PEPTIDES DERIVED FROM THE C2 DOMAIN OF EPSILON PKC AND METHODS OF USE, THEREOF

Abstract

e C2 regions of εPKC and methods of use, thereof, are described. These peptides modulate the activity of εPKC in an animal model of acute ischemic heart disease.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a Divisional application of U.S. patent application Ser. No. 11/906,645, filed Oct. 2, 2007, now allowed, which claims the benefit of U.S. Provisional Application No. 60/849,163, filed Oct. 2, 2006; U.S. Provisional Application No. 60/873,482, filed Dec. 6, 2006, all of which are incorporated herein by reference in its entirety.

TECHNICAL FIELD

[0003] The subject matter described herein relates to peptides derived from regions of the PKC polypeptide spanning the C2 domain. The peptides modulate the activity of PKC and are useful for reducing the severity of e.g., acute ischemic heart disease and stroke.

BACKGROUND

[0004] Protein kinase C(PKC) enzymes are a family of serine and threonine protein kinases involved in apoptosis (14,15), cell proliferation (16-18), secretion (19), and disease states, including ischemic heart disease (4, 20-22) and stroke (23,24). PKC activation involves binding to negatively charged phospholipids and phosphatidylserine. Different PKC isozymes have different sensitivities to other modulators, including Ca²+ and lipid-derived second messengers, such as diacylglycerol (25). Following activation by one or more binding events, PKC isozymes are known to translocate from the soluble cell fraction to the particulate cell fraction (26) containing cell membranes, nuclei, mitochondria, and other components (27,28).

[0005] The primary amino acid sequence of PKC can generally be separated into two domains: (i) the N-terminal regulatory domain and (ii) the conserved C-terminal catalytic domain. The regulatory domain includes the C1 and C2 domains, which mediate interactions with second messengers and phospholipids, and generally modulate inter and intramolecular protein-protein interactions. Differences in the primary amino acid sequence, order, and number of copies of signaling domains, contribute to the different activity and/or specificities of different PKC isozymes (25,30). C2 domains in at least one PKC subfamily were called region "V1" until homology to the C2 domains of other family members was recognized (29).

[0006] A small region of the C2 domain has been shown to modulate protein-protein interactions (2,5). In particular, several peptides derived from a region of the C2 domain were shown to act as competitive inhibitors of PKC (1). A peptide that interfered with interactions between εPKC and its anchoring protein, εRACK, inhibited εPKC activity (5). A peptide the interfered with auto-inhibitory intramolecular interactions increased PKC activity and reduced ischemic death in an animal model for ischemic heart disease (4, 5, 7). Related 6-10 amino acid peptides from the same region of C2 were identified (5, 22, 31-34) and shown to be selective and effective in regulating the biological activities of the corresponding PKC isozymes.

[0007] Such studies are limited to a small region of the C2 domain. The need exists to identify other regions of the C2 domain that modulate PKC activity, and to develop selective peptide inhibitors based on such regions. Such peptides are candidates for drug development (6, 9, 10, 12, 18, 23, 28, 35-41).

REFERENCES

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SUMMARY

[0072] The following aspects and embodiments thereof described and illustrated below are meant to be exemplary and illustrative, not limiting in scope.

[0073] In one aspect, a method for modulating ischemic cardiac damage in a mammalian subject is provided, comprising administering a therapeutically effective amount of a peptide comprising a contiguous amino acid sequence derived from the C2 domain of εPKC.

[0074] In some embodiments, the peptide does not include an amino acid sequence corresponding to amino acid residues 85-92 of the C2 domain of εPKC. In particular embodiments, the εPKC peptide is not ψεRACK (SEQ ID NO: 18).

[0075] In some embodiments, the εPKC peptide comprises a contiguous amino acid sequence corresponding to amino acid residues 28-35, 49-60, and 74-79, of εPKC. In some embodiments, the εPKC peptide comprises a contiguous amino acid sequence corresponding to a sequence selected from the group consisting of SEQ ID NO: 9, SEQ SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 16.

[0076] In particular embodiments, the εPKC peptide comprises a contiguous amino acid sequence corresponding to amino acid residues 28-35, 49-53, and 74-79, of εPKC. In particular embodiments, the εPKC peptide comprises a contiguous amino acid sequence corresponding to a sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 12, and SEQ ID NO: 16.

[0077] In some embodiments, the εPKC peptide corresponds to the beta-sandwich region of the C2 domain of εPKC.

[0078] In some embodiments, the peptide is conjugated to a peptide that increases cellular uptake of the peptide inhibitor. In particular embodiments, the peptide that increases cellular uptake of the peptide inhibitor is TAT.

[0079] In some embodiments, the mammalian subject is a heart transplant patient.

[0080] In another aspect, an isolated peptide is provided, comprising a contiguous amino acid sequence derived from the C2 domain of a protein kinases C(PKC), wherein the peptide modulates the activity of the PKC. In some embodiments, the C2 domain of a protein kinases C (PKC) is the C2 domain of εPKC.

[0081] In some embodiments, the peptide does not include a sequence corresponding to amino acid residues 14-21 or 85-92 of the C2 domain of εPKC. In particular embodiments, the peptide is not peptide εV1-2 (SEQ ID NO: 7) or ψεRACK (SEQ ID NO: 18).

[0082] In some embodiments, the peptide is an activator of εPKC.

[0083] In some embodiments, the amino acid sequence derived from the C2 domain of a PKC corresponds to amino acid residues 28-35, 49-60, and 74-79, of εPKC. In some embodiments, the amino acid sequence derived from the C2 domain of a PKC is an amino acid sequence corresponding to a sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 16.

[0084] In particular embodiments, the amino acid sequence derived from the C2 domain of a PKC corresponds to amino acid residues 28-35, 49-53, and 74-79, of εPKC. In particular embodiments, the amino acid sequence derived from the C2 domain of a PKC is an amino acid sequence corresponding to a sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 12, and SEQ ID NO: 16.

[0085] In some embodiments, the peptide reduces ischemic cardiac damage in a mammalian subject. In particular embodiments, the peptide reduces ischemic cardiac damage in a mammalian subject.

[0086] In some embodiments, the peptide is an inhibitor of εPKC.

[0087] In some embodiments, the amino acid sequence derived from the C2 domain of a PKC corresponds to amino acid residues 40-47, 62-67, and 119-124, of εPKC. In some embodiments, the amino acid sequence derived from the C2 domain of a PKC is an amino acid sequence corresponding to a sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 14, and SEQ ID NO: 21.

[0088] In particular embodiments, the peptide is conjugated to a peptide that increases cellular uptake of the peptide inhibitor. In particular embodiments, the peptide that increases cellular uptake of the peptide inhibitor is TAT.

[0089] In addition to the exemplary aspects and embodiments described above, further aspects and embodiments will become apparent by reference to the drawings and by study of the following descriptions.

BRIEF DESCRIPTION OF THE DRAWINGS

[0090] FIGS. 1A and 1B depict a space-filling model of the C2 domain of εPKC, showing surface mapping of residues based on function. FIGS. 1C and 1D show corresponding α-carbon trace structures of the C2 domain. The structures on the right (FIGS. 1B and 1D) are rotated 180° relative to the structures on the left (FIGS. 1A and 1C).

