530 535 540161724PRTHomo sapiens 16Met Val Met Gly Ile Phe Ala Asn Cys Ile Phe Cys Leu Lys Val Lys1 5 10 15Tyr Leu Pro Gln Gln Gln Lys Lys Lys Leu Gln Thr Asp Ile Lys Glu 20 25 30Asn Gly Gly Lys Phe Ser Phe Ser Leu Asn Pro Gln Cys Thr His Ile 35 40 45Ile Leu Asp Asn Ala Asp Val Leu Ser Gln Tyr Gln Leu Asn Ser Ile 50 55 60Gln Lys Asn His Val His Ile Ala Asn Pro Asp Phe Ile Trp Lys Ser65 70 75 80Ile Arg Glu Lys Arg Leu Leu Asp Val Lys Asn Tyr Asp Pro Tyr Lys 85 90 95Pro Leu Asp Ile Thr Pro Pro Pro Asp Gln Lys Ala Ser Ser Ser Glu 100 105 110Val Lys Thr Glu Gly Leu Cys Pro Asp Ser Ala Thr Glu Glu Glu Asp 115 120 125Thr Val Glu Leu Thr Glu Phe Gly Met Gln Asn Val Glu Ile Pro His 130 135 140Leu Pro Gln Asp Phe Glu Val Ala Lys Tyr Asn Thr Leu Glu Lys Val145 150 155 160Gly Met Glu Gly Gly Gln Glu Ala Val Val Val Glu Leu Gln Cys Ser 165 170 175Arg Asp Ser Arg Asp Cys Pro Phe Leu Ile Ser Ser His Phe Leu Leu 180 185 190Asp Asp Gly Met Glu Thr Arg Arg Gln Phe Ala Ile Lys Lys Thr Ser 195 200 205Glu Asp Ala Ser Glu Tyr Phe Glu Asn Tyr Ile Glu Glu Leu Lys Lys 210 215 220Gln Gly Phe Leu Leu Arg Glu His Phe Thr Pro Glu Ala Thr Gln Leu225 230 235 240Ala Ser Glu Gln Leu Gln Ala Leu Leu Leu Glu Glu Val Met Asn Ser 245 250 255Ser Thr Leu Ser Gln Glu Val Ser Asp Leu Val Glu Met Ile Trp Ala 260 265 270Glu Ala Leu Gly His Leu Glu His Met Leu Leu Lys Pro Val Asn Arg 275 280 285Ile Ser Leu Asn Asp Val Ser Lys Ala Glu Gly Ile Leu Leu Leu Val 290 295 300Lys Ala Ala Leu Lys Asn Gly Glu Thr Ala Glu Gln Leu Gln Lys Met305 310 315 320Met Thr Glu Phe Tyr Arg Leu Ile Pro His Lys Gly Thr Met Pro Lys 325 330 335Glu Val Asn Leu Gly Leu Leu Ala Lys Lys Ala Asp Leu Cys Gln Leu 340 345 350Ile Arg Asp Met Val Asn Val Cys Glu Thr Asn Leu Ser Lys Pro Asn 355 360 365Pro Pro Ser Leu Ala Lys Tyr Arg Ala Leu Arg Cys Lys Ile Glu His 370 375 380Val Glu Gln Asn Thr Glu Glu Phe Leu Arg Val Arg Lys Glu Val Leu385 390 395 400Gln Asn His His Ser Lys Ser Pro Val Asp Val Leu Gln Ile Phe Arg 405 410 415Val Gly Arg Val Asn Glu Thr Thr Glu Phe Leu Ser Lys Leu Gly Asn 420 425 430Val Arg Pro Leu Leu His Gly Ser Pro Val Gln Asn Ile Val Gly Ile 435 440 445Leu Cys Arg Gly Leu Leu Leu Pro Lys Val Val Glu Asp Arg Gly Val 450 455 460Gln Arg Thr Asp Val Gly Asn Leu Gly Ser Gly Ile Tyr Phe Ser Asp465 470 475 480Ser Leu Ser Thr Ser Ile Lys Tyr Ser His Pro Gly Glu Thr Asp Gly 485 490 495Thr Arg Leu Leu Leu Ile Cys Asp Val Ala Leu Gly Lys Cys Met Asp 500 505 510Leu His Glu Lys Asp Phe Ser Leu Thr Glu Ala Pro Pro Gly Tyr Asp 515 520 525Ser Val His Gly Val Ser Gln Thr Ala Ser Val Thr Thr Asp Phe Glu 530 535 540Asp Asp Glu Phe Val Val Tyr Lys Thr Asn Gln Val Lys Met Lys Tyr545 550 555 560Ile Ile Lys Phe Ser Met Pro Gly Asp Gln Ile Lys Asp Phe His Pro 565 570 575Ser Asp His Thr Glu Leu Glu Glu Tyr Arg Pro Glu Phe Ser Asn Phe 580 585 590Ser Lys Val Glu Asp Tyr Gln Leu Pro Asp Ala Lys Thr Ser Ser Ser 595 600 605Thr Lys Ala Gly Leu Gln Asp Ala Ser Gly Asn Leu Val Pro Leu Glu 610 615 620Asp Val His Ile Lys Gly Arg Ile Ile Asp Thr Val Ala Gln Val Ile625 630 635 640Val Phe Gln Thr Tyr Thr Asn Lys Ser His Val Pro Ile Glu Ala Lys 645 650 655Tyr Ile Phe Pro Leu Asp Asp Lys Ala Ala Val Cys Gly Phe Glu Ala 660 665 670Phe Ile Asn Gly Lys His Ile Val Gly Glu Ile Lys Glu Lys Glu Glu 675 680 685Ala Gln Gln Glu Tyr Leu Glu Ala Val Thr Gln Gly His Gly Ala Tyr 690 695 700Leu Met Ser Gln Asp Ala Pro Asp Val Phe Thr Val Ser Val Gly Asn705 710 715 720Leu Pro Pro Lys Ala Lys Val Leu Ile Lys Ile Thr Tyr Ile Thr Glu 725 730 735Leu Ser Ile Leu Gly Thr Val Gly Val Phe Phe Met Pro Ala Thr Val 740 745 750Ala Pro Trp Gln Gln Asp Lys Ala Leu Asn Glu Asn Leu Gln Asp Thr 755 760 765Val Glu Lys Ile Cys Ile Lys Glu Ile Gly Thr Lys Gln Ser Phe Ser 770 775 780Leu Thr Met Ser Ile Glu Met Pro Tyr Val Ile Glu Phe Ile Phe Ser785 790 795 800Asp Thr His Glu Leu Lys Gln Lys Arg Thr Asp Cys Lys Ala Val Ile 805 810 815Ser Thr Met Glu Gly Ser Ser Leu Asp Ser Ser Gly Phe Ser Leu His 820 825 830Ile Gly Leu Ser Ala Ala Tyr Leu Pro Arg Met Trp Val Glu Lys His 835 840 845Pro Glu Lys Glu Ser Glu Ala Cys Met Leu Val Phe Gln Pro Asp Leu 850 855 860Asp Val Asp Leu Pro Asp Leu Ala Ser Glu Ser Glu Val Ile Ile Cys865 870 875 880Leu Asp Cys Ser Ser Ser Met Glu Gly Val Thr Phe Leu Gln Ala Lys 885 890 895Gln Ile Ala Leu His Ala Leu Ser Leu Val Gly Glu Lys Gln Lys Val 900 905 910Asn Ile Ile Gln Phe Gly Thr Gly Tyr Lys Glu Leu Phe Ser Tyr Pro 915 920 925Lys His Ile Thr Ser Asn Thr Met Ala Ala Glu Phe Ile Met Ser Ala 930 935 940Thr Pro Thr Met Gly Asn Thr Asp Phe Trp Lys Thr Leu Arg Tyr Leu945 950 955 960Ser Leu Leu Tyr Pro Ala Arg Gly Ser Arg Asn Ile Leu Leu Val Ser 965 970 975Asp Gly His Leu Gln Asp Glu Ser Leu Thr Leu Gln Leu Val Lys Arg 980 985 990Ser Arg Pro His Thr Arg Leu Phe Ala Cys Gly Ile Gly Ser Thr Ala 995 1000 1005Asn Arg His Val Leu Arg Ile Leu Ser Gln Cys Gly Ala Gly Val 1010 1015 1020Phe Glu Tyr Phe Asn Ala Lys Ser Lys His Ser Trp Arg Lys Gln 1025 1030 1035Ile Glu Asp Gln Met Thr Arg Leu Cys Ser Pro Ser Cys His Ser 1040 1045 1050Val Ser Val Lys Trp Gln Gln Leu Asn Pro Asp Val Pro Glu Ala 1055 1060 1065Leu Gln Ala Pro Ala Gln Val Pro Ser Leu Phe Leu Asn Asp Arg 1070 1075 1080Leu Leu Val Tyr Gly Phe Ile Pro His Cys Thr Gln Ala Thr Leu 1085 1090 1095Cys Ala Leu Ile Gln Glu Lys Glu Phe Arg Thr Met Val Ser Thr 1100 1105 1110Thr Glu Leu Gln Lys Thr Thr Gly Thr Met Ile His Lys Leu Ala 1115 1120 1125Ala Arg Ala Leu Ile Arg Asp Tyr Glu Asp Gly Ile Leu His Glu 1130 1135 1140Asn Glu Thr Ser His Glu Met Lys Lys Gln Thr Leu Lys Ser Leu 1145 1150 1155Ile Ile Lys Leu Ser Lys Glu Asn Ser Leu Ile Thr Gln Phe Thr 1160 1165 1170Ser Phe Val Ala Val Glu Lys Arg Asp Glu Asn Glu Ser Pro Phe 1175 1180 1185Pro Asp Ile Pro Lys Val Ser