IL185500A - Vdac1 compositions and use thereof for preparation of medicaments for inducing apoptosis - Google Patents

Description

185500/2 VDACI COMPOSITIONS AND METHODS OF USE THEREOF FOR INDUCING APOPTOSIS 185500 i?-Ji I 453582 v>?waifi i) ti vyn ona JIM?>\£M VDACI v\i n BGU/002 IL 185500/2 VDACl COMPOSITIONS AND METHODS OF USE THEREOF FOR INDUCING APOPTOSIS FIELD OF THE INVENTION The present invention relates generally to the mitochondrial protein, voltage-dependent anion channel (VDAC), polynucleotides encoding same and variants thereof, as well as peptide fragments, peptide derivatives and analogs. In particular, the present invention is directed to VDACl fragments and specific amino acid and polynucleotide sequences thereof useful in inducing apoptosis and to pharmaceutical compositions comprising same useful in the treatment of diseases associated with aberrant apoptosis.

BACKGROUND OF THE INVENTION VDAC Voltage-dependent anion channel (VDAC; mitochondrial porin) is a pore-forming protein found in the outer mitochondrial membrane in all eukaryotic cells controlling the fluxes of ions and metabolites between the mitochondria and the cytosol. VDAC is recognized as a key protein in mitochondria-mediated apoptosis due to its function in the release of apoptotic proteins located in the inter-membranal space and its interaction with apoptotic proteins. VDAC also serves as binding sites for several cytosolic enzymes and mitochondrial intermembranal space proteins, including hexokinase, creatine kinase and glycerol kinase. 185500/2 Three mammalian isoforms of VDAC are known, VDACl , VDAC2, VDAC3, where VDACl is the major isoform expressed in mammalian cells. Kayser et al. (Biol. Chem Hoppe-Seyler 1991 370: 1265-1278) and Blachly-Dysion et al (1993) disclosed the cloning of human VDACl and Blachly-Dysion et al disclose the functional expression in yeast of two human VDAC isoforms, VDACl and VDAC2. Human VDAC 1 was shown to specifically bind hexokinase. US Patent No. 5,780,235 discloses two novel VDAC sequences, which were named HACH (human voltage-dependent anion channel), subsequently identified as VDAC2 and VDAC3. That patent provides genetically engineered expression vectors, host cells containing the vector, a method for producing HACH and a method for identifying pharmaceutical compositions inhibiting the 185500/2 expression and activity of HACH and for the use of such compositions for the treatment of cancer and proliferative diseases.

Apoptosis Apoptosis, also known as programmed cell death, plays a central role in, inter alia, development, immune cell regulation and tissue homeostasis in multicellular organisms. Genetic and molecular analysis from various species has indicated that the apoptotic pathway is highly conserved. In addition to being essential for normal development and maintenance, apoptosis is important in the defense against viral infection and in preventing cancer.

Mitochondria play an important role in the regulation of apoptotic cell death. The release of apoptogenic intermediates such as cytochrome c from the intermembranal space into the cytoplasm of a cell initiates a cascade of caspase activation that executes the cell death program. Substantial evidence links VDAC to apoptosis and suggests that VDAC is a critical player in the release of apoptogenic proteins from mitochondria in mammalian cells (Shoshan-Barmatz and Gincel, 2003; Shimizu et al., J. Cell Biolo. 2001 152(2):237-250).

Lawen A et al. (Int. J. of Biochem. and Cell Biol. 2005 37:277-282) describe structure predictions of VDAC1, highlighting the presence of several amphipathic β-strands possibly organized in a β-burrel. Lawen et al, further preset few models of VDAC's anti-apoptotic function. It is shown that COS-7 cells and Daudi cells transfected with pl-VDACl-GFP construct are partially protected from apoptosis induced by the anti cancer drugs didemnin and etoposide, and by the oxidase inhibitors capsaicin and resiniferatoxin.

Anti-apoptotic and Pro-apoptotic Proteins Diverse intrinsic cell death signals emanating from various subcellular organelles can induce the release of cytochrome c from mitochondria. The Bcl-2 family of pro- and anti-apoptotic proteins constitutes a decisive control point for apoptosis. Proteins in the Bcl-2 family are major regulators of apoptosis (reviewed in Kim, 2005). Members of this family include both pro- and anti-apoptotic proteins and share homology in up to four conserved regions termed Bcl-2 homology (BH) 1-4 domains. The family can be divided 185500/2 into three main sub-classes: anti-apoptotic proteins, pro-apoptotic proteins and BH3-only proteins.

The anti-apoptotic proteins, which include hexokinase-I (HK-I), Bcl-2 and Bcl-xL, share homology throughout all four BH domains. The pro-apoptotic proteins can be further subdivided and include multidomain proteins, such as Bax and Bak, which possess sequence homology in BH1-3 domains.

The more distantly related BH3-only proteins appear to be only pro-apoptotic and share sequence homology within the BH3 region, which is required for their apoptotic function. The BH3-only proteins include, for example, BID, NOXA, PUMA and BAD. Sugiyama et al. (Oncogene 2002 21 :2944-4956) showed that Bim, one of the BH3-only proteins, has the ability to induce apoptosis via direct interaction with VDAC, inducing its activity via interactions like Bax/Bak.

It is currently held that anti-apoptotic members of the Bcl-2 family of proteins, such as HK-I, HK-II, Bcl-2 and Bcl-xL, act to promote cell survival by interacting with VDAC. Conversely, pro-apoptotic members of the Bcl-2 family of proteins, including Bak and Bax, may interact with VDAC to promote release of cytochrome c. Because of the pivotal role that mitochondria play in apoptotic cell death, mitochondrial proteins serve as potential targets for apoptosis regulating therapies.

One major obstacle in cancer chemotherapy is inherent, or acquired, resistance, apparently due to the suppression of apoptosis. Hexokinase-I (HK-I) is an anti-apoptotic mitochondrial protein that binds to VDAC. Many tumor cells exhibit a high glycolytic rate, which is correlated with a high level of HK-I expression. It is believed that the overexpression of anti-apoptotic proteins such as HK-I in cancer cells is a self-defense mechanism of those cells and is related to the cell's resistance to chemotherapy. It would be useful to develop agents that overcome apoptosis suppression in cancer cells, including HK-I suppression.

Certain compositions related to VDAC and use thereof for either inhibiting or inducing apoptosis are known in the art. US Patent Application Publication No. 2005/0085420 discloses methods of inhibiting apoptosis by promoting formation of a BAK/VDAC2 complex, and methods of promoting apoptosis by disrupting formation of a BAK/VDAC2 complex. The VDAC2/BAK inhibitor compound is, for example, a BH3 domain peptide, a BH3 domain-only mutein, an anti-VDAC2 antibody, a VDAC2 mutein and the like.

US Patent Application Publication No. 2005/02341 16 discloses small molecule compounds with utility as VDAC regulators, in particular as apoptosis suppressors.

There remains an unmet need for therapeutic agents effective in regulating apoptosis and specifically in inducing apoptosis in hyperproliferative disease and inhibiting apoptosis in neurodegenerative, cardiac and ophthalmic diseases. The art neither teaches nor suggests inducing apoptosis by expression of exogenous VDACl or the modulation of VDACl interaction with its associated proteins using VDACl derived peptides. 3a SUMMARY OF THE INVENTION The present invention provides, for the first time, VDACl molecules including polypeptide variants, peptide fragments thereof and derivatives, analogs and salts thereof, their nucleotide sequences, compositions comprising same and methods useful in treating diseases and disorders associated with aberrant apoptosis.

Unexpectedly, it is now shown that expression of exogenous VDACl amino acid sequence modulates apoptosis. Suitable sequences include VDACl polypeptide, polypeptide variant or an isolated peptide, or derivative or analog thereof, comprising an amino acid sequence derived from the mitochondrial VDACl protein modulates apoptosis. Specifically, the ectopic or exogenous expression of a VDACl amino acid sequence causes a dramatic increase in apoptosis of human cancer cell lines and in chemo and radio-resistant cancer cells.

The present invention is based in part on the discoveries that (a) overexpression of VDACl polypeptide or VDACl polypeptide variant triggers apoptosis in cancer cell lines; (b) HK-I, an anti-apoptosis protein, protects cells against apoptosis via its interaction with VDACl; and (c) an amino acid substitution in the cytosolic β-1οορ domains of VDACl eliminates HK-I or ruthenium red (RuR) protection against staurosporine-, curcumin-, As203- or VDACl overexpression-induced apoptosis.

Accordingly, the present invention provides VDACl amino acid sequences, pharmaceutical compositions comprising same and methods of use thereof. In particular, the present invention provides a VDACl molecule selected from a VDACl polypeptide variant, and a VDACl peptide, derivative, analog or salt thereof useful for inducing apoptotic cell death in cancer cells and in particular, in chemo- and radiotherapy-resistant cells.

The principles of the present invention are exemplified in both in vitro and in , vivo model systems of diseases associated with aberrant apoptosis. In various embodiments VDACl is selected from the group consisting of a rat VDACl isoform, a mouse VDACl isoform, a yeast VDACl isoform, a plant VDACl and a human VDACl isoform.

According to a first aspect, the present invention provides a VDACl molecule capable of modulating apoptosis in a cell, the molecule selected from: (i) an isolated 4 VDACl polypeptide variant having at least one amino acid substitution in an amino acid residue residing in a VDACl cytosolic domain and (ii) an isolated VDACl peptide fragment, analog, chemical derivative and a salt thereof, wherein the peptide fragment, analog, chemical derivative and a salt thereof is derived from a VDACl cytosolic domain or partial sequence thereof.

In some embodiments the molecule induces apoptosis or enhances sensitivity to an apoptosis-inducing reagent. Non-limiting examples of apoptosis-inducing reagents include STS, curcumin, doxorubicin and Taxol®.

In one embodiment the VDACl molecule is a peptide fragment comprising an amino acid sequence of a VDACl cytosolic domain or a partial sequence thereof.

The VDACl cytosolic domains include the N-terminus (amino acids M 1 to about D9) and three β-loops, β-loop 1 (from amino acid W63 to about D78) β-1οορ 2 (from amino acid SIOO to about Rl 19), β-1οορ 4 (from amino acid Gl 86 to about 1226). The β-loop 3 is exposed to the intermembrane space (from amino acid E l 57 to about T174).

A peptide fragment derived from a cytosolic domain may include the amino acid sequence of the entire cytosolic domain or a partial sequence thereof. The peptide fragment derived from a cytosolic domain may comprise up to about 30 consecutive amino acids, up to about 25 amino acids, up to about 20 amino acids, or up to about 10 amino acid residues.