[0091] FIG. 2A shows immunoblot analyses and resulting graphs of εPKC translocation in cells. FIG. 2B shows a histogram summarizing the effects of various peptides on εPKC translocation in cells.

[0092] FIGS. 3A-3C show analyses of the effects of C2 derived peptides on MARCKS phosphorylation in primary muscle cells.

[0093] FIGS. 4A and 4B show analyses of the effects of C2 derived peptides in an ex vivo model of ischemia and reperfusion. (A) Cross-sections of treated and control hearts stained with TTC to assess tissue infarction. (B) Graph showing relative infarct size based on staining.

[0094] FIGS. 5A-5D show immunoblot analyses and resulting graphs of εPKC translocation in heart tissue following in vivo treatment with C2-derived peptides from εPKC (A), δPKC (B), αPKC(C), and ζPKC (D).

DETAILED DESCRIPTION

I. Peptides Derived from the C2 Domain of εPKC

[0095] Two peptides derived from a portion of the C2 domain of εPKC were previously shown to selectively modulate εPKC activity. One peptide (εV1-2), interfered with interactions between εPKC and its anchoring protein, εRACK (5). The other peptide interfered with PKC auto-inhibitory, intramolecular interactions (4). The latter peptide was named ψεRACK because it increased PKC activity (as does binding to εRACK). It was heretofore unknown whether other regions of the C2 domain modulated PKC activity, and whether peptides with biological activity could be derived from the amino acid sequences of these regions.

[0096] The present compositions and methods are based on the identification of biologically active peptides corresponding to (i.e., derived from) amino acid sequences of the entire εPKC C2 domain (i.e., C2-derived peptides). FIGS. 1A-1D show the structure of the εC2 domain (49) in space filling (FIGS. 1A and 1B) and ribbon (FIGS. 1C and 1D) format. The numbering of the different regions in FIGS. 1C and 1D corresponds to the numbers shown in the following primary amino acid sequence of the εC2 domain (SEQ ID NO: 1).

##STR00001##

[0097] In the above sequence, "E" indicates β-strand regions and "H" indicates helical regions. Residues in unordered loop regions are shown as gaps (i.e., without E or H annotation). The underlined sequences correspond to peptides used in the present experiments. Shading and boxes represent sequences present in overlapping peptides. The location of the previously described εV1-2 and ψεRACK peptides are also indicated. Residues 1-136 of εPKC are shown. The εC2 region corresponds to residues 1-131 (i.e., Met-1 to Leu-131).

[0098] Most activating C2-derived peptides mapped to one face of the beta-sandwich (medium grey color), while three of the four inhibiting C2-derived peptides mapped to the dark grey regions (FIGS. 1A and 1B). No biologically active C2-derived peptides mapped to the light-grey regions. C2-derived peptides corresponding to regions 1, 2, 3, 5, 6, and 9, and the ΨεRACK region, were all εPKC-specific activators (FIGS. 1C and 1D). C2-derived peptides corresponding to regions 11 and 13 were non-specific PKC activators. C2-derived peptides corresponding to the εV1-2 region, and regions 4, 7, and 12, were εPKC-specific inhibitors. C2-derived peptides corresponding to regions 8 and 10 produced inconclusive results. These results are summarized in Table 1. The various peptides are described in more detail in the text and in Tables 2 and 3.

[0099] Without being limited to a theory, it is suggested that the C2 domain participates in important protein-protein interactions and that peptides derived from the C2 domain are useful for modulating intramolecular and/or intermolecular interactions with εPKC binding proteins. Peptides corresponding to regions of the C2 domain involved in intramolecular interactions with an inhibitory domain, or in intermolecular interactions with inhibitory proteins, are agonists of εPKC, as they disrupt inhibitory interactions. Peptides corresponding to binding sites for C2 activating proteins, such as εRACK, are antagonists.

[0100] Because the fold of the C2 domains of other PKC isozymes is structurally similar to that of the C2 domain of PKC, peptides derived from homologous positions of other isozymes are likely to have similar biological activities with respect to their cognate PKC isozyme. To illustrate the homology between different PKC isozymes, an alignment of several PKC C2 domains is shown below (49, 57, 58). Isolated peptides shown to be isozyme-specific activators are highlighted in light grey. Isolated peptides shown to be isozyme-specific inhibitors are highlighted in dark grey. Isolated peptides shown to be non-specific activators are indicated by bold lettering.

TABLE-US-00001 Alignment of the C2 domain of several PKC isozymes EEEEEEEEEEEEE EEEEEE epsil --------------MVVFNGLLKIKICEAVSLKPTAWSLRHAVGPRPQTFLLDPYIALNVDD------- --- eta ----------MSSGTMKFNGYLRVRIGEAVGLQPTRWSLRHSLFKKG-HQLLDPYLTVSVDQ--------- - delta ---------------MAPFLRISFNSYELGSLQ-------AEDEAN------QPFCAVKMKEALSTER- GKT alpha HTEKRGRIYLKAEVTD---EKLHVTVRDAKNLIPMDPNGLS-----------DPYVKLKLIPDPKNES- -K- beta SMERRGRIYIQAHIDR---EVLIVVVRDAKNLVPMDPNGLS-----------DPYVKLKLIPDPKSES-- K- EEEEE EEEEEEE EEEEEEEE EEEEEE EE epsil SRIGQTATKQKTNSPAWHDEFVTDVCNG---RKIELAVFH-DAPIGYDDFVANCTIQFEELLQN--GS- R eta VRVGQTSTKQKTNKPTYNEEFCANVTDG---GHLELAVFH-ETPLGYD-EVANCTLQFQELVGTTGASD delta --LVQKKPTM---YPEWKSTFDAHIYEG---RVIQIVLMRAAE----EPMSEVTVGVSVLAERCKKNN- G alpha ---QKTKTIRSTLNPQWNESFTFKLKPSDKDRRLSVEIWDWDRTTRNDFMGSLSEGVSELMKMP----- - beta ---QKTKTIKCSLNPEWNETERFQLKESDKDRRLSVEIWDWDLTSRNDFMGSLSFGISELQKAG----- EEEEEEE EEEEEEEEEEE epsil HFEDWIDLEPE--------------GKVYVIIDLSGSSG (SEQ ID NO: 1) eta TFEGWVDLEPE--------------GKVFVVITLT---- (SEQ ID NO: 2) delta KAEFWLDLQPQ--------------AKVLMSVQYFLED- (SEQ ID NO: 3) alpha AS-GWYKLLNQEEGEYYNVPIPEG GRIYLKAEV (SEQ ID NO: 4) beta V-DGWFKLLSQEEGEYFNVP GRIYIQAHI (SEQ ID NO: 5)

[0101] Since homologs of the C2 domain are present in over 60 different proteins (59), many of which are signaling proteins, the results of the present study are also likely to apply to proteins other than PKC, including but not limited to 5-lipoxygenase (60), pleckstrin (61), and members of the tumor necrosis factor family (62).

[0102] Experiments performed in support of the present peptides and methods are described, below.