Glu Leu Ile Ala Lys Glu Asp Val 1190 1195 1200Asp Phe Leu Pro Tyr Met Ser Trp Gln Gly Glu Pro Gln Glu Ala 1205 1210 1215Val Arg Asn Gln Ser Leu Leu Ala Ser Ser Glu Trp Pro Glu Leu 1220 1225 1230Arg Leu Ser Lys Arg Lys His Arg Lys Ile Pro Phe Ser Lys Arg 1235 1240 1245Lys Met Glu Leu Ser Gln Pro Glu Val Ser Glu Asp Phe Glu Glu 1250 1255 1260Asp Gly Leu Gly Val Leu Pro Ala Phe Thr Ser Asn Leu Glu Arg 1265 1270 1275Gly Gly Val Glu Lys Leu Leu Asp Leu Ser Trp Thr Glu Ser Cys 1280 1285 1290Lys Pro Thr Ala Thr Glu Pro Leu Phe Lys Lys Val Ser Pro Trp 1295 1300 1305Glu Thr Ser Thr Ser Ser Phe Phe Pro Ile Leu Ala Pro Ala Val 1310 1315 1320Gly Ser Tyr Leu Pro Pro Thr Ala Arg Ala His Ser Pro Ala Ser 1325 1330 1335Leu Ser Phe Ala Ser Tyr Arg Gln Val Ala Ser Phe Gly Ser Ala 1340 1345 1350Ala Pro Pro Arg Gln Phe Asp Ala Ser Gln Phe Ser Gln Gly Pro 1355 1360 1365Val Pro Gly Thr Cys Ala Asp Trp Ile Pro Gln Ser Ala Ser Cys 1370 1375 1380Pro Thr Gly Pro Pro Gln Asn Pro Pro Ser Ser Pro Tyr Cys Gly 1385 1390 1395Ile Val Phe Ser Gly Ser Ser Leu Ser Ser Ala Gln Ser Ala Pro 1400 1405 1410Leu Gln His Pro Gly Gly Phe Thr Thr Arg Pro Ser Ala Gly Thr 1415 1420 1425Phe Pro Glu Leu Asp Ser Pro Gln Leu His Phe Ser Leu Pro Thr 1430 1435 1440Asp Pro Asp Pro Ile Arg Gly Phe Gly Ser Tyr His Pro Ser Ala 1445 1450 1455Ser Ser Pro Phe His Phe Gln Pro Ser Ala Ala Ser Leu Thr Ala 1460 1465 1470Asn Leu Arg Leu Pro Met Ala Ser Ala Leu Pro Glu Ala Leu Cys 1475 1480 1485Ser Gln Ser Arg Thr Thr Pro Val Asp Leu Cys Leu Leu Glu Glu 1490 1495 1500Ser Val Gly Ser Leu Glu Gly Ser Arg Cys Pro Val Phe Ala Phe 1505 1510 1515Gln Ser Ser Asp Thr Glu Ser Asp Glu Leu Ser Glu Val Leu Gln 1520 1525 1530Asp Ser Cys Phe Leu Gln Ile Lys Cys Asp Thr Lys Asp Asp Ser 1535 1540 1545Ile Leu Cys Phe Leu Glu Val Lys Glu Glu Asp Glu Ile Val Cys 1550 1555 1560Ile Gln His Trp Gln Asp Ala Val Pro Trp Thr Glu Leu Leu Ser 1565 1570 1575Leu Gln Thr Glu Asp Gly Phe Trp Lys Leu Thr Pro Glu Leu Gly 1580 1585 1590Leu Ile Leu Asn Leu Asn Thr Asn Gly Leu His Ser Phe Leu Lys 1595 1600 1605Gln Lys Gly Ile Gln Ser Leu Gly Val Lys Gly Arg Glu Cys Leu 1610 1615 1620Leu Asp Leu Ile Ala Thr Met Leu Val Leu Gln Phe Ile Arg Thr 1625 1630 1635Arg Leu Glu Lys Glu Gly Ile Val Phe Lys Ser Leu Met Lys Met 1640 1645 1650Asp Asp Ala Ser Ile Ser Arg Asn Ile Pro Trp Ala Phe Glu Ala 1655 1660 1665Ile Lys Gln Ala Ser Glu Trp Val Arg Arg Thr Glu Gly Gln Tyr 1670 1675 1680Pro Ser Ile Cys Pro Arg Leu Glu Leu Gly Asn Asp Trp Asp Ser 1685 1690 1695Ala Thr Lys Gln Leu Leu Gly Leu Gln Pro Ile Ser Thr Val Ser 1700 1705 1710Pro Leu His Arg Val Leu His Tyr Ser Gln Gly 1715 1720171327PRTHomo sapiens 17Met Ala Ala Ser Arg Arg Ser Gln His His His His His His Gln Gln1 5 10 15Gln Leu Gln Pro Ala Pro Gly Ala Ser Ala Pro Pro Pro Pro Pro Pro 20 25 30Pro Pro Leu Ser Pro Gly Leu Ala Pro Gly Thr Thr Pro Ala Ser Pro 35 40 45Thr Ala Ser Gly Leu Ala Pro Phe Ala Ser Pro Arg His Gly Leu Ala 50 55 60Leu Pro Glu Gly Asp Gly Ser Arg Asp Pro Pro Asp Arg Pro Arg Ser65 70 75 80Pro Asp Pro Val Asp Gly Thr Ser Cys Cys Ser Thr Thr Ser Thr Ile 85 90 95Cys Thr Val Ala Ala Ala Pro Val Val Pro Ala Val Ser Thr Ser Ser 100 105 110Ala Ala Gly Val Ala Pro Asn Pro Ala Gly Ser Gly Ser Asn Asn Ser 115 120 125Pro Ser Ser Ser Ser Ser Pro Thr Ser Ser Ser Ser Ser Ser Pro Ser 130 135 140Ser Pro Gly Ser Ser Leu Ala Glu Ser Pro Glu Ala Ala Gly Val Ser145 150 155 160Ser Thr Ala Pro Leu Gly Pro Gly Ala Ala Gly Pro Gly Thr Gly Val 165 170 175Pro Ala Val Ser Gly Ala Leu Arg Glu Leu Leu Glu Ala Cys Arg Asn 180 185 190Gly Asp Val Ser Arg Val Lys Arg Leu Val Asp Ala Ala Asn Val Asn 195 200 205Ala Lys Asp Met Ala Gly Arg Lys Ser Ser Pro Leu His Phe Ala Ala 210 215 220Gly Phe Gly Arg Lys Asp Val Val Glu His Leu Leu Gln Met Gly Ala225 230 235 240Asn Val His Ala Arg Asp Asp Gly Gly Leu Ile Pro Leu His Asn Ala 245 250 255Cys Ser Phe Gly His Ala Glu Val Val Ser Leu Leu Leu Cys Gln Gly 260 265 270Ala Asp Pro Asn Ala Arg Asp Asn Trp Asn Tyr Thr Pro Leu His Glu 275 280 285Ala Ala Ile Lys Gly Lys Ile Asp Val Cys Ile Val Leu Leu Gln His 290 295 300Gly Ala Asp Pro Asn Ile Arg Asn Thr Asp Gly Lys Ser Ala Leu Asp305 310 315 320Leu Ala Asp Pro Ser Ala Lys Ala Val Leu Thr Gly Glu Tyr Lys Lys 325 330 335Asp Glu Leu Leu Glu Ala Ala Arg Ser Gly Asn Glu Glu Lys Leu Met 340 345 350Ala Leu Leu Thr Pro Leu Asn Val Asn Cys His Ala Ser Asp Gly Arg 355 360 365Lys Ser Thr Pro Leu His Leu Ala Ala Gly Tyr Asn Arg Val Arg Ile 370 375 380Val Gln Leu Leu Leu Gln His Gly Ala Asp Val His Ala Lys Asp Lys385 390 395 400Gly Gly Leu Val Pro Leu His Asn Ala Cys Ser Tyr Gly His Tyr Glu 405 410 415Val Thr Glu Leu Leu Leu Lys His Gly Ala Cys Val Asn Ala Met Asp 420 425 430Leu Trp Gln Phe Thr Pro Leu His Glu Ala Ala Ser Lys Asn Arg Val 435 440 445Glu Val Cys Ser Leu Leu Leu Ser His Gly Ala Asp Pro Thr Leu Val 450 455 460Asn Cys His Gly Lys Ser Ala Val Asp Met Ala Pro Thr Pro Glu Leu465 470 475 480Arg Glu Arg Leu Thr Tyr Glu Phe Lys Gly His Ser Leu Leu Gln Ala 485 490 495Ala Arg Glu Ala Asp Leu Ala Lys Val Lys Lys Thr Leu Ala Leu Glu 500 505 510Ile Ile Asn Phe Lys Gln Pro Gln Ser His Glu Thr Ala Leu His Cys 515 520 525Ala Val Ala Ser Leu His Pro Lys Arg Lys Gln Val Thr Glu Leu Leu 530 535 540Leu Arg Lys Gly Ala Asn Val Asn Glu Lys Asn Lys Asp Phe Met Thr545 550 555 560Pro Leu His Val Ala Ala Glu Arg Ala His Asn Asp Val Met Glu Val 565 570 575Leu His Lys His Gly Ala Lys Met Asn Ala Leu Asp Thr Leu Gly Gln 580 585 590Thr Ala Leu His Arg Ala Ala Leu Ala Gly His Leu Gln Thr Cys Arg 595 600 605Leu Leu Leu Ser Tyr Gly Ser Asp Pro Ser Ile Ile Ser Leu Gln Gly 610 615 620Phe Thr Ala Ala Gln Met Gly Asn Glu Ala Val Gln Gln Ile Leu Ser625 630 635 640Glu Ser Thr Pro Ile Arg Thr Ser Asp Val Asp Tyr