In one embodiment the VDACl peptide fragment comprises an amino acid sequence that modulates the interaction between VDACl and a mitochondrial anti-apoptotic protein. Certain mitochondrial anti-apoptotic proteins are known in the art and include hexokinase-I (HK-I), HK-II, Bcl2, Bcl2Al, Bcl2Ll, Bcl2L10, Bcl2Lll, Bcl2L2, Bcl-xL and Bcl-W.

In one embodiment the VDACl peptide of the invention comprises a VDACl amino acid sequence up to about 70 amino acid residues. In some embodiment the VDACl peptide comprises an amino acid sequence up to about 60 amino acid residues, up to about 40 amino acid residues, up to about 30 amino acid residues, and preferably from about 15 to about 30 amino acid residues.

In some embodiments the VDACl peptide is selected from the group of peptides having amino acid sequence as set forth in SEQ ID NO: l , SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:5. In certain embodiments the VDACl peptide comprises an amino acid sequence as set forth in SEQ ID NO. 2.

In some embodiments the peptide of the invention is linked to a cell penetrating peptide (CPP). In one exemplary embodiment the CPP is an amino acid sequence comprising the Drosophila antennapedia (ANTP) domain or a fragment thereof. In certain embodiments the VDAC peptides further comprise an ANTP amino acid sequence as set forth in SEQ ID NO:39. In one embodiment the VDAC peptide further comprises tryptophan zipper amino acid sequences and a fragment of the ANTP amino acid domain. In some embodiments the peptide is selected from the group of peptides having amino acid sequence as set forth in SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 15. In one embodiment the peptide comprises an amino acid sequence as set forth in SEQ ID NO: 12.

In other embodiments the peptide is selected from a VDACl peptide derivative and VDACl peptide analog. In one embodiment the VDACl peptide derivative is an amidated VDAC 1 peptide.

In certain embodiments the VDACl molecule is selected from a VDACl polypeptide variant having at least one amino acid substitution in an amino acid residue residing in a VDACl protein domain exposed to the cytosol. The cytosolic domains of VDACl have been identified as the N-terminus, first, second and fourth β-loop domains of VDACl. In one embodiment the amino acid substitution replaces a charged amino acid selected from aspartate (D), glutamate (E) and lysine (K) with a non-charged amino acid. In one embodiment the VDACl polypeptide variant is selected from any one of SEQ ID NO:43-SEQ ID NO:48. The VDACl polypeptide variant may comprise further structural changes including additional amino acid substations, amino acid deletions and additions. Accordingly the present invention provides a polypeptide variant having about 80%, about 90% about 95%, about 98% or about 99% homology to any one of SEQ ID NO:43-SEQ IDNO:48.

In another aspect, the present invention provides isolated nucleotide sequences encoding a VDACl polypeptide variant or peptide capable of modulating apoptosis in a cell. In one embodiment a nucleotide sequence is selected from the group consisting of the sequences set forth in any one of SEQ ID NO:6-SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO:16-SEQ ID NO: 18 or SEQ ID NO:20.

In another aspect the present invention provides a polynucleotide construct comprising a nucleic acid sequence encoding a VDACl polypeptide variant or peptide derivative, analog or salt thereof capable of modulating apoptosis in a cell. In one embodiment the VDACl polypeptide variant or peptide induces apoptosis. In some embodiments the construct comprises a nucleotide sequence selected from the group consisting of sequences set forth in any one of SEQ ID NO:6-SEQ ID NO:8, SEQ ID NO-.10, SEQ ID NO: 16-SEQ ID NO: 18 or SEQ ID NO:20.

In some embodiments the polynucleotide construct is an expression vector.

Further provided is a host cell comprising a polynucleotide construct comprising a nucleotide sequence encoding a VDACl polypeptide variant or peptide, derivative, analog or salt thereof capable of modulating apoptosis in a cell. In one embodiment the VDACl polypeptide variant or peptide, derivative, analog or salt thereof can induce apoptosis.

According to another aspect the present invention provides a pharmaceutical composition comprising a VDACl sequence. In one embodiment the VDACl sequence is an amino acid sequence selected from the group consisting of a VDACl polypeptide, a VDACl polypeptide variant, a VDACl peptide, derivative, analog or salt thereof and a pharmaceutically acceptable diluent or excipient. In some embodiments VDACl is human VDACl. In one embodiment, the pharmaceutical composition comprises a VDACl peptide comprising amino acid sequence as set forth in anyone of SEQ ID NO: 1-SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO: 1 1-SEQ ID NO: 14 and SEQ ID NO: 15. In certain embodiments the VDACl peptide is selected from the group consisting of a peptide comprising an amino acid sequence set forth in any one of SEQ ID NO:2 and SEQ ID NO: 12, encoded by a nucleotide set forth in SEQ ID NO:7 and SEQ ID NO : 17, respectively.

In one embodiment the pharmaceutical composition of the invention provides a VDACl amino acid sequence selected from a VDACl polypeptide or VDACl polypeptide variant. In one embodiment the polypeptide is selected from SEQ ID NO:21, SEQ ID NO:24 and SEQ ID NO:27. In one preferred embodiment the polypeptide has SEQ ID NO:21. In one embodiment the VDACl polypeptide variant comprises at least one amino acid substitution within a cytosolic β-loop domain of VDACl. In one embodiment the VDACl polypeptide variant comprises an amino acid substitution of a charged amino acid residue. In one embodiment the VDACl polypeptide variant is selected from any one of SEQ ID NO:43-SEQ ID NO:48.

In one embodiment the present invention further provides a pharmaceutical composition comprising an isolated nucleic acid sequence encoding a VDACl polypeptide, VDACl polypeptide variant or a VDACl peptide of the invention. In one embodiment the nucleic acid has a sequence selected from SEQ ID NO:6-SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO:16-SEQ ID NO: 18 , SEQ ID NO:20, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:36 and SEQ ID NO:51-58. The present invention further encompasses polynucleotide constructs comprising the nucleic acid sequences according to the invention and host cells comprising said constructs.

The present invention further provides a pharmaceutical composition comprising a polynucleotide construct comprising an isolated nucleic acid sequence selected from any one of SEQ ID N0.6-SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 16-SEQ ID NO: 18, SEQ ID NO:20, ,SEQ ID NO:30-36 and SEQ ID NO:51-56. In some embodiments the polynucleotide construct comprises an expression vector. In exemplary embodiments the pharmaceutical composition comprises a polynucleotide construct comprising a nucleotide sequence selected from the group consisting of SEQ ID NO.7, SEQ ID NO: 17, SEQ ID NO:30 and SEQ ID NO:50.

In certain embodiments the pharmaceutical composition comprises a VDACl amino acid sequence according to the present invention and a shielding particle. In certain embodiments the shielding particle comprises PEI, PEG and lipids.

In certain embodiments the pharmaceutical composition comprises an encapsulated VDACl amino acid sequence according to the present invention. In certain embodiments the VDACl amino acid sequence is encapsulated into a vesicle, or into immunoliposomes.

In yet another aspect the present invention provides a method for treating a disease associated with aberrant apoptosis, the method comprising administering to a subject in need thereof a composition comprising a therapeutically effective amount of an agent selected from the group consisting of a) a VDACl polypeptide, b) a VDACl polypeptide variant, c) at least one VDACl peptide, a derivative or analog of thereof and d) a VDACl nucleic acid encoding a VDACl polypeptide, VDACl polypeptide variant 8 or a VDACl peptide. In some embodiments the disease is selected from cancer including chemo- and radiotherapy-resistant cancer.

In yet another aspect the present invention provides a method for inducing apoptosis in a cell or enhancing the effect of an apoptosis-inducing reagent in a cell comprising the step of upregulating VDACl expression in the cell under conditions sufficient to induce apoptosis of the cell.

In one embodiment upregulating VDACl expression comprises the step of providing an exogenous VDACl molecule to the cell under conditions sufficient for said VDACl amino acid sequence to induce apoptosis of the cell. In some embodiments the VDACl molecule is selected from a VDACl polypeptide, a VDACl polypeptide variant and a VDACl peptide, derivative analog or salt thereof according to the principles of the present invention. In other embodiments VDACl molecule is selected from a VDACl nucleotide sequence according to the principles of the present invention.

Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE FIGURES Figures I A shows that the interaction between HK-I and the mitochondrial VDACl reconstituted into a planar lipid bilayer induced decrease in the channel conductance. Figure IB shows SDS-PAGE of purified VDACl and HK-I. Figure 1C shows inhibition of cytochrome c release by HK-I.

Figures 2A-2D shows the effects of HK-I on the permeability transition pore (PTP) opening, as monitored by Ca2+ accumulation (Fig. 2A) and swelling of energized mitochondria (Fig. 2C-2D). HK-I inhibited PTP opening and this inhibition was prevented by an antibody specific to the N-terminal of HK-I (Fig. 2C). The failure of non-binding species of HK, such as yeast HK, or chymotrypsin-treated HK-I (Fig. 2D) to 9 prevent PTP opening, confirmed the specificity of the HK-I effect, and of the requirement of the HK-I N-terrninal region for its interaction with VDAC.

Figure 3A shows leukemic U937 cells that were transformed to express green fluorescent protein (GFP) and VDAC1-GFP. Figure 3B shows an immunoblot using anti-VDAC or anti-GFP antibodies of extracts from cell transfected with VDAC1, VDAC1-GFP, mutated E72QVDAC1 or mutated E72QVDAC 1 -GFP. Figures 3C-3D show that over-expression of recombinant HK-I protects against apoptotic cell death induced by over-expression of native, but not of E72Q or E202Q VDAC1 variants. Figure 3E shows the results of experiments confirming that overexpression of recombinant HK-I protects cells from apoptosis induced by staurosporine (STS).

Figure 4A and 4B show that over-expression of recombinant HK-I protects against apoptotic cell death induced by over-expression of native and some, but not in all mutated versions of VDAC1.

Figure 5 shows that RuR protects against apoptotic cell death induced by over-expression of native, but not of certain VDAC1 polypeptide variants. Figures 5B and 5C show the protective effect of RuR against apoptosis induced by exogenous expression of native and some VDAC1 variants but not of E72Q- or E65Q-VDAC1 variants. Figure 5D shows that RuR increases the total amount of VDAC1 protein in cells expressing native mVDACl, but not in cells expressing E72Q-mVDACl. Figure 5E shows that RuR inhibited recombinant native mVDACl channel activity and stabilized the channel in an almost completely closed state. In contrast, the channel activity of E72Q-mVDACl was not inhibited by RuR.