TABLE-US-00002 TABLE 1 The biological activities of the εPKC C2- derived peptidcs and controls used in the study. εPKC MARCKS MARCKS Cardio- PKC translocation phosphorylation phosphorylation protective translocation, Peptide in cells in WT Cells in εKO Cells ex vivo in vivo 2 activator activator no effect yes NT^a 5 activator activator no effect yes activates ε and not δ α, ζPKC 6 activator activator weak activator yes NT^a 9 activator activator no effect yes NT^a ψεRACK activator activator no effect yes activates and not δ α, ζPKC 1 activator activator no effect yes† activates ε and not δ α, ζPKC 3 activator activator no effect yes.dagger-dbl. NT^a 8 activator not conclusive not conclusive no NT^a 10 activator activator not conclusive no NT^a 11 trend activator activator no non specific; towards activates activator ε, α, and δPKC 13 activator activator activator no NT^a εV1-2 inhibitor inhibitor no effect no Inhibits only εPKC^b 4 inhibitor inhibitor NT^a NT^a NT^a 7 inhibitor inhibitor NT^a no NT^a 12 inhibitor inhibitor NT^a NT^a NT^a scrambled activator no effect no effect no no εV1-2 scrambled NT^a NT^a NT^a no NT^a ψεRACK TAT no effect no effect no effect no NT^a carrier ^aNT--not tested ^bPrevious study (48) †trend towards cardioprotective (n = 5) .dagger-dbl.trend towards cardioprotective (n = 3)

II. Experiments Performed in Support of the Invention

[0103] Thirteen 5-9-residue peptides derived from amino sequences that span the C2 domain (also known as the V1 domain (29); residues 1-131) of εPKC were synthesized and tested for their ability to modulate εPKC activation using several in vitro and in vivo biological assays. In addition to the indicated C2-derived peptides, three peptides were synthesized as controls, in particular, scrambled versions of a previously established peptide activator (ψεRACK) and inhibitor (εV1-2), and the TAT carrier peptide alone. As previously described, these peptides were introduced into cells as "cargo" by conjugating them to a cell-penetrating "carrier" peptide (i.e., TAT_47-57) (43,44). Tables 2 and 3 lists all the peptides tested in experiments in support of the present peptides and methods, including the amino acid sequences, SEQ ID NOs, and corresponding region of the C2 (V1) domain.

[0104] Four assays were used to investigate the effect of the TAT-conjugated peptides on εPKC: (i) εPKC translocation in cultured cells (26), (ii) phosphorylation of the PKC substrate MARCKS in cultured cells (41,50), (iii) a cardiac protection assay (4, 6, 7) carried out using an intact heart, ex vivo, and (iv) in vivo translocation of PKC after a single intraperitoneal injection (48).

[0105] A. εPKC Translocation in Cells

[0106] Upon activation, PKC translocates from the soluble to the particulate cell fraction, which translocation can be monitored by immunoblot analysis (26). It was previously determined that the effect of peptides on PKC translocation is better observed when the cells are treated with a sub-maximal concentration of PMA (22,42). Larger concentrations of PMA, a non-physiological activator, stimulate a greater degree of PKC translocation over translocation stimulated by peptide activators alone (51). PKC translocation upon peptide treatment, although less than that seen with large amounts of PMA, has been shown to better correlate with other experimental data (4, 6, 22)).

[0107] Pre-treatment with most peptides prior to PMA-stimulation increased the amount of εPKC in the pellet fraction and a decreased the amount in the soluble fraction, indicating that the peptides stimulated (activated) εPKC translocation. FIG. 2A shows representative immunoblot analyses and accompanying graphs of the data. A representative activator (peptide 2) or inhibitor (peptide 7) was preincubated at 500 nM for 15 minutes with cells before sub-optimal stimulation with 3 nM PMA for 7 minutes.

TABLE-US-00003 TABLE 2 TAT-conjugated peptides εPKC C2-derived peptides and controls used in the study assays. Primary SEQ ID Origin of Peptide Sequence NO: Sequence 1 CFNGLLKIKI^a 6 εC2/V1_4-12 εV1-2^b CEAVSLKPT^a 7 εC2/V1_14-21 scrambled CLSETKPAV^a 8 scrambled εV1-2^d εC2/V1_14-21 2 CAVGPRPQT^a 9 εC2/V1_28-35 3 CFLLDPY^a 10 εC2/V1_36-41 4 CPYIALNVD^a 11 εC2/V1_40-47 5 CSRIGQ^a 12 εC2/V1_49-53 6 CTATKQKT^a 13 εC2/V1_54-60 7 CPAWHD^a 14 εC2/V1_62-67 8 CEFVTDV^a 15 εC2/V1_68-73 9 CNGRKI^a 16 εC2/V1_74-79 10 CIELAVF^a 17 εC2/V1_79-84 ψεRACK^c CHDAPIGYD^a 18 εC2/V1_85-92 scrambled CPDYHDAGI^a 19 scrambled ψεRACK^d εC2/V1_85-92 11 CHFEDWID^a 20 εC2/V1_112-118 12 CLEPEGK^a 21 εC2/V1_119-124 13 CVYVIIDL^a 22 εC2/V1_125-131 TAT carrier^d CYGRKKRRQRRR 23 TAT_47-57 ^aPeptides conjugated by Cys (underlined) S-S bond to TAT_47-57 carrier peptide. ^bPreviously published εPKC-specific inhibitor, εV1-2 (5). ^cPreviously published εPKC-specific activator, ψεRACK (4). ^dUsed as a control peptide; corresponds to SEQ ID NO: 31 with an additional, N-terminal C.

TABLE-US-00004 TABLE 3 Additional peptides with known PKC-modulating acivity Peptide Isozyme Sequence† SEQ ID NO: Activity Ref. ψβRACK βPKC SVEIWD 24 activate all the (31) classical PKC isozymes βC2-4 βPKC SLNPEWNET 25 inhibit of all the (31) classical PKC isozymes βC2-2 βPKC MDPNGLSDPYVKL 26 inhibit of all the (31) classical PKC isozymes δψRACK δPKC MRAAEDPM 27 agonist, selective for (22) δPKC δV1-1 δPKC SFNSYELGSL 28 antagonist, selective for (22) δPKC -- ηPKC EAVGLQPT 29 inhibitor (1, 63) -- ηPKC HETPLGYD 30 activator (1, 63) †Shown without the additional N-terminal C residue used for conjugation.

[0108] Pretreatment with peptide 2 increased the amount of εPKC translocation by approximately 30% compared to pre-treatment with the TAT control peptide (n=5, p<0.05). Pretreatment with peptide 7 decreased the amount of translocated εPKC by approximately 50% compared to pre-treatment with the TAT control peptide (n=3, p<0.01). Statistical significance was determined by Students t-Tcst using Microsoft Excel.

[0109] The histogram shown in FIG. 2B summarizes the effects of the various peptides on εPKC translocation in cells. As noted above, increased translocation indicates activation of PKC, while decrease translocation indicates inhibition. All the peptides altered εPKC translocation. Pre-treatment with four peptides, i.e., peptides 4, 7, 12, and εV1-2 (which was previously reported to be an εPKC-selective inhibitor) before PMA stimulation resulted in a decrease in the amount of εPKC in the pellet fraction relative to control, indicating that these peptides may act as inhibitors of εPKC translocation. The scrambled εV1-2 peptide (LSETKPAV)) also stimulated εPKC translocation in CHO cells, although this result was not apparent from other assays described, herein. Pre-treatment with TAT carrier peptide alone had no effect on PKC translocation. All data were statistically significant (n>3, p<0.05), as determined by Students t-Test using Microsoft Excel.