Arg Leu Leu Glu 645 650 655Ala Ser Lys Ala Gly Asp Leu Glu Thr Val Lys Gln Leu Cys Ser Ser 660 665 670Gln Asn Val Asn Cys Arg Asp Leu Glu Gly Arg His Ser Thr Pro Leu 675 680 685His Phe Ala Ala Gly Tyr Asn Arg Val Ser Val Val Glu Tyr Leu Leu 690 695 700His His Gly Ala Asp Val His Ala Lys Asp Lys Gly Gly Leu Val Pro705 710 715 720Leu His Asn Ala Cys Ser Tyr Gly His Tyr Glu Val Ala Glu Leu Leu 725 730 735Val Arg His Gly Ala Ser Val Asn Val Ala Asp Leu Trp Lys Phe Thr 740 745 750Pro Leu His Glu Ala Ala Ala Lys Gly Lys Tyr Glu Ile Cys Lys Leu 755 760 765Leu Leu Lys His Gly Ala Asp Pro Thr Lys Lys Asn Arg Asp Gly Asn 770 775 780Thr Pro Leu Asp Leu Val Lys Glu Gly Asp Thr Asp Ile Gln Asp Leu785 790 795 800Leu Arg Gly Asp Ala Ala Leu Leu Asp Ala Ala Lys Lys Gly Cys Leu 805 810 815Ala Arg Val Gln Lys Leu Cys Thr Pro Glu Asn Ile Asn Cys Arg Asp 820 825 830Thr Gln Gly Arg Asn Ser Thr Pro Leu His Leu Ala Ala Gly Tyr Asn 835 840 845Asn Leu Glu Val Ala Glu Tyr Leu Leu Glu His Gly Ala Asp Val Asn 850 855 860Ala Gln Asp Lys Gly Gly Leu Ile Pro Leu His Asn Ala Ala Ser Tyr865 870 875 880Gly His Val Asp Ile Ala Ala Leu Leu Ile Lys Tyr Asn Thr Cys Val 885 890 895Asn Ala Thr Asp Lys Trp Ala Phe Thr Pro Leu His Glu Ala Ala Gln 900 905 910Lys Gly Arg Thr Gln Leu Cys Ala Leu Leu Leu Ala His Gly Ala Asp 915 920 925Pro Thr Met Lys Asn Gln Glu Gly Gln Thr Pro Leu Asp Leu Ala Thr 930 935 940Ala Asp Asp Ile Arg Ala Leu Leu Ile Asp Ala Met Pro Pro Glu Ala945 950 955 960Leu Pro Thr Cys Phe Lys Pro Gln Ala Thr Val Val Ser Ala Ser Leu 965 970 975Ile Ser Pro Ala Ser Thr Pro Ser Cys Leu Ser Ala Ala Ser Ser Ile 980 985 990Asp Asn Leu Thr Gly Pro Leu Ala Glu Leu Ala Val Gly Gly Ala Ser 995 1000 1005Asn Ala Gly Asp Gly Ala Ala Gly Thr Glu Arg Lys Glu Gly Glu 1010 1015 1020Val Ala Gly Leu Asp Met Asn Ile Ser Gln Phe Leu Lys Ser Leu 1025 1030 1035Gly Leu Glu His Leu Arg Asp Ile Phe Glu Thr Glu Gln Ile Thr 1040 1045 1050Leu Asp Val Leu Ala Asp Met Gly His Glu Glu Leu Lys Glu Ile 1055 1060 1065Gly Ile Asn Ala Tyr Gly His Arg His Lys Leu Ile Lys Gly Val 1070 1075 1080Glu Arg Leu Leu Gly Gly Gln Gln Gly Thr Asn Pro Tyr Leu Thr 1085 1090 1095Phe His Cys Val Asn Gln Gly Thr Ile Leu Leu Asp Leu Ala Pro 1100 1105 1110Glu Asp Lys Glu Tyr Gln Ser Val Glu Glu Glu Met Gln Ser Thr 1115 1120 1125Ile Arg Glu His Arg Asp Gly Gly Asn Ala Gly Gly Ile Phe Asn 1130 1135 1140Arg Tyr Asn Val Ile Arg Ile Gln Lys Val Val Asn Lys Lys Leu 1145 1150 1155Arg Glu Arg Phe Cys His Arg Gln Lys Glu Val Ser Glu Glu Asn 1160 1165 1170His Asn His His Asn Glu Arg Met Leu Phe His Gly Ser Pro Phe 1175 1180 1185Ile Asn Ala Ile Ile His Lys Gly Phe Asp Glu Arg His Ala Tyr 1190 1195 1200Ile Gly Gly Met Phe Gly Ala Gly Ile Tyr Phe Ala Glu Asn Ser 1205 1210 1215Ser Lys Ser Asn Gln Tyr Val Tyr Gly Ile Gly Gly Gly Thr Gly 1220 1225 1230Cys Pro Thr His Lys Asp Arg Ser Cys Tyr Ile Cys His Arg Gln 1235 1240 1245Met Leu Phe Cys Arg Val Thr Leu Gly Lys Ser Phe Leu Gln Phe 1250 1255 1260Ser Thr Met Lys Met Ala His Ala Pro Pro Gly His His Ser Val 1265 1270 1275Ile Gly Arg Pro Ser Val Asn Gly Leu Ala Tyr Ala Glu Tyr Val 1280 1285 1290Ile Tyr Arg Gly Glu Gln Ala Tyr Pro Glu Tyr Leu Ile Thr Tyr 1295 1300 1305Gln Ile Met Lys Pro Glu Ala Pro Ser Gln Thr Ala Thr Ala Ala 1310 1315 1320Glu Gln Lys Thr 1325181166PRTHomo sapiens 18Met Ser Gly Arg Arg Cys Ala Gly Gly Gly Ala Ala Cys Ala Ser Ala1 5 10 15Ala Ala Glu Ala Val Glu Pro Ala Ala Arg Glu Leu Phe Glu Ala Cys 20 25 30Arg Asn Gly Asp Val Glu Arg Val Lys Arg Leu Val Thr Pro Glu Lys 35 40 45Val Asn Ser Arg Asp Thr Ala Gly Arg Lys Ser Thr Pro Leu His Phe 50 55 60Ala Ala Gly Phe Gly Arg Lys Asp Val Val Glu Tyr Leu Leu Gln Asn65 70 75 80Gly Ala Asn Val Gln Ala Arg Asp Asp Gly Gly Leu Ile Pro Leu His 85 90 95Asn Ala Cys Ser Phe Gly His Ala Glu Val Val Asn Leu Leu Leu Arg 100 105 110His Gly Ala Asp Pro Asn Ala Arg Asp Asn Trp Asn Tyr Thr Pro Leu 115 120 125His Glu Ala Ala Ile Lys Gly Lys Ile Asp