Figure 6 is a model illustrating the proposed VDAC1 transmembrane topology and potential cytosolic, transmembrane and intermembranal domains, where the peptides and mutations are labeled.

Figure 7 is a schematic illustration of a peptide comprising amino acid sequences derived from the cytosolic β-ΐοορ 1 domain (LP1) peptide (SEQ ID NO:2) comprising the tryptophan zipper sequences (SEQ ID NOS :41 and 42) and an antennapedia homeodomain (SEQ ID NO:39).

Figure 8 shows the alignment of the polypeptide sequences of the human, mouse and rat VDAC1 3 2 and 3 isoforms and an alignment of two cytosolic β-loop domains from those sequences. It should be noted that the according to the numbering in this figure, the amino acid count includes the first methionine, therefore E72 is presented as E73 , etc.

Figure 9 is a graph depicting the LP1 peptide (SEQ ID NO:2) induced detachment of brain mitochondrial bound HK-I.

Figures 10 are graphs showing that LP1 peptide eliminates the HK-I inhibition of the permeability transition pore (PTP) opening.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to VDACl polypeptides, peptides, derivatives and analogs thereof, compositions comprising same and methods useful for modulating apoptotic cell death in diseases associated with aberrant apoptosis.

In one embodiment there is provided a peptide useful for the treatment of proliferative disease, including cancer and in particular for the stimulation of apoptosis, in cancer cells and other diseases exhibiting aberrant apoptosis. In particular the invention relates to VDACl specific amino acid sequences useful in inducing apoptosis. The VDACl peptides can induce apoptosis in primary cancers as well as in resistant cells and improve the efficacy of chemotherapeutic drugs.

Without wishing to be bound to theory the VDACl -based peptides are designed to minimize the self-defense mechanisms in cancer cells, which involves over-expression of anti-apoptotic proteins. The present invention takes advantage of the central role that mitochondria and VDACl, as the gatekeeper, play in apoptosis to generate potent, specific and effective VDACl -based cancer therapies that diminish the activities of pro-survival proteins, promote apoptosis and increase chemotherapy effectiveness. Anti-apoptotic proteins are expressed in the majority of tumors, therefore the VDACl peptides are active in tumors regardless of the signaling pathway that controls apoptosis in the different tumor types.

Definitions For convenience certain terms employed in the specification, examples and claims are described herein. 11 The term "VDAC1" as used herein and in the claims refers to the VDAC1 isoform of a highly conserved family of mitochondrial porin and the corresponding polynucleotides. Four VDAC isoforms, encoded by three genes, are known to date.

The term "VDAC1 cytosolic domains" refers to amino acid sequences that are exposed to the cytosol, as opposed to those exposed to the intermembranal space embedded within the membrane. The cytosolic domains include the VDAC1 N-terminal amino acids M1-D9, β-loopl amino acids T64-D78, β-ΐοορ 2 amino acids S103-R1 19, and β-1οορ 4 amino acids G 1 86-1226. A peptide derived from a cytosolic domain may include the amino acid sequence of the entire cytosolic domain or a fragment thereof. The peptide fragment derived from a cytosolic domain may comprise up to about 25 consecutive amino acids, up to about 20 amino acids, or up to about 15 amino acids. In some embodiments the exemplary peptide sequences are set forth in SEQ ID NOs: 1-3 and SEQ ID NO:5.

As used herein the term "apoptosis" or "apoptotic cell death" refers to programmed cell death which can be characterized by cell shrinkage, membrane blebbing and chromatin condensation culminating in cell fragmentation. Cells undergoing apoptosis also display a characteristic pattern of DNA cleavage. Alternatively, apoptosis can be characterized indirectly by changes in the activity or expression of members of the apoptotic pathway, e.g. increased mitochondrial release of cytochrome c. A non-limiting example of apoptosis-inducing reagents includes actinomycin D, antibiotic A-23187, b-lapachone, Camptothecin, ceramide, curcumin, dexamethasone, etoposide (Etopophos®, Vepesid®), Hypericin, prostaglandin A2, S-Nitrosoglutathione, staurosporin, sulindac sulfide, sulindac sulfone, Taxol , vinblastine sulfate, vincristine sulfate, 15(S)-HPETE, 4-hydroxyphenyl retinamide, betulinic acid and the like.

As used herein, the term "modulates apoptosis" includes either upregulation or downregulation of apoptosis in a cell. Furthermore modulation of apoptosis includes enhancement of apoptosis in a cell that has previously received a signal that induces apoptosis, e.g., the cell has been subjected to a signal or chemical that induces apoptosis. Modulation of apoptosis is discussed in more detail below and can be useful in ameliorating various disorders, e.g., cancer and viral diseases. 12 The term "expression vector" and "recombinant expression vector" as used herein refers to a DNA molecule containing a desired coding sequence and appropriate nucleic acid sequences necessary for the expression of the operably linked coding sequence in a particular host organism. The expression vector may comprise sequences encoding heterologous domains including but not limited to protein detection, purification or cleavage sequences that may be fused at the N- or C-terminus to the desired coding sequence, to yield a fusion protein. It is contemplated that the present invention encompasses expression vectors that are integrated into host cell genomes, as well as vectors that remain unintegrated into the host genome.

Any vector selected can be constructed such that it is capable of being transferred into the cells of interest either with or without VDAC1 amino acid sequence. Methods for manipulating the vector nucleic acid are well known in the art and include for example direct cloning and site-specific recombination using recombinases. In this manner, an expression vector can be constructed such that it can be replicated in any desired cell, expressed in any desired cell, and can even become integrated into the genome of any desired cell.

The term "expression product" is used herein to denote a VDAC1 amino acid sequence. A VDAC1 expression product is preferably a translation product, or a fragment of translation product from any one or more of the VDAC1 polypeptides, polypeptide variants or peptides. .

The term "exogenous" is used herein refers to a VDAC1 amino acid sequence which is introduced into a cell. For example, "exogenous VDAC1 amino acid sequence" should be construed to include a VDAC1 amino acid sequence expressed from a nucleic acid, which has been introduced into a cell using recombinant technology, a VDAC1 amino acid sequence that is added to a cell and any and all combinations thereof. Therefore, the term should not be construed to be limited solely to the addition of VDAC1 amino acid sequence to a cell per se, but should be expanded to include the expression of VDAC1 amino acid sequence in a cell when the VDAC1 amino acid sequence is expressed from a nucleic acid, which has been introduced into the cell.

The protein sequences of the human, mouse and rat VDAC isoforms are provided in the sequence listing herein below. 13 Human VDACl set forth in SEQ ID N0:21 , (NP_003365) is a 283 amino acid protein. The numbering of the VDACl mutation begins at the alanine following the first methionine; Human VDAC2 set forth in SEQ ID NO: 22 (NP 003366) is a 294 amino acid protein; Human VDAC3 set forth in SEQ ID NO:23 (NP_005653) is a 283 amino acid protein.

Mouse VDACl is set forth in SEQ ID NO:24 (NPJB5824); Mouse VDAC2 is set forth in SEQ ID NO:25 (NP_035825); Mouse VDAC3 is set forth in SEQ ID NO:26 (NP_035826).

Rat VDACl is set forth in SEQ ID NO:27 (NP_1 12643); Rat VDAC2 is set forth in SEQ ID NO:28 (NP_1 12644); Rat VDAC3 is set forth in SEQ ID NO:29 (NPJ 12645).

Alignment of the human amino acid sequences is shown in figure 8. The corresponding polynucleotide sequences are set forth in SEQ ID NO:30-SEQ ID NO:38. The polynucleotide sequences of Human VDACl, VDAC2 and VDAC3 having accession numbers NM_003374, NM_003375 and NM_005662 are set forth in SEQ ID NO:30-SEQ ID NO:32, respectively. The polynucleotide sequences of mouse VDACl, VDAC2 and VDAC3 having accession numbers NM O 1 1694, NM O 1 1695 and NM_01 1696 are set forth in SEQ ID NO:33-SEQ ID NO:35, respectively. The polynucleotide sequences of rat VDACl, VDAC2 and VDAC3 having accession numbers NM_031353, NM_031354 and NM_031355 are set forth in SEQ ID NC-.36-SEQ ID NO:38, respectively.

Throughout the specification and the claims that follow, the terms "VDACl polypeptide", "VDACl peptide", "VDACl" peptidomimetic" "VDACl peptide analog" and the like refers to molecules having a sequence which is a VDACl amino acid or a variant thereof, or a sequence derived from the VDACl family of proteins. Within the context of the present invention, a VDACl amino acid sequence can be or comprise one or more amino acid residue insertions, deletions, or substitutions. Preferably, any substitution is conservative in that it minimally disrupts the biochemical properties of the VDACl amino acid sequence polypeptide. Given the properties of the subject amino acids comprising the disclosed protein products, some rational substitutions will be recognized by the skilled worker. Amino acid substitutions, i.e. "conservative substitutions," may be made, for instance, on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved. Additionally, all or a part of the amino acids may be substituted with the D isoform of amino acids.

The VDAC1 polypeptide variants according to the invention comprise a native VDAC1 polypeptide sequence having an amino acid substitution in one or more of the cytosolic domains. The cytosolic, transmembrane and intermembrane domains of VDAC1 are schematically shown in figure 6 (modified from Colombini, 2004).

The present invention also provides pharmaceutical formulations, both for veterinary and for human medical use, which comprise as the active agent one or more of the VDAC1 polypeptide, VDAC1 polypeptide variants, VDAC1 peptides, derivatives, analogs or salts thereof or a polynucleotide thereof described in the invention, for the manufacture of a medicament for the treatment or prophylaxis of the conditions variously described herein.

Polypeptides, Peptides, Peptide Derivatives and Peptide Analogs Expression of exogenous VDAC1 protein and variants thereof in human cancer cell lines was shown to cause a dramatic increase in apoptosis in those cells. These results demonstrate that modulation of VDAC1 levels, either directly or indirectly, can be used to modulate apoptosis.

An "amino acid sequence", as used herein, refers to an oligopeptide, peptide, or polypeptide sequence, and fragments thereof, and to naturally occurring, recombinant or synthetic molecules. The term "polypeptide" refers to any chain of amino acids, regardless of length or post-translational modification (for example, glycosylation or phosphorylation) .

The term "variant" as used herein refers to a polypeptide sequence that possesses some modified sequence of the wild type or parent protein. For example, the variant may have amino acids deleted, inserted or substituted.