[0110] B. PKC Substrate Phosphorylation in Cells

[0111] While translocation is a rapid and convenient in vitro assay, it is not strictly indicative of PKC activation (26). A more direct and sensitive measure of PKC activation is PKC substrate phosphorylation. Because there are no known εPKC-specific substrates, myristoylated alanine-rich C kinase substrate (MARCKS) was used. MARCKS is a widely distributed actin cross-linking protein that is highly phosphorylated on serine residues (50,52) following PKC activation.

[0112] The level of MARCKS phosphorylation was determined using antibodies specific for phosphorylated MARCKS, following incubation of primary mouse muscle cells from wild-type (WT, FIGS. 3A and 3C) or εPKC knock-out (KO, FIG. 3B) mice with 1 μM each peptide for 15 minutes (FIGS. 3A and 3B) or 30 minutes (FIG. 3C) prior to cell lysis. This assay has been used before to show that integrin-induced ε, α and δPKC activation leads to MARCKS phosphorylation (41). The data shown are from several experiments (indicated in each column) and statistical significance was determined by Student's t-Test using Microsoft Excel.

[0113] Dark gray bars indicate no significant difference compared to control; light gray bars in FIG. 3B indicate a trend towards an effect that was not statistically significant; white bars indicate a significant effect. Actin was used as a loading control. In total, 11 of the peptides increased MARCKS phosphorylation compared to no peptide treatment or TAT-control treatment (FIG. 3A and Table 2), indicating that these peptides were PKC activators. No change in MARCKS phosphorylation was observed following treatment with the control peptides scrambled εV1-2 or scrambled ψεRACK. Only peptide 8 had no conclusive effect on MARCKS phosphorylation.

[0114] The availability of εPKC-knock out (KO) mice and cells derived therefrom, increased the utility of the MARCKS phosphorylation assay. Changes in the levels of phosphorylated MARCKS in cells obtained from both WT and εPKC KO mice indicated that the effects of the TAT-conjugated peptides were not selective for εPKC. However, changes only in cells obtained from WT mice indicated that the effects of the TAT-conjugated peptides were specific to the εPKC isozyme Most of the peptides (six out of eleven activators: peptides 1, 2, 3, 5, 9, and ψεRACK) increased MARCKS phosphorylation in WT but not in KO muscle cells and are therefore εPKC-selective activators (FIGS. 3A and 3B, Table 2). A few peptides increased MARCKS phosphorylation (e.g., peptides 11 and 13) in both cell types and are therefore not specific for εPKC. In no case did a peptide affect MARCKS phosphorylation in KO and not WT cells. The MARCKS phosphorylation assay was primarily used to show a gain of function (i.e., PKC activation); nonetheless, peptides 4, 7, 12, and EV1-2 inhibited MARCKS phosphorylation in WT cells.

[0115] This phosphorylation assay suggested that several peptides derived from the C2 domain of εPKC affected the activity of PKC, and that a subset of these peptides were isozyme-selective.

[0116] C. Ex Vivo Cardiac Protection

[0117] An ex vivo model of ischemia and reperfusion (47) was used to further characterize the C2-derived peptides. Studies have demonstrated that activation of εPKC in hearts prior to an ischemic event leads to reduced damage of the myocardium (4, 7, 53-55). Pre-treatment of hearts subjected to ischemia and reperfusion ex vivo (using a Langendorff apparatus) with the ψεRACK peptide led to reduced damage (infarction) (4,43). Because other PKC isozymes have no effect on cardiac protection (e.g., βPKC (43)), or even have opposing effects to εPKC activation (e.g., δPKC activation increases damage (22)), changes in cardiac protection following perfusion with a εC2-derived peptide indicates only εPKC modulation.

[0118] FIGS. 4A and 4B show analyses of the effects of C2 derived peptides in an ex vivo model of ischemia and reperfusion. FIG. 4A shows representative cross-sections of treated and control hearts stained with 2,3,5-triphenyltetrazolium chloride (TTC) to assess tissue infarction. Live tissue stained darkly, while dead tissue remained white. FIG. 4B shows quantitation of infarct size based on the staining. The data are an average of several experiments (indicated in each column) and statistical significance was determined by Student's t-Test using Microsoft Excel. Bar shading is the same as in FIG. 3. With the exception of peptide εV1-2, none of the peptides that demonstrated inhibitory activity in the translocation or MARCKS phosphorylation assays (i.e., peptides 4, 7 and 12) were tested in the ex vivo assay.

[0119] Based on the level of damage observed by TTC staining (FIG. 4A) and release of CPK (a cytosolic enzyme that is released from the myocytes when the muscle is damaged; FIG. 4B), it was concluded that peptides 2, 5, 6, and 9, as well as the previously characterized εPKC-selective agonist, ψεRACK, were cardioprotective, indicating that these peptides are εPKC-selective activators. Peptides 1 and 3 showed a trend towards cardioprotection, but the data were not statistically significant. The four peptides that did not induce MARCKS phosphorylation in WT and KO skeletal muscle cells, i.e., peptide 8, and the three control peptides (scrambled εV1-2, scrambled ψεRACK, and TAT), were not cardioprotective. Peptides 11 and 13, which induced MARCKS phosphorylation in both WT and KO cells were also not cardioprotective, possibly because in addition to εPKC they activate the opposing isozyme δPKC (22). Note that treatment with peptide 9 involved hearts subjected to only 30 minutes of ischemia instead of 40 minutes. These results of the ex vivo cardiac protection assay are summarized in Table 2.

[0120] D. In vivo PKC Translocation

[0121] In a further in vivo study, peptide activity and selectivity were examined using representative peptides injected into mice via intraperitoneal (IP) injection, and the amount of εPKC, δPKC, αPKC, and ζPKC translocation in hearts was determined. These four PKC isozymes represent each of the three major sub-families of PKC. In particular, εPKC and δPKC are members of the novel PKC family (calcium insensitive, diacylglycerol sensitive), αPKC is a classical PKC (calcium and diacylglycerol sensitive), and ζPKC is an atypical PKC (calcium and diacylglycerol insensitive). A previous study demonstrated the effects of PKC regulating peptides on PKC translocation in hearts and other organs after a single intraperitoneal injection (48).

[0122] Levels of εPKC, δPKC, αPKC, and ζPKC in the particulate fraction of mouse hearts were assayed by immunoblot 15 minutes following IP injection of 20 nmol each peptide (FIGS. 5A-5D). The accompanying graphs show quantitation based on two experiments, calculated as fold increase (+/-standard error) in the particulate fraction/total fraction, normalized to the actin loading control, and relative to the control TAT peptide. The ψεRACK peptide, peptide 1, and peptide 5 caused translocation of εPKC, but not δPKC, ζPKC, or αPKC, indicating that these peptides were selective εPKC activators. Since peptides 11 and 13 caused MARCKS phosphorylation in myocytes from the εPKC knockout mice (FIG. 3), it is concluded that they activated several PKC isozymes. Further, because these peptides did not protect the heart from ischemic damage, it is believed that they activate both the cardiac protective isozyme, εPKC (4, 22, 56), and one or more isozymes that mediates cardiac damage, such as δPKC (22,38). IP injection of peptide 11 caused a substantial translocation of δPKC, αPKC, and εPKC (FIG. 5C), consistent with other observations suggesting that peptide 11 is a promiscuous activator of PKC.