Val Cys Ile Val Leu Leu 130 135 140Gln His Gly Ala Glu Pro Thr Ile Arg Asn Thr Asp Gly Arg Thr Ala145 150 155 160Leu Asp Leu Ala Asp Pro Ser Ala Lys Ala Val Leu Thr Gly Glu Tyr 165 170 175Lys Lys Asp Glu Leu Leu Glu Ser Ala Arg Ser Gly Asn Glu Glu Lys 180 185 190Met Met Ala Leu Leu Thr Pro Leu Asn Val Asn Cys His Ala Ser Asp 195 200 205Gly Arg Lys Ser Thr Pro Leu His Leu Ala Ala Gly Tyr Asn Arg Val 210 215 220Lys Ile Val Gln Leu Leu Leu Gln His Gly Ala Asp Val His Ala Lys225 230 235 240Asp Lys Gly Asp Leu Val Pro Leu His Asn Ala Cys Ser Tyr Gly His 245 250 255Tyr Glu Val Thr Glu Leu Leu Val Lys His Gly Ala Cys Val Asn Ala 260 265 270Met Asp Leu Trp Gln Phe Thr Pro Leu His Glu Ala Ala Ser Lys Asn 275 280 285Arg Val Glu Val Cys Ser Leu Leu Leu Ser Tyr Gly Ala Asp Pro Thr 290 295 300Leu Leu Asn Cys His Asn Lys Ser Ala Ile Asp Leu Ala Pro Thr Pro305 310 315 320Gln Leu Lys Glu Arg Leu Ala Tyr Glu Phe Lys Gly His Ser Leu Leu 325 330 335Gln Ala Ala Arg Glu Ala Asp Val Thr Arg Ile Lys Lys His Leu Ser 340 345 350Leu Glu Met Val Asn Phe Lys His Pro Gln Thr His Glu Thr Ala Leu 355 360 365His Cys Ala Ala Ala Ser Pro Tyr Pro Lys Arg Lys Gln Ile Cys Glu 370 375 380Leu Leu Leu Arg Lys Gly Ala Asn Ile Asn Glu Lys Thr Lys Glu Phe385 390 395 400Leu Thr Pro Leu His Val Ala Ser Glu Lys Ala His Asn Asp Val Val 405 410 415Glu Val Val Val Lys His Glu Ala Lys Val Asn Ala Leu Asp Asn Leu 420 425 430Gly Gln Thr Ser Leu His Arg Ala Ala Tyr Cys Gly His Leu Gln Thr 435 440 445Cys Arg Leu Leu Leu Ser Tyr Gly Cys Asp Pro Asn Ile Ile Ser Leu 450 455 460Gln Gly Phe Thr Ala Leu Gln Met Gly Asn Glu Asn Val Gln Gln Leu465 470 475 480Leu Gln Glu Gly Ile Ser Leu Gly Asn Ser Glu Ala Asp Arg Gln Leu 485 490 495Leu Glu Ala Ala Lys Ala Gly Asp Val Glu Thr Val Lys Lys Leu Cys 500 505 510Thr Val Gln Ser Val Asn Cys Arg Asp Ile Glu Gly Arg Gln Ser Thr 515 520 525Pro Leu His Phe Ala Ala Gly Tyr Asn Arg Val Ser Val Val Glu Tyr 530 535 540Leu Leu Gln His Gly Ala Asp Val His Ala Lys Asp Lys Gly Gly Leu545 550 555 560Val Pro Leu His Asn Ala Cys Ser Tyr Gly His Tyr Glu Val Ala Glu 565 570 575Leu Leu Val Lys His Gly Ala Val Val Asn Val Ala Asp Leu Trp Lys 580 585 590Phe Thr Pro Leu His Glu Ala Ala Ala Lys Gly Lys Tyr Glu Ile Cys 595 600 605Lys Leu Leu Leu Gln His Gly Ala Asp Pro Thr Lys Lys Asn Arg Asp 610 615 620Gly Asn Thr Pro Leu Asp Leu Val Lys Asp Gly Asp Thr Asp Ile Gln625 630 635 640Asp Leu Leu Arg Gly Asp Ala Ala Leu Leu Asp Ala Ala Lys Lys Gly 645 650 655Cys Leu Ala Arg Val Lys Lys Leu Ser Ser Pro Asp Asn Val Asn Cys 660 665 670Arg Asp Thr Gln Gly Arg His Ser Thr Pro Leu His Leu Ala Ala Gly 675 680 685Tyr Asn Asn Leu Glu Val Ala Glu Tyr Leu Leu Gln His Gly Ala Asp 690 695 700Val Asn Ala Gln Asp Lys Gly Gly Leu Ile Pro Leu His Asn Ala Ala705 710 715 720Ser Tyr Gly His Val Asp Val Ala Ala Leu Leu Ile Lys Tyr Asn Ala 725 730 735Cys Val Asn Ala Thr Asp Lys Trp Ala Phe Thr Pro Leu His Glu Ala 740 745 750Ala Gln Lys Gly Arg Thr Gln Leu Cys Ala Leu Leu Leu Ala His Gly 755 760 765Ala Asp Pro Thr Leu Lys Asn Gln Glu Gly Gln Thr Pro Leu Asp Leu 770 775 780Val Ser Ala Asp Asp Val Ser Ala Leu Leu Thr Ala Ala Met Pro Pro785 790 795 800Ser Ala Leu Pro Ser Cys Tyr Lys Pro Gln Val Leu Asn Gly Val Arg 805 810 815Ser Pro Gly Ala Thr Ala Asp Ala Leu Ser Ser Gly Pro Ser Ser Pro 820 825 830Ser Ser Leu Ser Ala Ala Ser Ser Leu Asp Asn Leu Ser Gly Ser Phe 835 840 845Ser Glu Leu Ser Ser Val Val Ser Ser Ser Gly Thr Glu Gly Ala Ser 850 855 860Ser Leu Glu Lys Lys Glu Val Pro Gly Val Asp Phe Ser Ile Thr Gln865 870 875 880Phe Val Arg Asn Leu Gly Leu Glu His Leu Met Asp Ile Phe Glu Arg 885 890 895Glu Gln Ile Thr Leu Asp Val Leu Val Glu Met Gly His Lys Glu Leu 900 905 910Lys Glu Ile Gly Ile Asn Ala Tyr Gly His Arg His Lys Leu Ile Lys 915 920 925Gly Val Glu Arg Leu Ile Ser Gly Gln Gln Gly Leu Asn Pro Tyr Leu 930 935 940Thr Leu Asn Thr Ser Gly Ser Gly Thr Ile Leu Ile Asp Leu Ser Pro945 950 955 960Asp Asp Lys Glu Phe