The peptides, derivatives analogs and salts thereof comprise a VDAC1 amino acid sequence of up to about 70 amino acid residues, up to about 60 amino acid residues, up to about 40 amino acid residues, preferably from about 15 to about 30 amino acid residues in length.

The terms "peptide" or "peptide fragment" as used herein are meant to encompass natural, non-natural and/or chemically modified amino acid residues connected one to the other by peptide or non-peptide bonds. Therefore the term "peptide" includes a fragment, analog, derivative or a salt thereof. The amino acid residues are represented throughout the specification and claims by either one or three-letter codes, as is commonly known in the art. The compounds of the invention include linear and cyclic peptides and derivatives and analogs thereof.

The terms "analog" and "derivative" refer to a peptide comprising at least one altered amino acid residue by an amino acid substitution, addition, deletion, or chemical modification, as compared with the native peptide. Peptide analogs particularly include amino acid substitutions and/or additions with non-natural amino acid residues, and chemical modifications, which do not occur in nature. Peptide analogs include peptide variants and peptide mimetics. A peptide mimetic or "peptidomimetic" is a molecule that mimics the biological activity of a peptide but is not completely peptidic in nature. Whether completely or partially non-peptide, peptidomimetics according to this invention provide a spatial arrangement of chemical moieties that closely resembles the three-dimensional arrangement of groups in the peptide on which the peptidomimetic is based. As a result of this similar active-site geometry, the peptidomimetic has effects on biological systems, which are similar to the biological activity of the peptide.

Peptide derivatives particularly include amino acid substitutions and/or additions with naturally occurring amino acid residues, and chemical modifications such as, for example, enzymatic modifications, typically present in nature. Accordingly, the present invention encompasses both peptide derivatives and analogs of the VDAC peptides as set forth in SEQ ID NO: l - SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:l 1 - SEQ ID NO: 13 and SEQ ID NO: 15.

The present invention encompasses VDAC peptide derivatives or analogs of which at least one amino acid has been chemically modified. Chemical modifications of amino acid residues include, but are not limited to, amidation, methylation, acetylation, glycosylation, oxidation, reduction, myristylation, sulfation, acylation, ADP-ribosylation, cyclization, hydroxy lation, iodination, derivatization by protecting/blocking groups, or any other derivatization method known in the art. Such alterations, which do not destroy, but may improve the VDAC peptide biological activity, can occur anywhere along the 16 sequence of the VDAC peptide, including at the peptide backbone, the amino acid side-chains, and at the amino or carboxyl termini.

By using "amino acid substitution", it is meant that an amino acid residue is substituted for a residue within the sequence resulting in a functionally equivalent or in a functionally different variant. The term "functionally equivalent" means, for example, a group of amino acids having similar polarity, similar charge, or similar hydrophobicity. For example, one or more amino acid residues within the sequence can be substituted by another amino acid of a similar polarity, which acts as a functional equivalent, resulting in a silent alteration. Substitutes for an amino acid within the sequence can be selected from other members of the class to which the amino acid belongs. For example, the non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. The positively charged (basic) amino acids include arginine, lysine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid (aspartate and glutamate). Such substitutions are known as conservative substitutions.

Additionally, a non-conservative substitution can be made in an amino acid that does not contribute to the biological activity of the peptide. Such non-conservative substitutions are also encompassed within the term "amino acid substitution", as used herein. It will be appreciated that the present invention further encompasses VDAC1 peptide derivatives or analogs, wherein at least one amino acid is substituted by another natural or non-natural amino acid to produce a peptide derivative or analog having increased stability or higher half life as compared to the native VDAC1 peptide fragment.

The present invention encompasses peptide hydrates and includes, but is not limited to, hemihydrate, monohydrate, dihydrate, trihydrate, and the like.

One aspect of the present invention provides a VDAC peptide further comprising a moiety that facilitates cell penetration. In one embodiment the moiety is a cell penetrating peptide (CPP). CPP's are typically amphipathic or cationic oligopeptides able to transport attached cargoes across cell membranes. In one embodiment the CPP comprises a fragment of the Drosophila antennapedia homeodomain (ANTP), comprising an amino acid sequence as set forth in SEQ ID NO: 39.

SEQ ID NO:39: 5' MRQIKIWFQNR MKWKK which is encoded by a nucleotide set forth in SEQ ID NO: 40 5' ATG CGT CAG ATT AAA ATT TGG TTT CAG AAT CGT CGT ATG AAA TGG AAA AAA.

The peptide or polypeptide of the invention can further comprise a tryptophan zipper pentamer or other sequence to further stabilize the peptide (Cochran, 2001). The amino acid sequences of the tryptophan zipper pentamer are set forth in SEQ ID NO: 4 1 and SEQ ID NO:42. SEQ ID NO:41 : SWTWE and SEQ ID NO:42: WTWK.

One aspect of the present invention provides for a peptide analog such as a peptidominietic, which mimics the structural features of the critical minimal epitope.

There are clear advantages for using a mimetic of a given peptide rather than the peptide itself, since peptide drugs often exhibit two undesirable properties: poor bioavailability and short duration of action. Peptide mimetics offer a route around these two major obstacles; since the molecules concerned have a long duration of action. Furthermore there are problems associated with stability, storage and immunoreactivity for peptides that are not experienced with peptide mimetics.

The design of the peptidomimetics may be based on the three-dimensional structure of VDAC alone with or in complex with another protein. Interaction with the peptidomimetic can either induce an interacting protein to cany out the normal function caused by such binding (agonist) or disrupts such function (antagonist, inhibitor).

A primary goal in the design of peptide mimetics has been to reduce the susceptibility of mimics to cleavage and inactivation by peptidases. In one approach, one or more amide bonds have been replaced in an essentially isosteric manner by a variety of chemical functional groups. In another approach, a variety of uncoded or modified amino acids such as D-amino acids and N-methyl amino acids have been used to modify mammalian peptides. Alternatively, a presumed bioactive conformation has been stabilized by a covalent modification, such as cyclization or by incorporation of γ-lactam or other types of bridges as disclosed for example in US Patent 5,811,392. In US Patent 5,552,534, non-peptide compounds are disclosed which mimic or inhibit the chemical and/or biological activity of a variety of peptides. Such compounds can be produced by appending to certain core species, such as the tetrahydropyranyl ring, chemical functional groups, which cause the compounds to be at least partially cross-reactive with the peptide. As will be recognized, compounds which mimic or inhibit 18 peptides are to varying degrees cross-reactive therewith. Other techniques for preparing peptidomimetics are disclosed in US Patents 5,550,251 and 5,288,707, for example. Non-limiting examples of the use of peptidomimetics in the art include inhibitors of protein isoprenyl transferases (particularly protein farnesyltransferase and geranylgeranyltransferase) and anti-cancer drugs (US Patent No. 5,965,539) inhibitors of p21 ras (US Patent No. 5,910,478) and inhibitors of neurotropin activity (US Patent No. 6,291,247).

Whenever VDAC1 peptide fragments are mentioned in the invention, also salts and functional derivatives thereof are contemplated, as long as they are able to induce apoptosis of a target cell. Thus, the present invention encompasses peptides containing non-natural amino acid derivatives or non-protein side chains.

The term "derivative" includes any chemical derivative of the peptide fragments of the invention having one or more residues chemically derivatized by reaction of side chains or functional groups. Such derivatized molecules include, for example, those molecules in which free amino groups have been derivatized to form amine hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups. For example, free carboxyl groups may be derivatized to form salts, methyl and ethyl esters or other types of esters or hydrazides; free hydroxyl groups may be derivatized to form O-acyl or O-alkyl derivatives; the imidazole nitrogen of histidine may be derivatized to form N-im-benzylhistidine. The C-terminus of the peptide of the invention may be presented in the free carboxy form or, preferably, it is ami dated to facilitate the synthesis and increase the stability of the peptide, for example to increase the resistance of the peptides to enzymatic cleavage in the organism, or modified in a way that increases its solubility.

Also included as chemical derivatives are those peptides, which contain one or more naturally occurring amino acid derivatives of the twenty standard amino acid residues. For example: 4-hydroxyproline may be substituted for proline; 5 -hydroxy lysine may be substituted for lysine; 3-methylhistidine may be substituted for histidine; homoserine may be substituted for serine; and ornithine may be substituted for lysine.

In addition, a VDAC peptide can differ from the natural sequence of the peptides of the invention by chemical modifications including, but are not limited to, terminal-NH2 acylation, acetylation, or thioglycolic acid amidation, and by terminal- 19 carboxlyamidation, e.g., with ammonia, methylamine, and the like. Peptides can be linear, cyclic or branched and the like, which conformations can be achieved using methods well known in the art.

Addition of amino acid residues may be performed at either terminus of the polypeptides or peptides of the invention for the purpose of providing a "linker" by which the peptides of this invention can be conveniently bound to a carrier. Such linkers are usually of at least one amino acid residue and can be of 40 or more residues, more often of 1 to 10 residues. Typical amino acid residues used for linking are tyrosine, cysteine, lysine, glutamic and aspartic acid, or Ihe like.

Peptide Synthesis The peptides, peptide derivatives and peptide analogs of the invention may be synthesized using any recombinant or synthetic method known in the art, including, but not limited to, solid phase (e.g. Boc or f-Moc chemistry) and solution phase synthesis methods. For solid phase peptide synthesis, a summary of the many techniques may be found in Stewart and Young, 1963; and Meienhofer, 1973. For a review of classical solution synthesis, see Schroder and Lupke, 1965.

The amino acid residues described herein are preferred to be in the "L" isomeric form. However, residues in the "D" isomeric form can be substituted for any L-amino acid residue, as long as the peptide substantially retains the desired functional property. Use of "D" amino acids may be used as is known in the art to increase the stability or half-life of the resultant peptide.

Nucleic acids In another aspect, the invention provides nucleic acid molecules encoding the native or mutated VDAC1 polypeptide or peptides of the invention.

The nucleic acid molecules may include DNA, RNA, or derivatives of either DNA or RNA. An isolated nucleic acid sequence encoding a peptide can be obtained from its natural source, for example as a portion of a gene. A nucleic acid molecule can also be produced using recombinant DNA technology (e.g., polymerase chain reaction (PCR) amplification, cloning) or chemical synthesis. Nucleic acid sequences include natural nucleic acid sequences and homologs thereof, comprising, but not limited to, natural allelic variants and modified nucleic acid sequences in which nucleotides have been inserted, deleted, substituted, and/or inverted in such a manner that such modifications do not substantially interfere with the nucleic acid molecule's ability to encode a functional peptide of the present invention.