[0123] Atypical ζPKC, present only in the soluble cell fraction, did not translocate in response to any peptide tested (FIG. 5D). Control peptide (scrambled εV1-2) did not cause an increase in either εPKC or δPKC (FIG. 5C).

[0124] E. Summary of Results

[0125] The peptides tested in the above experiments encompassed almost all the β-sheet regions and parts of the top and bottom loop regions of the εC2 domain (FIGS. 1A-1D). The biological activity of each peptide is shown in Table 1. All the peptides derived from the C2 domain of εPKC modulated the activity of εPKC in the two cell-based assays (i.e., translocation and MARCKS phosphorylation; FIGS. 2 and 3). Of these, 10 peptides were εPKC activators (peptides 1, 2, 3, 5, 6, 9, 10, ψεRACK, 11, and 13) as demonstrated by an increase in εPKC translocation and an increase in MARCKS phosphorylation. These peptides correspond to εPKC residues 4-12, 28-41, 49-60, 74-92, 112-118, and 125-131. Five peptides that activated εPKC in the two cell-based assays were cardioprotective, i.e., peptides 2, 5, 6, 9, and ψεRACK, corresponding to εPKC residues 28-35, 49-60, 74-79, and 85-92; FIG. 4. Of these five peptides, four (peptides 2, 5, 9, and ψεRACK; i.e., residues 28-35, 49-53, 74-79, and 85-92) were selective for εPKC as determined by MARCKS phosphorylation using cells obtained from WT or εPKC KO mice (FIG. 3).

[0126] Two of the peptides (peptides 11 and 13) activated PKC in vitro but were not selective for εPKC (FIGS. 2 and 3). These peptides correspond to εPKC residues 112-118 and 125-131. The two peptides did not induce cardiac protection from ischemia and reperfusion (FIG. 4), suggesting that they affect PKC isozymes (e.g., δPKC) that increases cardiac damage by ischemia (22). Translocation studies in vivo confirmed that peptide 11 activated δ and εPKC as well as αPKC (FIG. 5).

[0127] Four peptides (peptides εV1-2, 4, 7, and 12) were εPKC inhibitors (FIGS. 2 and 3). These peptides correspond to εPKC residues 14-21, 40-47, 62-67, and 119-124.

[0128] Together, these results demonstrate the ability of εC2 domain-derived peptides to modulate εPKC biological activity as activators or inhibitors, depending on the particular region of the C2 domain represented by the peptide.

III. Treatment Methods

[0129] The data described herein show that activator peptides derived from the C2 region of εPKC are cardioprotective in an animal model for ischemic heart damage. Such peptides are useful for slowing or inhibiting the progression of heart failure following ischemia, prolonging survival, reducing fractional shortening, reducing left ventricular weight to body weight ratio, reducing fibrosis, causing the EKG/ECG of a subject to more closely resemble that of a healthy animal, and/or combinations thereof. The peptides may be of particular value in protecting a heart from ischemic damage during a transplantation procedure.

[0130] C2-derived peptides for administration to humans or other animals (preferably mammals) may include natural amino acids, such as the L-amino acids or non-natural amino acids, such as b-amino acids. The amino acids in the peptide may be linked by peptide bonds or, in modified peptides described herein, by non-peptide bonds. A wide variety of modifications to the amide bonds which link amino acids may be made and are known in the art. Such modifications are discussed in general reviews (e.g., Freidinger, R. M. (2003) J. Med. Chem. 46:5553; Ripka, A. S. and Rich, D. H. (1998) Curr. Opin. Chem. Biol. 2:441). These modifications are designed to improve the properties of the peptide by increasing the potency of the peptide or by increasing the half-life of the peptide.

[0131] The potency of the peptide may be increased by restricting conformational flexibility. This may be achieved by, for example, including the placement of additional alkyl groups on the nitrogen or alpha-carbon of the amide bond, such as the peptoid strategy of Zuckerman et al. and the alpha modifications of Goodman et. al. (see, e.g., (1996) Pure Appl. Chem. 68:1303). The amide nitrogen and alpha carbon may be linked together to provide additional constraint (Scott et al. (2004) Org. Letts. 6:1629-32).

[0132] The half-life of the peptide may be increased by introducing non-degradable moieties to the peptide chain. This may be achieved by, for example, replacement of the amide bond by a urea residue (Patil et al. (2003) J. Org. Chem. 68:7274-80) or an aza-peptide link (Zega and Urleb (2002) Acta Chim. Slov. 49:649-62). Other examples of non-degradable moieties that may be introduced to the peptide chain include introduction of an additional carbon "beta peptide" (Gellman, S. H. (1998) Acc. Chem. Res. 31:173) or ethene unit (Hagihara et al. (1992) J. Am. Chem. Soc. 114:6568) to the chain, or the use of hydroxyethylene moieties (Patani, G. A. and Lavoie, E. J. (1996) Chem. Rev. 96:3147-76) and are also well known in the art. Additionally, one or more amino acids may be replaced by an isosteric moiety such as, for example, the pyrrolinones of Hirschmann et al ((2000) J. Am. Chem. Soc. 122:11037), or tetrahydropyrans (Kulesza, A. et al. (2003) Org. Letts. 5:1163). A common modification to increase half-life is pegylation.

[0133] Although the peptides are described primarily with reference to amino acid sequences from Rattus norvegicus, it is understood that the peptides are not limited to the specific amino acid sequences set forth herein. Skilled artisans will recognize that, through the process of mutation and/or evolution, polypeptides of different lengths and having different constituents, e.g., with amino acid insertions, substitutions, deletions, and the like, may arise that are related to, or sufficiently similar, to, a sequence set forth herein by virtue of amino acid sequence homology and advantageous functionality as described herein.

[0134] The peptides described herein encompass amino acid sequences similar to the amino acid sequences set forth herein that have at least about 50% identity thereto and function in a similar manner. Preferably, the amino acid sequences have at least about 60% identity, at least about 70% identity, at least about 80% identity, at least about 90% identity, or at least about 95% identity to described sequences. Exemplary percent identity values are at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%. Percent identity may be determined, for example, by comparing sequence information using the advanced BLAST computer program, including version 2.2.9, available from the National Institutes of Health. The BLAST program is based on the alignment method of Karlin and Altschul. ((1990) Proc. Nat'l. Acad. Sci. U.S.A. 87:2264-68) and as discussed in Altschul et al. ((1990) J. Mol. Biol. 215:403-10); Karlin and Altschul ((1993) Proc. Nat'l. Acad. Sci. U.S.A. 90:5873-77); and Altschul et al. ((1997) Nucleic Acids Res. 25:3389-3402).