Gln Ser Val Glu Glu Glu Met Gln Ser Thr Val 965 970 975Arg Glu His Arg Asp Gly Gly His Ala Gly Gly Ile Phe Asn Arg Tyr 980 985 990Asn Ile Leu Lys Ile Gln Lys Val Cys Asn Lys Lys Leu Trp Glu Arg 995 1000 1005Tyr Thr His Arg Arg Lys Glu Val Ser Glu Glu Asn His Asn His 1010 1015 1020Ala Asn Glu Arg Met Leu Phe His Gly Ser Pro Phe Val Asn Ala 1025 1030 1035Ile Ile His Lys Gly Phe Asp Glu Arg His Ala Tyr Ile Gly Gly 1040 1045 1050Met Phe Gly Ala Gly Ile Tyr Phe Ala Glu Asn Ser Ser Lys Ser 1055 1060 1065Asn Gln Tyr Val Tyr Gly Ile Gly Gly Gly Thr Gly Cys Pro Val 1070 1075 1080His Lys Asp Arg Ser Cys Tyr Ile Cys His Arg Gln Leu Leu Phe 1085 1090 1095Cys Arg Val Thr Leu Gly Lys Ser Phe Leu Gln Phe Ser Ala Met 1100 1105 1110Lys Met Ala His Ser Pro Pro Gly His His Ser Val Thr Gly Arg 1115 1120 1125Pro Ser Val Asn Gly Leu Ala Leu Ala Glu Tyr Val Ile Tyr Arg 1130 1135 1140Gly Glu Gln Ala Tyr Pro Glu Tyr Leu Ile Thr Tyr Gln Ile Met 1145 1150 1155Arg Pro Glu Gly Met Val Asp Gly 1160 116519630PRTHomo sapiens 19Met Asp Ile Lys Gly Gln Phe Trp Asn Asp Asp Asp Ser Glu Gly Asp1 5 10 15Asn Glu Ser Glu Glu Phe Leu Tyr Gly Val Gln Gly Ser Cys Ala Ala 20 25 30Asp Leu Tyr Arg His Pro Gln Leu Asp Ala Asp Ile Glu Ala Val Lys 35 40 45Glu Ile Tyr Ser Glu Asn Ser Val Ser Ile Arg Glu Tyr Gly Thr Ile 50 55 60Asp Asp Val Asp Ile Asp Leu His Ile Asn Ile Ser Phe Leu Asp Glu65 70 75 80Glu Val Ser Thr Ala Trp Lys Val Leu Arg Thr Glu Pro Ile Val Leu 85 90 95Arg Leu Arg Phe Ser Leu Ser Gln Tyr Leu Asp Gly Pro Glu Pro Ser 100 105 110Ile Glu Val Phe Gln Pro Ser Asn Lys Glu Gly Phe Gly Leu Gly Leu 115 120 125Gln Leu Lys Lys Ile Leu Gly Met Phe Thr Ser Gln Gln Trp Lys His 130 135 140Leu Ser Asn Asp Phe Leu Lys Thr Gln Gln Glu Lys Arg His Ser Trp145 150 155 160Phe Lys Ala Ser Gly Thr Ile Lys Lys Phe Arg Ala Gly Leu Ser Ile 165 170 175Phe Ser Pro Ile Pro Lys Ser Pro Ser Phe Pro Ile Ile Gln Asp Ser 180 185 190Met Leu Lys Gly Lys Leu Gly Val Pro Glu Leu Arg Val Gly Arg Leu 195 200 205Met Asn Arg Ser Ile Ser Cys Thr Met Lys Asn Pro Lys Val Glu Val 210 215 220Phe Gly Tyr Pro Pro Ser Pro Gln Ala Gly Leu Leu Cys Pro Gln His225 230 235 240Val Gly Leu Pro Pro Pro Ala Arg Thr Ser Pro Leu Val Ser Gly His 245 250 255Cys Lys Asn Ile Pro Thr Leu Glu Tyr Gly Phe Leu Val Gln Ile Met 260 265 270Lys Tyr Ala Glu Gln Arg Ile Pro Thr Leu Asn Glu Tyr Cys Val Val 275 280 285Cys Asp Glu Gln His Val Phe Gln Asn Gly Ser Met Leu Lys Pro Ala 290 295 300Val Cys Thr Arg Glu Leu Cys Val Phe Ser Phe Tyr Thr Leu Gly Val305 310 315 320Met Ser Gly Ala Ala Glu Glu Val Ala Thr Gly Ala Glu Val Val Asp 325 330 335Leu Leu Val Ala Met Cys Arg Ala Ala Leu Glu Ser Pro Arg Lys Ser 340 345 350Ile Ile Phe Glu Pro Tyr Pro Ser Val Val Asp Pro Thr Asp Pro Lys 355 360 365Thr Leu Ala Phe Asn Pro Lys Lys Lys Asn Tyr Glu Arg Leu Gln Lys 370 375 380Ala Leu Asp Ser Val Met Ser Ile Arg Glu Met Thr Gln Gly Ser Tyr385 390 395 400Leu Glu Ile Lys Lys Gln Met Asp Lys Leu Asp Pro Leu Ala His Pro 405 410 415Leu Leu Gln Trp Ile Ile Ser Ser Asn Arg Ser His Ile Val Lys Leu 420 425 430Pro Leu Ser Arg Leu Lys Phe Met His Thr Ser His Gln Phe Leu Leu 435 440 445Leu Ser Ser Pro Pro Ala Lys Glu Ala Arg Phe Arg Thr Ala Lys Lys 450 455 460Leu Tyr Gly Ser Thr Phe Ala Phe His Gly Ser His Ile Glu Asn Trp465 470 475 480His Ser Ile Leu Arg Asn Gly Leu Val Asn Ala Ser Tyr Thr Lys Leu 485 490 495Gln Leu His Gly Ala Ala Tyr Gly Lys Gly Ile Tyr Leu Ser Pro Ile 500 505 510Ser Ser Ile Ser Phe Gly Tyr Ser Gly Met Gly Lys Gly Gln His Arg 515 520 525Met Pro Ser Lys Asp Glu Leu Val Gln Arg Tyr Asn Arg Met Asn Thr 530 535 540Ile Pro Gln Thr Arg Ser Ile Gln Ser Arg Phe Leu Gln Ser Arg Asn545 550 555 560Leu Asn Cys Ile Ala Leu Cys Glu Val Ile Thr Ser Lys Asp Leu Gln 565 570 575Lys His Gly Asn Ile Trp Val Cys Pro Val Ser Asp His Val Cys Thr 580 585 590Arg Phe Phe Phe Val Tyr Glu Asp Gly Gln Val Gly Asp Ala Asn Ile 595 600 605Asn Thr Gln Asp Pro Lys Ile Gln Lys Glu Ile Met