A nucleic acid molecule homolog can be produced using a number of methods known to those skilled in the art (see, for example, Sambrook et al., 1989). For example, nucleic acid molecules can be modified using a variety of techniques including, but not limited to, classic mutagenesis techniques and recombinant DNA techniques, such as site-directed mutagenesis, chemical treatment of a nucleic acid molecule to induce mutations, restriction enzyme cleavage of a nucleic acid fragment, ligation of nucleic acid fragments, polymerase chain reaction (PCR) amplification and/or mutagenesis of selected regions of a nucleic acid sequence, synthesis of oligonucleotide mixtures and ligation of mixture groups to "build" a mixture of nucleic acid molecules and combinations thereof. Nucleic acid molecule homologs can be selected from a mixture of modified nucleic acids by screening for the function of the protein encoded by the nucleic acid with respect to the induction of an anti-viral response, for example by the methods described herein.

A polynucleotide or oligonucleotide sequence can be deduced from the genetic code of a protein, however, the degeneracy of the code must be taken into account, and nucleic acid sequences of the invention also include sequences, which are degenerate as a result of the genetic code, which sequences may be readily determined by those of ordinary skill in the art.

The terms "nucleic acid" and "polynucleotide" and "nucleotide sequence" as used herein refer to an oligonucleotide, polynucleotide or nucleotide and fragments or portions thereof, and to DNA or RNA of genomic or synthetic origin, which may be single- or double-stranded, and represent the sense or antisense strand. The term should also be understood to include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs, and, as applicable to the embodiment being described.

The term "oligonucleotide" refers to a nucleic acid sequence of at least about 6 nucleotides to about 60 nucleotides, preferably about 15 to 30 nucleotides, and more preferably about 20 to 25 nucleotides, which can be used in PCR amplification or a hybridization assay, or a microarray. As used herein, oligonucleotide is substantially 21 equivalent to the terms "amplimers", "primers", "oligomers", and "probes", as commonly defined in the art.

As used herein, highly stringent conditions are those, which are tolerant of up to about 5% to about 25% sequence divergence, preferably up to about 5% to about 15%. Without limitation, examples of highly stringent (-10°C below the calculated Tm of the hybrid) conditions use a wash solution of 0.1 X SSC (standard saline citrate) and 0.5% SDS at the appropriate Ti (incubation temperature) below the calculated Tm of the hybrid. The ultimate stringency of the conditions is primarily due to the washing conditions, particularly if the hybridization conditions used are those, which allow less stable hybrids to form along with stable hybrids. The wash conditions at higher stringency then remove the less stable hybrids. A common hybridization condition that can be used with the highly stringent to moderately stringent wash conditions described above is hybridization in a solution of 6 X SSC (or 6 X SSPE), 5 X Denhardt's reagent, 0.5% SDS, 100 g/ml denatured, fragmented salmon sperm DNA at an appropriate incubation temperature Ti. See generally Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d edition, Cold Spring Harbor Press (1989)) for suitable high stringency conditions.

Stringency conditions are a function of the temperature used in the hybridization experiment and washes, the molarity of the monovalent cations in the hybridization solution and in the wash solution(s) and the percentage of formamide in the hybridization solution. In general, sensitivity by hybridization with a probe is affected by the amount and specific activity of the probe, the amount of the target nucleic acid, the detectability of the label, the rate of hybridization, and the duration of the hybridization. The hybridization rate is maximized at a Ti of 20-25°C below Tm for DNA:DNA hybrids and 10-1 5°C below Tm for DNA:RNA hybrids. It is also maximized by an ionic strength of about 1.5M Na+. The rate is directly proportional to duplex length and inversely proportional to the degree of mismatching.

Specificity in hybridization, however, is a function of the difference in stability between the desired hybrid and "background" hybrids. Hybrid stability is a function of duplex length, base composition, ionic strength, mismatching, and destabilizing agents (if any). 22 The Tm of a perfect hybrid may be estimated for DNA:DNA hybrids using the equation of Meinkoth et al (1984), as Tm = 81.5°C + 16.6 (log M) + 0.41 (%GC) - 0.61 (% form) - 500/L and for DNArRNA hybrids, as Tm = 79.8°C + 18.5 (log M) + 0.58 (%GC) - 1 1.8 (%GC)2 - 0.56(% form) - 820/L where M , molarity of monovalent cations, 0.01 -0.4 M NaCl, %GC, percentage of G and C nucleotides in DNA, 30%-75%, % form, percentage formamide in hybridization solution, and L, length hybrid in base pairs.

Tm is reduced by 0.5-1.5°C (an average of 1°C can be used for ease of calculation) for each 1% mismatching. The Tm may also be determined experimentally. As increasing length of the hybrid (L) in the above equations increases the Tm and enhances stability, the full-length rat gene sequence can be used as the probe.

Filter hybridization is typically carried out at 68°C, and at high ionic strength (e.g., 5 - 6 X SSC), which is non-stringent, and followed by one or more washes of increasing stringency, the last one being of the ultimately desired high stringency. The equations for Tm can be used to estimate the appropriate Ti for the final wash, or the Tm of the perfect duplex can be determined experimentally and Ti then adjusted accordingly.

The present invention further includes a nucleic acid sequence of the present invention operably linked to one or more transcription control sequences to form a recombinant molecule. The phrase "operably linked" refers to linking a nucleic acid sequence to a transcription control sequence in a manner such that the molecule is able to be expressed when transfected (i.e., transformed, transduced or transfected) into a host cell. Transcription control sequences are sequences, which control the initiation, elongation, and termination of transcription. Particularly important transcription control sequences are those which control transcription initiation, such as promoter, enhancer, operator and repressor sequences. Suitable transcription control sequences include any transcription control sequence that can function in at least one of the recombinant cells of the present invention. A variety of such transcription control sequences are known to those skilled in the art. Preferred transcription control sequences include those which function in animal, bacteria, helminth, yeast and insect cells.

A nucleic acid molecule of the invention may be inserted into appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence.

Vectors can be introduced into cells or tissues by any one of a variety of known methods within the art, including in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such methods are generally described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York 1989, 1992; in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. 1989.

A recombinant cell of the present invention comprises a cell transfected with a nucleic acid molecule that encodes a polypeptide or peptide of the invention. A variety of expression vector/host systems may be utilized to contain and express sequences encoding the viral antigens of the invention. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (e.g., baculovirus); plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems. The invention is not meant to be limited by the host cell employed. The expression of the construct according to the present invention within the host cell may be transient or it may be stably integrated in the genome thereof.

Upregulation of Activity/Expression level of VDACl In one embodiment VDACl expression can be upregulated using a method that comprises the step of providing an exogenous VDACl molecule to the cell under conditions sufficient for said VDACl amino acid sequence to induce apoptosis of the cell. In some embodiments the VDACl molecule is selected from a VDACl polypeptide, a VDACl polypeptide variant and a VDACl peptide, derivative analog or salt thereof according to the principles of the present invention. In other embodiments VDACl molecule is selected from a VDACl nucleotide sequence according to the principles of the present invention. 24 In addition to administering a VDAC polypeptide, VDAC polypeptide variant, or polynucleotide directly to the cell, the upregulation VDACl level can be effected at one of three levels within a cell: at the genomic level for example by activation of transcription by means of tissue specific or cell specific promoters, enhancers, or other regulatory elements; at the transcript level for example by polyadenylation or activation of translation, or at the protein level for example via post-translational modifications or interaction with inhibitory proteins and the like.

Following is a non-comprehensive list of agents capable of upregulating the expression level and/or activity of VDACl.

An agent capable of upregulating expression of a VDACl may be an exogenous polynucleotide sequence designed and constructed to express VDACl, VDACl variant or at least a functional fragment thereof. Accordingly, the exogenous polynucleotide sequence may be a DNA or RNA sequence encoding a VDACl molecule, capable of regulating apoptosis.

The phrase "functional fragment" as used herein refers to part of the VDACl protein (i.e., a peptide) that exhibits functional properties of the protein, such as inducing or preventing apoptosis in a cell. According to preferred embodiments of the present invention, the functional portion of VDACl is a polypeptide or peptide having a sequence selected from SEQ ID NO:21, SEQ ID NO:24 or SEQ ID NO:27, or a variant form thereof which retains the ability to induce apoptosis can be used. VDACl has been cloned from human, rat, mouse, yeast and plant sources, therefore coding sequence information for VDACl is available from gene and protein databases, including the GenBank database (http://www4.ncbi.nlm.nih.gov/).

To express exogenous VDACl in mammalian cells, a polynucleotide sequence encoding a VDACl or a variant SEQ ID NO:30, SEQ ID N0.33, SEQ ID NO:36, SEQ ID NO: 5 1-SEQ ID NO: 56) is preferably ligated into a nucleic acid construct suitable for mammalian cell expression. Such a nucleic acid construct includes a promoter sequence for directing transcription of the polynucleotide sequence in the cell in a constitutive or inducible manner. it will be appreciated that the nucleic acid construct of the present invention can also utilize VDACl homologues, which exhibit the desired activity (i.e. modulation of apoptosis) can be, for example, at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to a nucleotide sequence encoding a protein of the invention (e.g., to the entire length of the nucleotide sequence encoding the protein), or a biologically active portion or complement of any of these nucleotide sequences as determined using the BestFit software of the Wisconsin sequence analysis package, utilizing the Smith and Waterman algorithm.

Constitutive promoters suitable for use with the present invention are promoter sequences that are active under most environmental conditions and most types of cells, such as the cytomegalovirus (CMV) and Rous sarcoma virus (RSV). Inducible promoters suitable for use with the present invention include for example the tetracycline-inducible promoter (Zabala, M . et al., 2004).

The nucleic acid construct (also referred to herein as an "expression vector") of the present invention includes additional sequences that render this vector suitable for replication and integration in prokaryotes, eukaryotes, or preferably both (e.g., shuttle vectors). A typical cloning vector may also contain transcription and translation initiation sequences, transcription and translation terminators, and a polyadenylation signal.