[0135] Conservative amino acid substitutions may be made in the amino acid sequences to obtain derivatives of the peptides that may advantageously be utilized in the present invention. Conservative amino acid substitutions, as known in the art and as referred to herein, involve substituting amino acids in a protein with amino acids having similar side chains in terms of, for example, structure, size and/or chemical properties. For example, the amino acids within each of the following groups may be interchanged with other amino acids in the same group: amino acids having aliphatic side chains, including glycine, alanine, valine, leucine and isoleucine; amino acids having non-aromatic, hydroxyl-containing side chains, such as serine and threonine; amino acids having acidic side chains, such as aspartic acid and glutamic acid; amino acids having amide side chains, including glutamine and asparagine; basic amino acids, including lysine, arginine and histidine; amino acids having aromatic ring side chains, including phenylalanine, tyrosine and tryptophan; and amino acids having sulfur-containing side chains, including cysteine and methionine. Additionally, aspartic acid, glutamic acid and their amides, are also considered interchangeable herein.

[0136] In some embodiments, the C2-derived peptide is conjugated to a cell permeable carrier protein or peptide that may increase cellular uptake of the peptide inhibitor. In one example, a Drosophila Antennapedia homeodomain-derived sequence (CRQIKIWFQNRRMKWKK; SEQ ID NO: 32), is attached (i.e., conjugated) to the C2-derived peptide by cross-linking via an N-terminal Cys-Cys bond as discussed in Theodore, L. et al. ((1995), J. Neurosci. 15:7158-67) and Johnson, J. A. et al. ((1996) Circ. Res 79:1086). In some embodiments, the C2-derived peptides are conjugated to cell permeable carrier peptides between cysteine residues on the amino or carboxy-termini of the peptides. Such cysteine residues may be part of the naturally-occurring peptide sequence or may be added to the naturally-occurring peptide sequence.

[0137] The present peptides were modified by a Transactivating Regulatory Protein (Tat)-derived transport polypeptide (such as from amino acids 47-57 of Tat (YGRKKRRQRRR; SEQ ID NO: 31) from the Human Immunodeficiency Virus, Type 1, as described in Vives et al. ((1997) J. Biol. Chem., 272:16010-17), U.S. Pat. No. 5,804,604, and Genbank Accession No. AAT48070; or with polyarginine as described in Mitchell et al. ((2000) J. Peptide Res. 56:318-25) and Rothbard et al. (2000) Nature Med. 6:1253-57). Such TAT derived cell permeable carrier peptides may be conjugated to C2-derived peptides between cysteine residues, as described immediately above.

[0138] C2-derived peptides having both PKC sequences and cell permeable carrier peptide sequences, with or without additional cysteine residues, may also be synthesized using standard methods, e.g., to produce a single peptide having both inhibitor peptide and cell permeable carrier peptide sequences. C2-derived peptides may be modified by other methods known to the skilled artisan in order to increase the cellular uptake of the peptides.

[0139] The C2-derived peptides may be advantageously administered in various forms. For example, the C2-derived peptides may be administered in tablet form for sublingual administration, in a solution or emulsion. The C2-derived peptides may also be mixed with a pharmaceutically-acceptable carrier or vehicle. The vehicle may be a liquid, suitable, for example, for parenteral administration, including water, saline or other aqueous solution, or may be an oil or aerosol. The carrier may be selected for intravenous or intraarterial administration, and may include a sterile aqueous or non-aqueous solution that may include preservatives, bacteriostats, buffers and antioxidants known to the art. In the aerosol form, the inhibitor may be used as a powder, with properties including particle size, morphology and surface energy known to the art for optimal dispersability. In tablet form, a solid carrier may include, for example, lactose, starch, carboxymethyl cellulose, dextrin, calcium phosphate, calcium carbonate, synthetic or natural calcium allocate, magnesium oxide, dry aluminum hydroxide, magnesium stearate, sodium bicarbonate, dry yeast or a combination thereof. The tablet preferably includes one or more agents which aid in oral dissolution. The C2-derived peptides may also be administered in forms in which other similar drugs known in the art are administered.

[0140] The C2-derived peptides may be administered to a patient by a variety of routes. For example, the peptides may be administered parenterally, including intraperitoneally, intravenously, intraarterially, subcutaneously, or intramuscularly. The C2-derived peptides may also be administered via a mucosal surface, including rectally, and intravaginally; intranasally, including by inhalation; sublingually; intraocularly and transdermally. Combinations of these routes of administration are also envisioned. A preferred mode of administration is by infusion or reperfusion through an artery, or an artery that is connected to such an occluded or partially-occluded artery. By "partially-occluded artery" it is meant herein an artery in which blood flow is reduced after an ischemic attack or other hypoxic event affecting the heart blood vessels when compared to blood flow prior to such event or attack.

[0141] In certain embodiments, C2-derived peptides may be co-administered in a composition with a second therapeutic agent. In this manner, one skilled in the art will recognize that C2-derived peptides individually, in combination, or combined with a second therapeutic agent, may be used to prepare a medicament for the slowing or inhibiting the progression of ischemic heart failure.

[0142] Further aspects and embodiments will be apparent to the skilled artisan in view of the present teachings. The foregoing description and the following examples are not intended to be limiting.

EXAMPLES

Experimental Procedures

[0143] Peptide Synthesis--Peptides were synthesized and conjugated to TAT carrier peptide (residues 47-57) via cysteine S--S bond by Anaspec, San Jose, Calif. Peptides are five to eight residues and represent sequences in the C2/V1 domain of εPKC. Three control peptides have also been synthesized: scrambled εV1-2, scrambled εψRACK, and TAT carrier peptide alone. See Table 1 for a summary of peptides synthesized, including sequences.

[0144] PKC Translocation in Cells--εPKC translocation from the soluble to the particulate fraction in CHO cells was used to assess the relative amount of activated (membrane-bound) εPKC, an assay that has been described previously (42). Evidence of translocation is either by an increase in the amount of PKC in the particulate fraction or by a decrease in the soluble fraction. Briefly, after stimulation, cells were washed with cold PBS, scraped in homogenization buffer, passed through a (25G5/8'') syringe needle, and spun at 100,000g for 30 min at 4° C. The pellet was then resuspended in homogenization buffer with 1% Triton X-100. Where applicable, the cells were pre-incubated with 500 nM peptide for 15 minutes prior to stimulation. PKC was stimulated with sub-maximal levels of the general PKC activator phorbol 12-myristate 13-acetate (PMA, 3 nM). The samples were then analyzed by Western blot. Antibodies against εPKC were obtained from Santa Cruz Biotechnology (Santa Cruz, Calif.) and used at 1:500 dilution. Antibodies against actin, used as a loading control, were obtained from Sigma-Aldrich (St. Louis, Mo.) and used at 1:1000 dilution.