Arg Val Ile Gly 610 615 620Thr Gln Val Tyr Thr Asn625 63020494PRTHomo sapiens 20Met Gln Thr Phe Val Thr Gln Gln Trp Lys Gln Ser Lys Glu Lys Ser1 5 10 15Asn Cys Leu His Asn Lys Lys Leu Ser Glu Lys Lys Val Lys Ser Pro 20 25 30Leu His Leu Phe Ser Thr Leu Arg Arg Ser Pro Ser Tyr Pro Pro Pro 35 40 45Gly Cys Gly Lys Ser Lys Ser Lys Leu Lys Ser Glu Gln Asp Gly Ile 50 55 60Ser Lys Thr His Lys Leu Leu Arg Arg Thr Cys Ser Ser Thr Val Lys65 70 75 80Thr Asp Asp Val Cys Val Thr Lys Ser His Arg Thr Phe Gly Arg Ser 85 90 95Leu Ser Ser Asp Pro Arg Ala Glu Gln Ala Met Thr Ala Ile Lys Ser 100 105 110His Lys Leu Leu Asn Arg Pro Cys Pro Ala Ala Val Lys Ser Glu Glu 115 120 125Cys Leu Thr Leu Lys Ser His Arg Leu Leu Thr Arg Ser Cys Ser Gly 130 135 140Asp Pro Arg Cys Glu His Asn Thr Asn Leu Lys Pro His Lys Leu Leu145 150 155 160Ser Arg Ser Tyr Ser Ser Asn Leu Arg Met Glu Glu Leu Tyr Gly Leu 165 170 175Lys Asn His Lys Leu Leu Ser Lys Ser Tyr Ser Ser Ala Pro Lys Ser 180 185 190Ser Lys Thr Glu Leu Phe Lys Glu Pro Asn Ala Glu Gly Arg Arg Leu 195 200 205Ser Leu Thr Ser Gly Leu Ile Gly Ile Leu Thr Pro Ser Ser Ser Ser 210 215 220Ser Ser Gln Leu Ala Pro Asn Gly Ala Lys Cys Ile Pro Val Arg Asp225 230 235 240Arg Gly Phe Leu Val Gln Thr Ile Glu Phe Ala Glu Gln Arg Ile Pro 245 250 255Val Leu Asn Glu Tyr Cys Val Val Cys Asp Glu Pro His Val Phe Gln 260 265 270Asn Gly Pro Met Leu Arg Pro Thr Val Cys Glu Arg Glu Leu Cys Val 275 280 285Phe Ala Phe Gln Thr Leu Gly Val Met Asn Glu Ala Ala Asp Glu Ile 290 295 300Ala Thr Gly Ala Gln Lys Lys Asn Tyr Asp Arg Val Met Lys Ala Leu305 310 315 320Asp Ser Ile Thr Ser Ile Arg Glu Met Thr Gln Ala Pro Tyr Leu Glu 325 330 335Ile Lys Lys Gln Met Asp Lys Gln Asp Pro Leu Ala His Pro Leu Leu 340 345 350Gln Trp Val Ile Ser Ser Asn Arg Ser His Ile Val Lys Leu Pro Val 355 360 365Asn Arg Gln Leu Lys Phe Met His Thr Pro His Gln Phe Leu Leu Leu 370 375 380Ser Ser Pro Pro Ala Lys Glu Ser Asn Phe Arg Ala Ala Lys Lys Leu385 390 395 400Phe Gly Ser Thr Phe Ala Phe His Gly Ser His Ile Glu Asn Trp His 405 410 415Ser Ile Leu Arg Asn Gly Leu Val Val Ala Ser Asn Thr Arg Leu Gln 420 425 430Leu His Gly Ala Met Tyr Gly Ser Gly Ile Tyr Leu Ser Pro Met Ser 435 440 445Ser Ile Ser Phe Gly Tyr Ser Gly Met Asn Lys Lys Gln Lys Val Ser 450 455 460Ala Lys Asp Glu Pro Ala Ser Ser Ser Lys Ser Ser Asn Thr Ser Gln465 470 475 480Ser Gln Lys Lys Arg Thr Ala Ile Pro Ile Pro Ala Lys Pro 485 49021819PRTHomo sapiens 21Met Asp Phe Ser Met Val Ala Gly Ala Ala Ala Tyr Asn Glu Lys Ser1 5 10 15Glu Thr Gly Ala Leu Gly Glu Asn Tyr Ser Trp Gln Ile Pro Ile Asn 20 25 30His Asn Asp Phe Lys Ile Leu Lys Asn Asn Glu Arg Gln Leu Cys Glu 35 40 45Val Leu Gln Asn Lys Phe Gly Cys Ile Ser Thr Leu Val Ser Pro Val 50 55 60Gln Glu Gly Asn Ser Lys Ser Leu Gln Val Phe Arg Lys Met Leu Thr65 70 75 80Pro Arg Ile Glu Leu Ser Val Trp Lys Asp Asp Leu Thr Thr His Ala 85 90 95Val Asp Ala Val Val Asn Ala Ala Asn Glu Asp Leu Leu His Gly Gly 100 105 110Gly Leu Ala Leu Ala Leu Val Lys Ala Gly Gly Phe Glu Ile Gln Glu 115 120 125Glu Ser Lys Gln Phe Val Ala Arg Tyr Gly Lys Val Ser Ala Gly Glu 130 135 140Ile Ala Val Thr Gly Ala Gly Arg Leu Pro Cys Lys Gln Ile Ile His145 150 155 160Ala Val Gly Pro Arg Trp Met Glu Trp Asp Lys Gln Gly Cys Thr Gly 165 170 175Lys Leu Gln Arg Ala Ile Val Ser Ile Leu Asn Tyr Val Ile Tyr Lys 180 185 190Asn Thr His Ile Lys Thr Val Ala Ile Pro Ala Leu Ser Ser Gly Ile 195 200 205Phe Gln Phe Pro Leu Asn Leu Cys Thr Lys Thr Ile Val Glu Thr Ile 210 215 220Arg Val Ser Leu Gln Gly Lys Pro Met Met Ser Asn Leu Lys Glu Ile225 230 235 240His Leu Val Ser Asn Glu Asp Pro Thr Val Ala Ala Phe Lys Ala Ala 245 250 255Ser Glu Phe Ile Leu Gly Lys Ser Glu Leu Gly Gln Glu Thr Thr Pro 260 265 270Ser Phe Asn Ala Met Val Val Asn Asn Leu Thr Leu Gln Ile Val Gln 275 280 285Gly His Ile Glu Trp Gln Thr Ala Asp Val Ile Val Asn Ser Val Asn 290 295 300Pro His Asp Ile Thr Val Gly Pro Val Ala Lys Ser Ile Leu Gln Gln305 310 315 320Ala Gly Val Glu Met Lys Ser Glu Phe Leu Ala Thr Lys Ala Lys Gln 325 330 335Phe Gln Arg Ser Gln Leu Val Leu Val Thr Lys Gly Phe Asn Leu Phe 340 345 350Cys Lys Tyr Ile Tyr His Val Leu Trp His Ser Glu Phe Pro Lys Pro 355 360 365Gln Ile Leu Lys His Ala Met Lys Glu Cys Leu Glu Lys Cys Ile Glu 370 375 380Gln Asn Ile Thr Ser Ile Ser Phe Pro Ala Leu Gly Thr Gly Asn Met385 390 395 400Glu Ile Lys Lys Glu Thr Ala Ala Glu Ile Leu Phe Asp Glu Val Leu 405 410 415Thr Phe Ala Lys Asp His Val Lys His Gln Leu Thr Val Lys Phe Val 420 425 430Ile Phe Pro Thr Asp Leu Glu Ile Tyr Lys Ala Phe Ser Ser Glu Met 435 440 445Ala Lys Arg Ser Lys Met Leu Ser Leu Asn Asn Tyr Ser Val Pro Gln 450 455 460Ser Thr Arg Glu Glu Lys Arg Glu Asn Gly Leu Glu Ala Arg Ser Pro465 470 475 480Ala Ile Asn Leu Met Gly Phe Asn Val Glu Glu Met Tyr Glu Ala His 485 490 495Ala Trp Ile Gln Arg Ile Leu Ser Leu Gln Asn His His Ile Ile Glu 500 505 510Asn Asn His Ile Leu Tyr Leu Gly Arg Lys Glu His Asp Ile Leu Ser 515 520 525Gln Leu Gln Lys Thr Ser Ser Val Ser Ile Thr Glu Ile Ile Ser Pro 530 535 540Gly Arg Thr Glu Leu Glu Ile Glu Gly Ala Arg Ala Asp Leu Ile Glu545 550 555 560Val Val Met Asn Ile Glu Asp Met Leu Cys Lys Val Gln Glu Glu Met 565 570 575Ala Arg Lys Lys Glu Arg Gly Leu Trp Arg Ser Leu Gly Gln Trp Thr 580 585 590Ile Gln Gln Gln Lys Thr Gln Asp Glu Met Lys Glu Asn Ile Ile Phe 595 600 605Leu Lys Cys Pro Val Pro Pro Thr Gln Glu Leu Leu Asp Gln Lys Lys 610 615 620Gln Phe Glu Lys Cys Gly Leu Gln Val Leu Lys Val Glu Lys Ile Asp625 630 635 640Asn Glu Val Leu Met Ala Ala Phe Gln Arg Lys Lys Lys Met Met Glu 645 650 655Glu Lys Leu His Arg Gln Pro Val Ser His Arg Leu Phe Gln Gln Val 660 665 670Pro Tyr Gln Phe Cys Asn Val Val Cys Arg Val Gly Phe Gln Arg Met 675 680 685Tyr Ser Thr Pro Cys Asp Pro Lys Tyr Gly Ala Gly Ile Tyr Phe Thr 690 695 700Lys Asn Leu Lys Asn Leu Ala Glu Lys Ala Lys Lys Ile Ser Ala Ala705 710 715 720Asp Lys Leu Ile Tyr Val Phe