Eukaryotic promoters typically contain two types of recognition sequences, the TATA box and upstream promoter elements. The TATA box, located 25-30 base pairs upstream of the transcription initiation site, is thought to be involved in directing RNA polymerase to begin RNA synthesis. The other upstream promoter elements determine the rate at which transcription is initiated. Enhancer elements can stimulate transcription from linked homologous or heterologous promoters. Enhancers are active when placed downstream or upstream from the transcription initiation site. Many enhancer elements derived from viruses have a broad host range and are active in a variety of tissues. For example, the SV40 early gene enhancer is suitable for many cell types. Other enhancer/promoter combinations that are suitable for the present invention include those derived from polyoma virus or human or murine cytomegalovirus (CMV) and the long tandem repeats (LTRs) from various retroviruses, such as murine leukemia virus, murine or Rous sarcoma virus, and HIV. In constructing the expression vector, the promoter can be accommodated at various distances from the transcription start site without loss of promoter function. 26 Polyadenylation sequences can also be added to the expression vector in order to increase the efficiency of VDAC1 mRNA translation, including a GU- or U-rich sequence located downstream from the polyadenylation site and a highly conserved sequence of six nucleotides, namely AAUAAA, located 1 1-30 nucleotides upstream of the site. Termination and polyadenylation signals suitable for the present invention include those derived from SV40.

In addition to the embodiments already described, the expression vector of the present invention may also contain other specialized elements intended to increase the level of expression of cloned nucleic acids or to facilitate the identification of cells that carry the recombinant DNA. For example, a number of animal viruses contain DNA sequences that promote extra-chromosomal replication of the viral genome in permissive cell types. Plasmids bearing these viral replicons are replicated episomally as long as the appropriate factors are provided by genes either carried on the plasmid or with the genome of the host cell.

The expression vector of the present invention may or may not include a eukaryotic replicon. If a eukaryotic replicon is present, the vector is capable of amplification in eukaryotic cells using the appropriate selectable marker. If the vector does not comprise a eukaryotic replicon, no episomal amplification is possible. Instead, the recombinant DNA integrates into the genome of the engineered cell, where the promoter directs expression of the desired nucleic acid.

The expression vector of the present invention may further comprise polynucleotide sequences that allow, for example, the translation of several proteins from a single mRNA, such as an internal ribosome entry site (IRES) and sequences for genomic integration of the promoter-chimeric polypeptide.

Mammalian expression vectors are commercially available.

Viruses are very specialized infectious agents that have evolved, in many cases, to elude host defense mechanisms. Typically, viruses infect and propagate in specific cell types. The targeting specificity of viral vectors utilizes its natural specificity to specifically target predetermined cell types and thereby introduce a recombinant gene into the infected cell. Thus, the type of vector used by the present invention will depend on the cell type transformed. The ability to select suitable vectors according to the cell 27 type transformed is well within the capabilities of the ordinarily skilled artisan and as such, no general description of selection considerations is provided herein.

Recombinant viral vectors are useful for in vivo expression of VDACl since they offer advantages such as lateral infection and targeting specificity of targeted cells.

Introduction of nucleic acids by viral infection offers several advantages over other methods such as lipofection and electroporation, since higher transfection efficiency can be obtained due to the infectious nature of viruses.

An agent capable of upregulating a VDACl may also comprise any compound capable of increasing the transcription and/or translation of an endogenous DNA or mRNA encoding the VDACl and thus increasing endogenous VDACl activity.

An agent capable of upregulating a VDACl may also comprise an exogenous polypeptide, including at least a functional portion (as described hereinabove) of the VDACl Pharmaceutical Compositions The present invention provides pharmaceutical compositions comprising a polypeptide or nucleic acid as described above and a physiologically acceptable carrier.

Depending on the location of the tissue of interest, VDACl amino acid sequence can be supplied in any manner suitable for the provision of VDACl amino acid sequence. Thus, for example, a composition containing a source of VDACl amino acid sequence (i.e., a VDACl polypeptide or a VDACl expression vector, or cells expressing a VDACl amino acid sequence, as described herein) can be introduced into tissue of interest (e.g., injected, or pumped as a continuous infusion, or as a bolus within a tumor or intercutaneous or subcutaneous site, applied to all or a portion of the surface of the skin, dropped onto the surface of the eye, etc.).

Apart from other considerations, the fact that the novel active ingredients of the invention are polypeptides, peptides, peptide analogs or peptidomimetics, dictates that the formulation be suitable for delivery of these types of compounds. In general, peptides are less suitable for oral administration due to susceptibility to digestion by gastric acids or intestinal enzymes, but it is now disclosed that the compositions according to the present invention may be administered orally. The pharmaceutical composition of this invention may be administered by any suitable means, such as topically, or parenterally 28 including intranasal, subcutaneous, intramuscular, intravenous, intra-arterial, intraarticular, or intralesional administration. Ordinarily, intravenous (i.v.), intraarticular or oral administration will be preferred.

The molecules of the present invention as active ingredients are dissolved, dispersed or admixed in a diluent or excipient that is pharmaceutically acceptable and compatible with the active ingredient as is well known. Suitable excipients are, for example, water, saline, phosphate buffered saline (PBS), dextrose, glycerol, ethanol, or the like and combinations thereof. Other suitable carriers are well known to those in the art. (See, for example, Ansel et al., 1990 and Gennaro, 1990). In addition, if desired, the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents.

Pharmaceutical compositions of the present invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, grinding, pulverizing, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the present invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active compounds into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.

For injection, the compounds of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants for example, polyethylene glycol are generally known in the art.

Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses. 29 Pharmaceutical compositions, which can be used orally, include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. AU formulations for oral administration should be in dosages suitable for the chosen route of administration. For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

For administration by inhalation, the variants for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the peptide and a suitable powder base such as lactose or starch.

Pharmaceutical compositions for parenteral administration include aqueous solutions of the active ingredients in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions.

Suitable natural or synthetic carriers are well known in the art (Pillai et al., 2001).

Optionally, the suspension may also contain suitable stabilizers or agents, which increase the solubility of the compounds, to allow for the preparation of highly concentrated solutions. Alternatively, the active ingredient may be in powder form for reconstitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use.

The compounds of the present invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.

Pharmaceutical compositions suitable for use in context of the present invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of a compound effective to prevent, delay, alleviate or ameliorate symptoms of a disease of the subject being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art.

Toxicity and therapeutic efficacy of the polypeptides, fragments and analogs described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the IC50 (the concentration which provides 50% inhibition) and the LD50 (lethal dose causing death in 50 % of the tested animals) for a subject compound. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition (e.g. Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. l p.l).

Depending on the severity and responsiveness of the condition to be treated, dosing can also be a single administration of a slow release composition, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved. The amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, and all other relevant factors.

In one particularly preferred embodiment according to the present invention, the peptides are administered orally (e.g. as a syrup, capsule, or tablet). In certain embodiments, peptide delivery can be enhanced by the use of protective excipients. This is typically accomplished either by complexing the peptide with a composition to render it resistant to acidic and enzymatic hydrolysis or by packaging the polypeptide in an appropriately resistant carrier such as a liposome. Means of protecting polypeptides for oral delivery are well known in the art (see, e.g., U.S. Patent 5,391 ,377 describing lipid based formulations for oral delivery of therapeutic agents).

Elevated serum half-life can be maintained by the use of sustained-release protein "packaging" systems. Such sustained release systems are well known to those of skill in the art. In one preferred embodiment, a biodegradable microsphere delivery system for proteins and peptides such as the ProLease® system (reviewed in Tracy, 1998) a dry 31 powder composed of biodegradable polymeric microspheres containing the peptide in a polymer matrix that can be compounded as a dry formulation with or without other agents. Serum half-life can also be extended by conjugating the peptide or polypeptide of the invention to a moiety such as PEG using reagents and methods known to those with skill in the art.

The foregoing formulations and administration methods are intended to be illustrative and not limiting. It will be appreciated that, using the teaching provided herein, other suitable formulations and modes of administration can be readily devised.

A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active peptide(s) in a free-flowing form such as a powder or granules, optionally mixed with a binder, (e.g. povidone, gelatin, hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (e.g. sodium starch glycollate, cross-linked povidone, cross-linked sodium carboxymethyl cellulose), surface active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered peptide(s) moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile.

A syrup may be made by adding the active peptide(s) to a concentrated, aqueous solution of a sugar, for example, sucrose, to which may also be added any necessary ingredients. Such accessory ingredients may include flavorings, an agent to retard crystallization of the sugar or an agent to increase the solubility of any other ingredients, such as a polyhydric alcohol, for example, glycerol or sorbitol.

In addition to the aforementioned ingredients, the formulations of this invention may further include one or more accessory ingredient(s) selected from diluents, buffers, flavoring agents, binders, surface active agents, thickeners, lubricants, preservatives, (including antioxidants) and the like.

According to some embodiments of the invention, the therapeutically effective amount of the VDAC1 polypeptide, polypeptide variant, peptide, derivative or analog is a dosage in a range from about 0.02 mg/kg to about 10 mg/kg. Preferably, the dosage of the VDAC1 amino acid sequence according to the present invention is in a range from about 0.05 mg/kg to about 2 mg/kg, more preferably, the dosage of the VDAC1 peptide, derivative or analog is in a range from about 0.1 mg/kg to about 1 mg/kg. It will be understood that the dosage may be an escalating dosage so that low dosage may be administered first, and subsequently higher dosages may be administered until an appropriate response is achieved. Also, the dosage of the composition can be administered to the subject in multiple administrations in the course of the treatment period in which a portion of the dosage is administered at each administration.

Without wishing to be bound to theory, the peptide, derivative or analog thereof is designed to interfere with VDAC1 protein-protein interactions. Preferably, the peptide, derivative or analog thereof is designed to inhibit interactions between VDAC1 protein and a mitochondrial anti-apoptosis protein.

The art discloses examples of peptides and analogs thereof designed to interfere with protein-protein interactions. For example, SAHB (stabilized a helix of BCL2 domains) is a helical, protease resistant, cell permeable peptidomimetic useful for activation of apoptosis in cancer cells (Walensky et si., 2004). Another example is a peptidomimetic designed to mimic the protein-protein interactions of an apoptotic activator, SMAC (Li et al, 2004).

It will be apparent to those of ordinary skill in the art that the therapeutically effective amount of the molecule according to the present invention will depend, inter alia upon the administration schedule, the unit dose of molecule administered, whether the molecule is administered in combination with other therapeutic agents, the immune status and health of the patient, the therapeutic activity of the molecule administered and the judgment of the treating physician. As used herein, a "therapeutically effective amount" refers to the amount of a molecule required to alleviate one or more symptoms associated with a disorder being treated over a period of time.