[0145] PKC Substrate Phosphorylation in Cells--Phosphorylation of myristoylated alanine-rich C kinase substrate (MARCKS), a general PKC substrate, was monitored by Western blot of cell lysates. To assess PKC activation by the peptides and their specificity for εPKC, MARCKS phosphorylation in wild-type primary skeletal muscle cells was compared with MARCKS phosphorylation in primary skeletal muscle cells isolated from PKC knockout mice (45), as we previously described (46). Primary skeletal muscle cells were prepared as previously published (46). εKO cells were prepared from εPKC knockout mice (45). For the εPKC inhibition assay, WT cells were treated with 1 μM peptide 7 for 15 minutes and then ψεRACK (500 nM) was added for 30 minutes prior to cell lysis. Antibodies against phosphorylated MARCKS were obtained from Cell Signaling (Danvers, Mass.) and used at 1:500 dilution. Antibodies against actin, used as a loading control, were obtained from Sigma-Aldrich (St. Louis, Mo.) and used at 1:1000 dilution.

[0146] Ex Vivo Cardiac Protection--Activation of εPKC prior to ischemia mediates cardiac protection in an ex vivo model of acute ischemic heart damage (13), an assay that has been described previously (47). Briefly, Wistar rats (300 to 350 g) were heparinized (1000 U/kg IP) and then anesthetized with sodium pentobarbital (100 mg/kg IP). Hearts were rapidly excised and then perfused with an oxygenated Krebs-Henseleit buffer containing NaCl (120 mmol/L), KCl (5.8 mmol/L), NaHCO3 (25 mmol/L), NaH2PO4 (1.2 mmol/L), MgSO4 (1.2 mmol/L), CaCl2 (1.0 mmol/L), and dextrose (10 mmol/L) at pH 7.4 and 37° C. in a Langendorff coronary perfusion system. A constant coronary flow rate of 10 mL/min was used. Hearts were submerged into a heat-jacketed organ bath at 37° C. Coronary effluent was collected to determine creatine phosphokinase (CPK) release. After 10 minutes of equilibration, the hearts were subjected to 40 minutes global ischemia and 60 minutes reperfusion. The hearts were perfused with 1 μM TAT-conjugated peptide for 10 minutes prior to ischemia. In addition to the relative amount of CPK released, a measure of cardiac myocyte lysis, tissue damage was assessed by triphenyl tetrazolium chloride (TTC) staining of heart cross-sections to quantitate the amount of infarcted (dead) tissue, as we previously described (38).

[0147] Isozyme Selectivity In Vivo--To determine that activator peptides causing MARCKS phosphorylation and decreasing ischemia-reperfusion damage in heart also induce εPKC translocation in vivo, we injected the respective peptides at 20 nmol in 200 μl saline into the peritoneum of 15-20 g mice, as previously reported (48). Fifteen minutes later, the mice were sacrificed and heart and brain were collected. Soluble and particulate fractions from mouse tissue were prepared as previously described (48). ε, ζ and δPKC translocation were determined by Western blot analysis using selective anti-PKC antibodies from Santa Cruz Biotechnology, Santa Cruz, Calif. and used at 1:500 dilution. Sarcomeric actin (1:1000, Sigma) was used as a loading control for all fractions.

[0148] While a number of exemplary aspects and embodiments have been discussed above, those of skill in the art will recognize certain modifications, permutations, additions and sub-combinations thereof. It is therefore intended that the following appended claims and claims hereafter introduced are interpreted to include all such modifications, permutations, additions and sub-combinations as are within their true spirit and scope.