Glu Ala Glu Val Leu Thr Gly Phe Phe 725 730 735Cys Gln Gly His Pro Leu Asn Ile Val Pro Pro Pro Leu Ser Pro Gly 740 745 750Ala Ile Asp Gly His Asp Ser Val Val Asp Asn Val Ser Ser Pro Glu 755 760 765Thr Phe Val Ile Phe Ser Gly Met Gln Ala Ile Pro Gln Tyr Leu Trp 770 775 780Thr Cys Thr Gln Glu Tyr Val Gln Ser Gln Asp Tyr Ser Ser Gly Pro785 790 795 800Met Arg Pro Phe Ala Gln His Pro Trp Arg Gly Phe Ala Ser Gly Ser 805 810 815Pro Val Asp22338PRTHomo sapiens 22Met Trp Glu Ala Asn Pro Glu Met Phe His Lys Ala Glu Glu Leu Phe1 5 10 15Ser Lys Thr Thr Asn Asn Glu Val Asp Asp Met Asp Thr Ser Asp Thr 20 25 30Gln Trp Gly Trp Phe Tyr Leu Ala Glu Cys Gly Lys Trp His Met Phe 35 40 45Gln Pro Asp Thr Asn Ser Gln Cys Ser Val Ser Ser Glu Asp Ile Glu 50 55 60Lys Ser Phe Lys Thr Asn Pro Cys Gly Ser Ile Ser Phe Thr Thr Ser65 70 75 80Lys Phe Ser Tyr Lys Ile Asp Phe Ala Glu Met Lys Gln Met Asn Leu 85 90 95Thr Thr Gly Lys Gln Arg Leu Ile Lys Arg Ala Pro Phe Ser Ile Ser 100 105 110Ala Phe Ser Tyr Ile Cys Glu Asn Glu Ala Ile Pro Met Pro Pro His 115 120 125Trp Glu Asn Val Asn Thr Gln Val Pro Tyr Gln Leu Ile Pro Leu His 130 135 140Asn Gln Thr His Glu Tyr Asn Glu Val Ala Asn Leu Phe Gly Lys Thr145 150 155 160Met Asp Arg Asn Arg Ile Lys Arg Ile Gln Arg Ile Gln Asn Leu Asp 165 170 175Leu Trp Glu Phe Phe Cys Arg Lys Lys Ala Gln Leu Lys Lys Lys Arg 180 185 190Gly Val Pro Gln Ile Asn Glu Gln Met Leu Phe His Gly Thr Ser Ser 195 200 205Glu Phe Val Glu Ala Ile Cys Ile His Asn Phe Asp Trp Arg Ile Asn 210 215 220Gly Ile His Gly Ala Val Phe Gly Lys Gly Thr Tyr Phe Ala Arg Asp225 230 235 240Ala Ala Tyr Ser Ser Arg Phe Cys Lys Asp Asp Ile Lys His Gly Asn 245 250 255Thr Phe Gln Ile His Gly Val Ser Leu Gln Gln Arg His Leu Phe Arg 260 265 270Thr Tyr Lys Ser Met Phe Leu Ala Arg Val Leu Ile Gly Asp Tyr Ile 275 280 285Asn Gly Asp Ser Lys Tyr Met Arg Pro Pro Ser Lys Asp Gly Ser Tyr 290 295 300Val Asn Leu Tyr Asp Ser Cys Val Asp Asp Thr Trp Asn Pro Lys Ile305 310 315 320Phe Val Val Phe Asp Ala Asn Gln Ile Tyr Pro Glu Tyr Leu Ile Asp 325 330 335Phe His23902PRTHomo sapiens 23Met Ala Asp Pro Glu Val Cys Cys Phe Ile Thr Lys Ile Leu Cys Ala1 5 10 15His Gly Gly Arg Met Ala Leu Asp Ala Leu Leu Gln Glu Ile Ala Leu 20 25 30Ser Glu Pro Gln Leu Cys Glu Val Leu Gln Val Ala Gly Pro Asp Arg 35 40 45Phe Val Val Leu Glu Thr Gly Gly Glu Ala Gly Ile Thr Arg Ser Val 50 55 60Val Ala Thr Thr Arg Ala Arg Val Cys Arg Arg Lys Tyr Cys Gln Arg65 70 75 80Pro Cys Asp Asn Leu His Leu Cys Lys Leu Asn Leu Leu Gly Arg Cys 85 90 95Asn Tyr Ser Gln Ser Glu Arg Asn Leu Cys Lys Tyr Ser His Glu Val 100 105 110Leu Ser Glu Glu Asn Phe Lys Val Leu Lys Asn His Glu Leu Ser Gly 115 120 125Leu Asn Lys Glu Glu Leu Ala Val Leu Leu Leu Gln Ser Asp Pro Phe 130 135 140Phe Met Pro Glu Ile Cys Lys Ser Tyr Lys Gly Glu Gly Arg Gln Gln145 150 155 160Ile Cys Asn Gln Gln Pro Pro Cys Ser Arg Leu His Ile Cys Asp His 165 170 175Phe Thr Arg Gly Asn Cys Arg Phe Pro Asn Cys Leu Arg Ser His Asn 180 185 190Leu Met Asp Arg Lys Val Leu Ala Ile Met Arg Glu His Gly Leu Asn 195 200 205Pro Asp Val Val Gln Asn Ile Gln Asp Ile Cys Asn Ser Lys His Met 210 215 220Gln Lys Asn Pro Pro Gly Pro Arg Ala Pro Ser Ser His Arg Arg Asn225 230 235 240Met Ala Tyr Arg Ala Arg Ser Lys Ser Arg Asp Arg Phe Phe Gln Gly 245 250 255Ser Gln Glu Phe Leu Ala Ser Ala Ser Ala Ser Ala Glu Arg Ser Cys 260 265 270Thr Pro Ser Pro Asp Gln Ile Ser His Arg Ala Ser Leu Glu Asp Ala 275 280 285Pro Val Asp Asp Leu Thr Arg Lys Phe Thr Tyr Leu Gly Ser Gln Asp 290 295 300Arg Ala Arg Pro Pro Ser Gly Ser Ser Lys Ala Thr Asp Leu Gly Gly305 310 315 320Thr Ser Gln Ala Gly Thr Ser Gln Arg Phe Leu Glu Asn Gly Ser Gln 325 330 335Glu Asp Leu Leu His Gly Asn Pro Gly Ser Thr Tyr Leu Ala Ser Asn 340 345 350Ser Thr Ser Ala Pro Asn Trp Lys Ser Leu Thr Ser Trp Thr Asn Asp 355 360 365Gln Gly Ala Arg Arg Lys Thr Val Phe Ser Pro Thr Leu Pro Ala Ala 370 375 380Arg Ser Ser Leu Gly Ser Leu Gln Thr Pro Glu Ala Val Thr Thr Arg385 390 395 400Lys Gly Thr Gly Leu Leu Ser Ser Asp Tyr Arg Ile Ile Asn Gly Lys 405 410 415Ser Gly Thr Gln Asp Ile Gln Pro Gly Pro Leu Phe Asn Asn Asn Ala 420 425 430Asp Gly Val Ala Thr Asp Ile Thr Ser Thr Arg Ser Leu Asn Tyr Lys 435 440 445Ser Thr Ser Ser Gly His Arg Glu Ile Ser Ser Pro Arg Ile Gln Asp 450 455 460Ala Gly Pro Ala Ser Arg Asp Val Gln Ala Thr Gly Arg Ile Ala Asp465 470 475 480Asp Ala Asp Pro Arg Val Ala Leu Val Asn Asp Ser Leu Ser Asp Val 485 490 495Thr Ser Thr Thr Ser Ser Arg Val Asp Asp His Asp Ser Glu Glu Ile 500 505 510Cys Leu Asp His Leu Cys Lys Gly Cys Pro Leu Asn Gly Ser Cys Ser 515 520 525Lys Val His Phe His Leu Pro Tyr Arg Trp Gln Met Leu Ile Gly Lys 530 535 540Thr Trp Thr Asp Phe Glu His Met Glu Thr Ile Glu Lys Gly Tyr Cys545 550 555 560Asn Pro Gly Ile His Leu Cys Ser Val Gly Ser Tyr Thr Ile Asn Phe 565 570 575Arg Val Met Ser Cys Asp Ser Phe Pro Ile Arg Arg Leu Ser Thr Pro 580 585 590Ser Ser Val Thr Lys Pro Ala Asn Ser Val Phe Thr Thr Lys Trp Ile 595 600 605Trp Tyr Trp Lys Asn Glu Ser Gly Thr Trp Ile Gln Tyr Gly Glu Glu 610 615 620Lys Asp Lys Arg Lys Asn Ser Asn Val Asp Ser Ser Tyr Leu Glu Ser625 630 635 640Leu Tyr Gln Ser Cys Pro Arg Gly Val Val Pro Phe Gln Ala Gly Ser 645 650 655Arg Asn Tyr Glu Leu Ser Phe Gln Gly Met Ile Gln Thr Asn Ile Ala 660 665 670Ser Lys Thr Gln Lys Asp Val Ile Arg Arg Pro Thr Phe Val Pro Gln 675 680 685Trp Tyr Val Gln Gln Met Lys Arg Gly Pro Asp His Gln Pro Ala Lys 690 695 700Thr Ser Ser Val Ser Leu Thr Ala Thr Phe Arg Pro Gln Glu Asp Phe705 710 715 720Cys Phe Leu Ser Ser Lys Lys Tyr Lys Leu Ser Glu Ile His His Leu 725 730 735His Pro Glu Tyr Val Arg Val Ser Glu His Phe Lys Ala Ser Met Lys 740 745 750Asn Phe Lys Ile Glu Lys Ile Lys Lys Ile Glu Asn Ser Glu Leu Leu 755 760 765Asp Lys Phe Thr Trp Lys Lys Ser Gln Met Lys Glu Glu Gly Lys Leu 770 775 780Leu Phe Tyr Ala Thr Ser Arg Ala Tyr Val Glu Ser Ile Cys Ser Asn785 790 795 800Asn Phe