In some embodiments the peptides and derivatives and analogs thereof of the present invention are delivered to cells as modified peptides. In one embodiment the peptides of the invention are linked to a cell penetrating peptide (CPP). In one preferred embodiment the CPP is an amino acid sequence comprising the Drosophila antennapedia (ANTP) domain or a fragment thereof. 33 In other embodiments the peptides of the present invention are delivered to cells as nucleic acids encoding the peptides in a non-viral gene delivery system. Particulate gene transfer systems are usually based on oligo- or polycationic carrier molecules which can condense nucleic acids by electrostatic interactions with their negatively charged phosphate backbone. The positive charge of cationic lipids leads to electrostatic interaction with the DNA, the lipidic moiety enables the hydrophobic collapse and the formation of so called 'lipoplexes'. Polycationic carrier molecules, like polylysine or polyethyleneimine (PEI) bind and condense DNA due to their high density of positive charges and result in the formation of so called 'polyplexes'. The surface of the delivery particle can be coated with hydrophilic polymers, e.g. polyethyleneglycol (PEG), which prevent binding to plasma proteins, blood cells and the RES and also enables a prolonged circulation time in the blood stream.

In yet another embodiment, the peptides of the present invention are delivered to cells in vesicles. Immunoliposomes have been described to allow targeted delivery of anticancer drugs into solid tumors (for review see Sapra and Allen et al, 2004).

Polypeptides and peptides, and polynucleotide sequences of the present invention can be administered to a subject following microencapsulation. Methods of preparing microcapsules are known in the art and include preparation from an assortment of materials including natural and synthetic materials.

Therapeutic Use Without wishing to be bound to theory, the mitochondrial pathway serves as an excellent target for apoptosis-inducing therapies for several reasons: Mitochondria are integrators of the apoptotic signal: they store apoptotic molecules and release them to the cytosol to initiate caspase cascades; Mitochondria-directed agents are predicted to be effective at low concentrations, since only a fraction of a cell's mitochondria need to be involved for apoptosis to ensue; Downstream effectors of mitochondrial apoptosis are present in all cell types, and seem to be conserved in tumor cells; and Mitochondrial apoptosis induction is known to kill cells effectively. 34 Accordingly, the present invention provides VDAC1 polypeptides, polypeptide variants, peptides, and analogs, derivatives and salts thereof useful in promoting or inhibiting apoptosis in the treatment of diseases associated with aberrant apoptosis.

As used herein, the phrase "diseases associated with aberrant apoptosis" includes diseases and disorders in which timely apoptosis does not ensue and cells proliferate without restraint, for example in proliferative diseases including cancer and tumor growth.

In one preferred embodiment the VDAC1 compounds of the invention are useful in preventing or alleviating a cell-proliferative disorder or a symptom of a cell-proliferative disorder. Cell-proliferative disorders and/or a symptom of a cell-proliferative disorder are prevented or alleviated by administering a VDAC1 polypeptide, polypeptide variant, or peptide to a subject. The cell-proliferative disorder is, for example, neoplasias including solid tumor cancers of the lung, pancreas, stomach, colon, rectum, kidney, breast, cervical/uterine, ovarian, testicular, melanoma, head and neck, and esophagus, as well as hematological cancers, such as leukemias and lymphomas, non-malignant neoplasias, cellular expansions due to DNA viruses such as Epstein-Barr virus, African swine fever virus and adenovirus, lymphoproliferative conditions, arthritis, inflammation, or autoimmune diseases.

Methods of Promoting Apoptosis The present invention provides compounds, compositions and methods of promoting, inducing or enhancing apoptosis. Promotion or induction of apoptosis is useful in the treatment of various disorders associated with aberrant cell proliferation, such as, for example, neoplasias including solid and non-solid cancers and non-malignant neoplasias. Diseases and disorders such as lymphoproliferative conditions, cancer including drug resistant cancer, arthritis, inflammation, autoimmune diseases and the like result from a defect in cell death regulation. Furthermore, certain viruses including Epstein-Barr virus and adenovirus, utilize the host cellular machinery to drive their own replication while repressing host cell death. Accordingly, it would be desirable to promote, induce or enhance apoptotic mechanisms in such disease conditions.

The present invention provides a method for inducing apoptotic cell death comprising contacting a cell with a VDAC1 peptide or VDAC1 polypeptide.

In one preferred embodiment of the invention, the compounds of the present invention are useful for the preparation of a medicament for inhibiting proliferative diseases or disorders including tumor growth and tumor progression. In another embodiment of the invention, the compounds are useful for preventing, treating or inhibiting a cell proliferative disease or disorder. The cell proliferative disease can be malignant or benign. The compositions are useful for the treatment or prevention of non-solid cancers, e.g. hematopoietic malignancies such as, but not being limited to, all types of leukemia, e.g. chronic myelogenous leukemia (CML), acute myelogenous leukemia (AML), mast cell leukemia, chronic lymphocytic leukemia and acute lymphocytic leukemia, lymphomas, and multiple myeloma, as well as of solid tumors such as, but not being limited to, mammary, ovarian, prostate, colon, cervical, gastric, esophageal, papillary thyroid, pancreatic, bladder, colorectal, melanoma, small-cell lung and non-small-cell lung cancers, granulosa cell carcinoma, transitional cell carcinoma, vascular tumors, all types of sarcomas, e.g. osteosarcoma, chondrosarcoma, Kaposi's sarcoma, myosarcoma, hemangiosarcoma, and glioblastomas.

It is to be understood that the terms "treating a proliferative disease or disorder" as used herein in the description and in the claims, are intended to encompass tumor formation, primary tumors, tumor progression or tumor metastasis.

In one preferred embodiment of the invention, the compounds of the present invention are useful for the preparation of a medicament for inhibiting proliferative diseases or disorders including tumor growth and tumor progression. In another embodiment of the invention, the compounds are useful for preventing, treating or inhibiting a cell proliferative disease or disorder. The cell proliferative disease can be malignant or benign. The compositions are useful for the treatment or prevention of non-solid cancers, e.g. hematopoietic malignancies such as, but not being limited to, all types of leukemia, e.g. chronic myelogenous leukemia (CML), acute myelogenous leukemia (AML), mast cell leukemia, chronic lymphocytic leukemia and acute lymphocytic leukemia, lymphomas, and multiple myeloma, as well as of solid tumors such as, but not being limited to, mammary, ovarian, prostate, colon, cervical, gastric, esophageal, papillary thyroid, pancreatic, bladder, colorectal, melanoma, small-cell lung and non-small-cell lung cancers, granulosa cell carcinoma, transitional cell carcinoma, vascular tumors, all types of sarcomas, e.g. osteosarcoma, chondrosarcoma, Kaposi's sarcoma, myosarcoma, hemangiosarcoma, and glioblastomas.

The following examples are presented in order to more folly illustrate some embodiments of the invention. They should, in no way be construed, however, as limiting the broad scope of the invention. One skilled in the art can readily devise many variations and modifications of the principles disclosed herein without departing from the scope of the invention.

EXAMPLES Materials and Methods Materials Carboxymethyl (CM)-cellulose, glucose-6-phosphate, glucose-6-phosphate dehydrogenase (G-6-PDH), HEPES, leupeptin, mannitol, PMSF, Ru360, soybean asolectin, staurosporine, sucrose and Tris were purchased from Sigma (USA). Cibacron blue-agarose was purchased from Amersham Biosciences (Sweden). n-Octyl-b-D-glucopyranoside (β-OG) was obtained from Bachem AG (Bubendorf, Switzerland). Lauryl-(dimethyl)-amineoxide (LDAO) and ruthenium red (97% pure) were obtained from Fluka (Switzerland). Hydroxy apatite (Bio-Gel HTP) was purchased from Bio-Rad Laboratories (USA) and Celite from the British Drug Houses (UK). Monoclonal anti-VDAC antibodies raised against the N-terminal region of 31HL human porin were purchased from Calbiochem (clone no. 173/045, Cat. No. 529538-B, Calbiochem-Novobiochem, UK). Monoclonal antibodies against actin and against the green fluorescence proteins (GFP) were obtained from Santa Cruz Biotechnology (USA). Polyclonal antibodies against HK were kindly provided by Dr. John E. Wilson (University of Michigan, Ann Arbor, MI). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibodies were obtained from Zymed (USA). HRP-conjugated anti-mouse antibodies were obtained from Promega (USA).

Plasmids and site-directed mutagenesis mVDACl (mouse VDAC1) cDNAs (obtained from WJ. Craigen, University of Houston, Texas) were subcloned into plasmid pEGFP-Nl (Clontech) for construction of the VDAC1-GFP or VDAC1 expressing mammalian vector with a neomycin resistant gene serving as marker. Rat muscle VDAC1 cDNA was cloned and sequenced (Shoshan-Barmatz, V. and Ashley R., unpublished). Site-directed mutagenesis of mVDACl was carried out in vitro by overlapping PCR amplification. Recombinant plasmid pSEYC58, 37 carrying wild type mVDACl gene, served as the template for amplification of the VDAC1 variant genes. The variant mVDACl genes were constructed using the T7 and T3 universal primers, together with the primers shown in table 1 to introduce the various point mutations.

Cell Types The U937 human monocytic cell line was grown under an atmosphere of 95% air and 5% C02 in RPMI 1640 supplemented with 10% fetal calf serum (FCS), 1 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin.

T-REx-293 cells: A transformed primary human embryonal kidney cell line (Invitrogen) was grown under an atmosphere of 95% air and 5% C02 in DMEM supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine, 1000 U/ml penicillin, 1 mg/ml streptomycin and 5 g/ml blasticidin. Other cell lines used were stably transfected derivatives of T-REx-293 that express the tetracycline repressor. hVDACl-shRNA T-REx-293 cells: T-REx-293 cells, stably transfected with the pSUPERretro plasmid encoding shRNA targeting hVDACl were grown with 0.5 g/ml puromycin and 5 ug ml blasticidin. The pc-mVDACl-hVDACl-shRNA T-REx-293 cells: hVDACl-shRNA-T-REx-293 cells were transfected with plasmid mVDACl- or E72Q-mVDACl-pcDNA4/TO, expressing mVDACl or E72Q-mVDACl under the tetracycline control. Cells were grown with 200 ug/ml zeocin, 0.5 μg/ml puromycin and 5 μg/ml blasticidin.

All constructs were confirmed by sequencing. The VDAC1 polypeptide variant sequences are denoted herein as follows: E72Q: SEQ ID NO:43; E87Q D88N: SEQ ID NO:44; E65Q-.SEQ ID NO:45; K73L:SEQ ID NO:46; D77N:SEQ ID NO:47; E202Q:SEQ ID NO:48; N75A:SEQ ID NO:49; G67A:SEQ ID NO:50. The corresponding nucleotide sequences are denoted herein SEQ ID NO:51- SEQ ID NO: 58, respectively. 38 Table 1 : Primers used in synthesizing: the VDAC1 variants Underlined nucleotides introduce the relevant mutation.