Sequence CWU 1

321136PRTArtificial Sequenceepsilon PKC C2 domain 1Met Val Val Phe Asn Gly Leu Leu Lys Ile Lys Ile Cys Glu Ala Val1 5 10 15Ser Leu Lys Pro Thr Ala Trp Ser Leu Arg His Ala Val Gly Pro Arg 20 25 30Pro Gln Thr Phe Leu Leu Asp Pro Tyr Ile Ala Leu Asn Val Asp Asp 35 40 45Ser Arg Ile Gly Gln Thr Ala Thr Lys Gln Lys Thr Asn Ser Pro Ala 50 55 60Trp His Asp Glu Phe Val Thr Asp Val Cys Asn Gly Arg Lys Ile Glu65 70 75 80Leu Ala Val Phe His Asp Ala Pro Ile Gly Tyr Asp Asp Phe Val Ala 85 90 95Asn Cys Thr Ile Gln Phe Glu Glu Leu Leu Gln Asn Gly Ser Arg His 100 105 110Phe Glu Asp Trp Ile Asp Leu Glu Pro Glu Gly Lys Val Tyr Val Ile 115 120 125Ile Asp Leu Ser Gly Ser Ser Gly 130 1352136PRTArtificial Sequenceeta PKC C2 domain 2Met Ser Ser Gly Thr Met Lys Phe Asn Gly Tyr Leu Arg Val Arg Ile1 5 10 15Gly Glu Ala Val Gly Leu Gln Pro Thr Arg Trp Ser Leu Arg His Ser 20 25 30Leu Phe Lys Lys Gly His Gln Leu Leu Asp Pro Tyr Leu Thr Val Ser 35 40 45Val Asp Gln Val Arg Val Gly Gln Thr Ser Thr Lys Gln Lys Thr Asn 50 55 60Lys Pro Thr Tyr Asn Glu Glu Phe Cys Ala Asn Val Thr Asp Gly Gly65 70 75 80His Leu Glu Leu Ala Val Phe His Glu Thr Pro Leu Gly Tyr Asp Phe 85 90 95Val Ala Asn Cys Thr Leu Gln Phe Gln Glu Leu Val Gly Thr Thr Gly 100 105 110Ala Ser Asp Thr Phe Glu Gly Trp Val Asp Leu Glu Pro Glu Gly Lys 115 120 125Val Phe Val Val Ile Thr Leu Thr 130 1353124PRTArtificial Sequencedelta PKC C2 domain 3Met Ala Pro Phe Leu Arg Ile Ser Phe Asn Ser Tyr Glu Leu Gly Ser1 5 10 15Leu Gln Ala Glu Asp Glu Ala Asn Gln Pro Phe Cys Ala Val Lys Met 20 25 30Lys Glu Ala Leu Ser Thr Glu Arg Gly Lys Thr Leu Val Gln Lys Lys 35 40 45Pro Thr Met Tyr Pro Glu Trp Lys Ser Thr Phe Asp Ala His Ile Tyr 50 55 60Glu Gly Arg Val Ile Gln Ile Val Leu Met Arg Ala Ala Glu Glu Pro65 70 75 80Met Ser Glu Val Thr Val Gly Val Ser Val Leu Ala Glu Arg Cys Lys 85 90 95Lys Asn Asn Gly Lys Ala Glu Phe Trp Leu Asp Leu Gln Pro Gln Ala 100 105 110Lys Val Leu Met Ser Val Gln Tyr Phe Leu Glu Asp 115 1204148PRTArtificial Sequencealpha PKC C2 domain 4His Thr Glu Lys Arg Gly Arg Ile Tyr Leu Lys Ala Glu Val Thr Asp1 5 10 15Glu Lys Leu His Val Thr Val Arg Asp Ala Lys Asn Leu Ile Pro Met 20 25 30Asp Pro Asn Gly Leu Ser Asp Pro Tyr Val Lys Leu Lys Leu Ile Pro 35 40 45Asp Pro Lys Asn Glu Ser Lys Gln Lys Thr Lys Thr Ile Arg Ser Thr 50 55 60Leu Asn Pro Gln Trp Asn Glu Ser Phe Thr Phe Lys Leu Lys Pro Ser65 70 75 80Asp Lys Asp Arg Arg Leu Ser Val Glu Ile Trp Asp Trp Asp Arg Thr 85 90 95Thr Arg Asn Asp Phe Met Gly Ser Leu Ser Phe Gly Val Ser Glu Leu 100 105 110Met Lys Met Pro Ala Ser Gly Trp Tyr Lys Leu Leu Asn Gln Glu Glu 115 120 125Gly Glu Tyr Tyr Asn Val Pro Ile Pro Glu Gly Gly Arg Ile Tyr Leu 130 135 140Lys Ala Glu Val1455144PRTArtificial Sequencebeta PKC C2 domain 5Ser Met Glu Arg Arg Gly Arg Ile Tyr Ile Gln Ala His Ile Asp Arg1 5 10 15Glu Val Leu Ile Val Val Val Arg Asp Ala Lys Asn Leu Val Pro Met 20 25 30Asp Pro Asn Gly Leu Ser Asp Pro Tyr Val Lys Leu Lys Leu Ile Pro 35 40 45Asp Pro Lys Ser Glu Ser Lys Gln Lys Thr Lys Thr Ile Lys Cys Ser 50 55 60Leu Asn Pro Glu Trp Asn Glu Thr Phe Arg Phe Gln Leu Lys Glu Ser65 70 75 80Asp Lys Asp Arg Arg Leu Ser Val Glu Ile Trp Asp Trp Asp Leu Thr 85 90 95Ser Arg Asn Asp Phe Met Gly Ser Leu Ser Phe Gly Ile Ser Glu Leu 100 105 110Gln Lys Ala Gly Val Asp Gly Trp Phe Lys Leu Leu Ser Gln Glu Glu 115 120 125Gly Glu Tyr Phe Asn Val Pro Gly Arg Ile Tyr Ile Gln Ala His Ile 130 135 140610PRTArtificial Sequenceepsilon C2/V1(4-12) 6Cys Phe Asn Gly Leu Leu Lys Ile Lys Ile1 5 1079PRTArtificial Sequenceepsilon V1-2 7Cys Glu Ala Val Ser Leu Lys Pro Thr1 589PRTArtificial Sequencescrambled epsilon V1-2 peptide 8Cys Leu Ser Glu Thr Lys Pro Ala Val1 599PRTArtificial Sequenceepsilon C2/V1(28-35) 9Cys Ala Val Gly Pro Arg Pro Gln Thr1 5107PRTArtificial Sequenceepsilon C2/V1 (36-41) 10Cys Phe Leu Leu Asp Pro Tyr1 5119PRTArtificial Sequenceepsilon C2/V1(40-47) 11Cys Pro Tyr Ile Ala Leu Asn Val Asp1 5126PRTArtificial Sequenceepsilon C2/V1(49-53) 12Cys Ser Arg Ile Gly Gln1 5138PRTArtificial Sequenceepsilon C2/V1(54-60) 13Cys Thr Ala Thr Lys Gln Lys Thr1 5146PRTArtificial Sequenceepsilon C2/V1(62-67) 14Cys Pro Ala Trp His Asp1 5157PRTArtificial Sequenceepsilon C2/V1(68-73) 15Cys Glu Phe Val Thr Asp Val1 5166PRTArtificial Sequenceepsilon C2/V1(74-79) 16Cys Asn Gly Arg Lys Ile1 5177PRTArtificial Sequenceepsilon C2/V1(79-84) 17Cys Ile Glu Leu Ala Val Phe1 5189PRTArtificial Sequencepseudoepsilon RACK peptide 18Cys His Asp Ala Pro Ile Gly Tyr Asp1 5199PRTArtificial Sequencescrambled pseudoepsilon RACK peptide 19Cys Pro Asp Tyr His Asp Ala Gly Ile1 5208PRTArtificial Sequenceepsilon C2/V1(112-118) 20Cys His Phe Glu Asp Trp Ile Asp1 5217PRTArtificial Sequenceepsilon C2/V1(119-124) 21Cys Leu Glu Pro Glu Gly Lys1 5228PRTArtificial Sequenceepsilon C2/V1(125-131) 22Cys Val Tyr Val Ile Ile Asp Leu1 52312PRTArtificial SequenceTAT carrier peptide with N-terminal cysteine 23Cys Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg1 5 10246PRTArtificial SequencepseudobetaRACK peptide 24Ser Val Glu Ile Trp Asp1 5259PRTArtificial Sequencebeta C2-4 peptide 25Ser Leu Asn Pro Glu Trp Asn Glu Thr1 52613PRTArtificial Sequencebeta C2-2 peptide 26Met Asp Pro Asn Gly Leu Ser Asp Pro Tyr Val Lys Leu1 5 10278PRTArtificial SequencedeltapseudoRACK peptide 27Met Arg Ala Ala Glu Asp Pro Met1 52810PRTArtificial Sequencepeptide delta V1-1 28Ser Phe Asn Ser Tyr Glu Leu Gly Ser Leu1 5 10298PRTArtificial Sequencepeptide derived from eta PKC 29Glu Ala Val Gly Leu Gln Pro Thr1 5308PRTArtificial Sequencepeptide derived from eta PKC 30His Glu Thr Pro Leu Gly Tyr Asp1 53111PRTArtificial SequenceTAT carrier protein 31Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg1 5 103217PRTArtificial SequenceAntennapedia homeodomain peptide 32Cys Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys1 5 10 15Lys

Claims

1. A method for modulating ischemic cardiac damage in a mammalian subject comprising administering a therapeutically effective amount of a peptide comprising a contiguous amino acid sequence derived from the C2 domain of εPKC.

2. The method of claim 1, wherein the peptide does not include an amino acid sequence corresponding to amino acid residues 85-92 of the C2 domain of εPKC.

3. The method of claim 1, wherein the εPKC peptide is not ψεRACK (SEQ ID NO:18).

4. The method of claim 1, wherein the εPKC peptide comprises a contiguous amino acid sequence corresponding to amino acid residues 28-35, 49-60, and 74-79, of εPKC.

5. The method of claim 1, wherein the εPKC peptide comprises a contiguous amino acid sequence corresponding to a sequence selected from the group consisting of SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:1.3, and SEQ ID NO:16.

6. The method of claim 4, wherein the εPKC peptide comprises a contiguous amino acid sequence corresponding to amino acid residues 28-35, 49-53, and 74-79, of εPKC.

7. The method of claim 5, wherein the εPKC peptide comprises a contiguous amino acid sequence corresponding to a sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 12, and SEQ ID NO: 16.

8. The method of claim 2, wherein the εPKC peptide corresponds to the beta-sandwich region of the C2 domain of εPKC.

9. The method of claim 1, wherein the peptide is conjugated to a carrier peptide.

10. The method of claim 9, wherein the carrier peptide comprises SEQ ID NO:31 or SEQ ID NO:32.

11. The method of claim 9, wherein the peptide is cross-linked to the carrier peptide by an N-terminal Cys-Cys bond.

12. The method of claim 1, wherein the mammalian subject is a heart transplant patient.

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