Asp Ser Phe Leu His Glu Thr His Glu Asn Lys Tyr Gly Lys 805 810 815Gly Ile Tyr Phe Ala Lys Asp Ala Ile Tyr Ser His Lys Asn Cys Pro 820 825 830Tyr Asp Ala Lys Asn Val Val Met Phe Val Ala Gln Val Leu Val Gly 835 840 845Lys Phe Thr Glu Gly Asn Ile Thr Tyr Thr Ser Pro Pro Pro Gln Phe 850 855 860Asp Ser Cys Val Asp Thr Arg Ser Asn Pro Ser Val Phe Val Ile Phe865 870 875 880Gln Lys Asp Gln Val Tyr Pro Gln Tyr Val Ile Glu Tyr Thr Glu Asp 885 890 895Lys Ala Cys Val Ile Ser 900

Claims

1. A method of identifying an inhibitor for Poly (ADP-ribose) polymerase (PARP), the method comprising: combining (i) a polypeptide comprising a PARP catalytic domain wherein the polypeptide is labeled with a donor fluorophore, (ii) a PARP probe, wherein the PARP probe is labeled with an acceptor fluorophore, and (iii) a test compound; exposing the donor fluorophore to excitation light; measuring a signal produced by the acceptor fluorophore; and identifying the test compound as an inhibitor for PARP based on the signal produced by the acceptor fluorophore.

2. The method of claim 1, wherein the PARP probe is a compound having the structure: ##STR00013## or a salt thereof.

3. The method of claim 1, further comprising identifying the test compound as an inhibitor for PARP if the signal produced by the acceptor fluorophore is decreased as compared to a reference level.

4. The method of claim 3, wherein the reference level is the signal produced by the acceptor fluorophore in the absence of the test compound.

5. The method of claim 1, wherein the PARP is PARP1, PARP2, PARP3, PARP4, PARP5a, PARP5b, PARP6, TIPARP, PARP8, PARP9, PARP10, PARP11, PARP12, PARP13, PARP14, PARP15, or PARP16.

6. The method of claim 1, wherein the PARP probe is biotinylated, and the acceptor fluorophore is attached to streptavidin.

7. The method of claim 1, wherein the polypeptide comprises a polyhistidine tag.

8. The method of claim 1, wherein the test compound is a small molecule, a nucleic acid, a polypeptide, or an antibody or antigen-binding fragment thereof.

9. A method of identifying an inhibitor for Poly (ADP-ribose) polymerase (PARP), the method comprising: combining (i) a polypeptide comprising a PARP catalytic domain, wherein the polypeptide is labeled with an acceptor fluorophore; (ii) a PARP probe, wherein the PARP probe is labeled with a donor fluorophore, and (iii) a test compound; exposing the donor fluorophore to excitation light; measuring a signal produced by the acceptor fluorophore in the presence of a test compound; and identifying the test compound as an inhibitor for PARP based on the signal produced by the acceptor fluorophore.

10. The method of claim 9, wherein the PARP probe is a compound having the structure: ##STR00014## or a salt thereof.

11. The method of claim 9, further comprising identifying the test compound as an inhibitor for PARP if the signal produced by the acceptor fluorophore is decreased as compared to a reference level.

12. The method of claim 11, wherein the reference level is the signal produced by the acceptor fluorophore in the absence of the test compound.

13. The method of claim 9, wherein the PARP is PARP1, PARP2, PARP3, PARP4 PARP5a, PARP5, PARP6, TIPARP, PARP8, PARP9, PARP10, PARP11, PARP12, PARP13, PARP14, PARP15, or PARP16.

14. The method of claim 9, wherein the test compound is a small molecule, a nucleic acid, a polypeptide, or an antibody or antigen-binding fragment thereof.

15. A method of identifying an inhibitor for Poly (ADP-ribose) polymerase (PARP), the method comprising: contacting a polypeptide comprising a PARP catalytic domain with a PARP probe in the presence of a test compound, wherein the PARP probe comprises a fluorophore; exposing the probe to polarized excitation light, thereby generating fluorescence; determining a fluorescence polarization value of the fluorescence; and identifying the test compound as an inhibitor for PARP based on the fluorescence polarization value of the fluorescence.

16. The method of claim 15, further comprising identifying the test compound as an inhibitor for PARP if the fluorescence polarization value of the fluorescence is decreased as compared to a reference level.

17. The method of claim 16, wherein the reference value is the fluorescence polarization value of the fluorescence in the absence of the test compound.

18. The method of claim 15, wherein the PARP probe is a compound having the structure: ##STR00015## or or a salt thereof.

19. The method of claim 15, wherein the PARP is PARP1, PARP2, PARP3, PARP4, PARP5a, PARP5b, PARP6, TIPARP, PARP8, PARP10, PARP1l, PARP12, PARP13, PARP14, PARP15, or PARP16.

20. The method of claim 15, wherein the test compound is a small molecule, a nucleic acid, a polypeptide, or an antibody or antigen-binding fragment thereof.

21. A method of identifying an inhibitor for Poly (ADP-ribose) polymerase (PARP), the method comprising: contacting a fusion polypeptide with a PARP probe that comprises a fluorophore, wherein the fusion polypeptide comprises a PARP catalytic domain and a luciferase enzyme; contacting the luciferase enzyme with a substrate to produce light, wherein the light can excite the fluorophore; measuring a signal produced by the fluorophore in the presence of a test compound; and identifying the test compound as an inhibitor for PARP based on the signal produced by the fluorophore.

22. The method of claim 21, further comprising identifying the test compound as an inhibitor for PARP if the signal produced by the fluorophore is decreased as compared to a reference level.

23. The method of claim 22, wherein the reference level is the signal produced by the fluorophore in the absence of the test compound.

24. The method of claim 21, wherein the PARP probe is a compound having the structure: ##STR00016## or a salt thereof.

25. The method of claim 21, wherein the fusion polypeptide comprises a sequence that is at least 85%, 90% or 95% identical to SEQ ID NO: 11.

26. The method of claim 22, wherein the PARP is PARP1, PARP2, PARP4, PARP5a, PARP5b, PARP3, PARP6, TIPARP, PARP8, PARP9, PARP10, PARP11, PARP12, PARP13, PARP14, PARP15, or PARP16.

27. The method of claim 22, wherein the test compound is a small molecule, a nucleic acid, a polypeptide, or an antibody or antigen-binding fragment thereof.

28. The method of claim 1, wherein the polypeptide comprising a PARP catalytic domain comprises a sequence that is at least 85%, 90% or 95% identical to amino acid residues of 456 to 657 of NP_056323.2 (SEQ ID NO: 1).

29. The method of claim 1, wherein the polypeptide comprising a PARP catalytic domain comprises a sequence that is at least 85%, 90% or 95% identical to amino acid residues of 808 to 1025 of NP_116178.2 (SEQ ID NO: 2).

30. The method of claim 1, wherein the polypeptide comprising a PARP catalytic domain comprises a sequence that is at least 85%, 90% or 95% identical to amino acid residues of 489 to 684 of NP_073587.1 (SEQ ID NO: 3).

31. The method of claim 1, wherein the polypeptide comprising a PARP catalytic domain comprises a sequence that is at least 85%, 90% or 95% identical to amino acid residues of 1611 to 1801 of NP_060024.2 (SEQ ID NO: 4).

32. The method of claim 1, wherein the polypeptide comprising a PARP catalytic domain comprises a sequence that is at least 85%, 90% or 95% identical to amino acid residues of 481 to 678 of NP_689828.1 (SEQ ID NO: 5).

33. The method of claim 1, wherein the polypeptide comprising a PARP catalytic domain comprises a sequence that is at least 85%, 90% or 95% identical to amino acid residues of 5 to 279 of NP_060321.3 (SEQ ID NO: 6).

34. A PARP probe having a structure according to Formula (I): ##STR00017## or a salt thereof, wherein: L is a linking group having 5-30 spacer atoms selected from C, N, O, and S connecting the N atom of the piperidinyl group of Formula (I) with group A; A is a fluorophore or an affinity tag.

35. The PARP probe of claim 34 wherein L is ##STR00018## a is 0, 1, or 2; b is 1-26; and c is 0, 1, or 2; wherein the sum of a+b+c is 1 to 26, or L is a chain of 5-30 atoms in length comprising --(CH.sub.2CH.sub.2O).sub.d-- wherein d is 2-10.

36. The PARP probe of claim 34 wherein A is: ##STR00019## or a salt thereof.

37. The PARP probe of claim 34 which is a compound having the structure: ##STR00020## or a salt thereof.