Mitochondrial preparation Mitochondria were isolated rat liver, brain (Johnson and Lardy 1967) or human leukemic U937 cells using published procedures (Bouregeron, et al, 1992).

Protein determination Mitochondrial protein content was determined by the Biuret method; hexokinase concentration was determined using the Bradford method with ovalbumin as standard.

Ca2+ accumulation Ca2+ uptake by freshly prepared rat liver mitochondria (0.5 mg/ml) was assayed for 1 to 20 minutes at 3C C in the presence of 225 mM mannitol, 75 mM sucrose, 120 39 μΜ CaCl2 (containing 3x1 O4 cpm/nmol [45Ca2+]), 5 mM HEPES/KOH, 5 niM succinate and 0.1 mM Pi, pH 7.0. Uptake of 45Ca2+ was terminated by rapid Millipore filtration followed by a wash with 5 ml of 150 mM KC1.

Mitochondrial swelling Ca^-induced mitochondria large amplitude swelling was assayed in freshly-prepared mitochondria under the same conditions as for Ca2+ accumulation, except that the temperature was 24°C . Cyclosporine A (CsA)-sensitive mitochondrial swelling was initiated upon addition of Ca2+ (0.2 mM) to the sample. Absorbance changes at 520 nm were monitored every 15-20 sec with an Ultraspec 2100 spectrophotometer.

Release of cytochrome C Mitochondria (0.5 mg/ml) were incubated with or without lU/ml of HK-I for 20 min in a solution containing 150 mM KC1, 25 mM NaHC03, 5 mM succinate, 1 mM MgCl2, 3 mM KH2P04, 20 mM HEPES, pH 7.4. Swelling was monitored after the addition of 200 μΜ Ca2+. Following complete swelling, the samples were centrifuged and mitochondrial pellets (25 μg) or supernatant (40 μΐ) were subjected to SDS-PAGE and Western blot analyses using monoclonal anti-cytochrome C antibodies.

Rat brain HK-I purification, activity and binding to mitochondria Rat brain HK-I was purified as described previously (Wilson, J. E., 1989; see also Azoulay-Zohar et al., 2004). HK-I activity was assayed by coupling NADH formation to the production of G-6-P by HK-I and its subsequent oxidation by G-6-PDH in a reaction mixture containing: 4 mM Mg-HEPES, 1 mM K-EDTA, 0.6 mM NAD+, 10 mM glucose, 1 mM ATP, 1 mg/ml BSA, 20 mM K-HEPES, pH 7.8. HK-I binding to mitochondria isolated from the different yeast strains (2 mg/ml) was carried out by a Ih incubation on ice of the mitochondria with HK-I (0-2 U/ml) in 0.1 ml of a solution containing 420 mM mannitol, 140 mM sucrose, 1 mg/ml BSA, 5 mM Mg-HEPES, pH 7.8. Soluble and mitochondria-bound HK-I-containing fractions were separated and analyzed for HK-I activity (Azoulay-Zohar et al., 2004). HK-I binding was defined as the percent of activity present in the bound fraction relative to the total activities found in the bound and free fractions combined. 40 Purification of VDAC Native and mutated mVDACl were extracted with lauryldimethylarnine N-oxide (LDAO) from mitochondria isolated from yeast expressing various VDACs, and purified by chromatography on hydroxyapatite followed by carboxymethyl (CM)-cellulose where LDAO was replaced by β-OG (β-octylglucoside).

VDAC channel recording and analysis Reconstitution of purified VDAC into a planar lipid bilayer (PLB) multi-channel current recording and data analyses were carried as follows. Briefly, PLB were prepared from soybean asolactin dissolved in n-decane (50 mg/ml) in a chamber containing 10 niM HEPES/KOH pH 7.4 and 0.5 or IM NaCl (cis/trans). Only PLBs with a resistance greater than 100 ϋΩ were used. Purified VDAC (about 1 ng) was added to the chamber defined as the cis side. After one or a few channels inserted into the PLB, excess protein was removed by perfusion of the cis chamber with 20 volumes of a solution (with the same composition as before perfusion), in order to prevent further protein incorporation. Currents were recorded under voltage-clamp mode using a Bilayer Clamp amplifier (Warner Instrument, USA). The currents were measured with respect to the trans side of the membrane (ground). The currents were low-pass filtered at 1 kHz, using a Bessal filter (Frequency Devices, USA) and digitized on-line using a Digidata 1200 interface board and pCLAMP 6 software (Axon Instruments). Sigma Plot 2000 scientific software was used for data analyses. Experiments were performed at 23-25 °C.

Gel electrophoresis and immunoblot analyses SDS-PAGE was performed according to Laemmli. Gels were stained with Coomassie Brilliant blue or electroblotted onto nitrocellulose membranes and immunostained using monoclonal anti-VDAC, anti-GFP, anti-actin or polyclonal anti-HK-I antibodies followed by incubation with HRP-conjugated anti-mouse IgG for anti-VDAC, anti-GFP or anti-actin antibodies, and HRP-conjugated goat anti-rabbit IgG for anti-HK-I as secondary antibodies. Antibody binding was detected using chemiluminescence (Santa Cruz Biotechnology, USA).

Xissue Culture and Cell transfection Logarithmically-growing U937 cells were resuspended in RPMI 1640 supplemented with 10% FCS, 100 U/ml penicillin and 100 mg/ml streptomycin at a 41 concentration of 2.5x1 07 cells/ml. Cells were transfected to express rat or murine VDAC1 using the vectors pEGFP, pEGFP-VDACl, pEGFP-E72Q-mVDACl, pE-mVDACl or pE-E72Q-mVDACl. Transfection of the U937 cells was performed by electroporation with a single pulse from a Bio-Rad micropulser II with a capacitance extender unit (200 V, 950 mF). Cells were incubated on ice for 10 min before and after transfection, and then resuspended in 20 ml of RPMI 1640 supplemented with 10% FCS, 1 mM 1-glutamine, 100 U/ml penicillin and 100 mg/ml streptomycin. Transfection efficiencies were 68-72%, as assessed by GFP expression. GFP-positive cells were viewed under a microscope using a blue filter.

Other cells were transfected using metafectene. In summary, cells (3 x 105) growing at 37°C in a C02 incubator were cultured to 30-50% confluence 24 h before transfection. For transfection, two separate solutions were prepared: solution A : 5μg DNA in 50μ1 of serum and antibiotics free DMEM medium; solution B : 2.4μ1 metafectene reagent (Biontex, Munich, DE) in 50μ1 serum and antibiotic-free DMEM medium. The two solutions were combined, mixed gently and incubate at room temperature for 20 min (DNA lipid complexes). After incubation, the cells were resuspended in 1 ml of serum and the DMEM medium, followed by addition of the DNA lipid complexes and gentle mixing. After 5 hours, the transfection mixture was replaced with a complete DMEM medium and cells were allowed to grow at 37°C in a C02 incubator.

Double-Transfection U937 cells were transfected with pcDNA3.1-HisA or pcDNA3.1 -HisA-HK-I (neomycin resistance; provided by Dr. J. E . Wilson, University of Michigan) by electroporation, and grown in complete RPMI 1640 with neomycin (400 mg/ml). After 12 days, the selected cells were transfected with pEGFP, pEGFP-mVDACl or pEGFP-E72Q-mVDACl and were grown in the presence of neomycin (400 mg/ml). The viability of double-transfected cells was analyzed during 48 to 76 nr.

Cell Treatment Cells were plated at a density of 5.4x1 04 cells/cm2 in 24-well plates, washed once with PBS and placed in serum-free medium. Cells were then incubated with or without RuR or Ru360. Cells were also treated with 1.25 mM staurosporine (STS) to induce 42 apoptosis and analyzed for viability 3, 5 and 7 hrs after STS addition. Treatments with curcumin (120 μΜ) or As203 (20 μΜ) were carried out for 48 hrs.

Acridine Orange fAcOrVEthidium Bromide .(EtBr) staining of cells To determine cell viability, cells were stained with 100 mg/ml acridine orange (AcOr) and 100 mg/ml ethidium bromide (EtBr) in PBS. Cells were centrifuged at 1500xg for 5 min at room temperature and resuspended in 25 ml of complete medium, to which 2 ml of AcOr EtBr solution were added. The cells were then visualized by fluorescence microscopy (Olympus 1X51) and images were recorded on an Olympus DP70 camera, using a SWB filter.

Example l . Mapping of the protein-protein docking surfaces in VDAC1 Recently, two domains of VDAC1 that are required for HK-I protection against cell death were identified. Using deletion analysis and site-directed mutagenesis of VDAC1, key contact surfaces on VDAC1 that are essential in its interaction with anti-apoptotic proteins were mapped. VDAC1 mutants were analyzed at the level of purified proteins reconstituted into planar lipid bilayers, isolated mitochondria and in cell culture, where the biological effects of the mutations on induction of apoptosis and protection against apoptosis by HK-I were determined.

The results presented herein in Figure 1 show that HK-I interacts directly with VDAC1 and induces its closure. Figures IA- 1C show that HK-I is able to close native VDAC1 but not mutated E72Q-VDAC1 (SEQ ID NO:43). Figure IA: Purified recombinant native or E72Q-mutated mVDACl was reconstituted into a PLB (planar lipid bilayer) in symmetrical solutions of 0.5 MNaCl. Currents through VDAC1 channel in response to a voltage step from zero to -20 mV were recorded before and 5 min after the addition of HK-I (28.6 mU/ml). The dashed lines indicate the zero-current level. Figure IB shows a Coomassie stain of purified VDAC1 (0.2 g) and HK-I (0.1 μ ) used in these studies. Figure 1C: Cytochrome c release was assayed, using a monoclonal anti-cytochrome c antibody, in the absence or the presence of HK-I ( 1 U/ml), chymotrypsin-treated HK-I ( 1 U/ml) or CsA (10 micro M).

Example 2 : HK-I prevents PTP opening and_release of cytochrome C Figure 2 shows the effects of HK-I on PTP opening, as monitored by Ca2+ accumulation and swelling of energized mitochondria, and by release of cytochrome c . 43 HK-I inhibited PTP opening and this inhibition was prevented by an antibody specific to the N-terminal of HK-I (Fig. 2C). The failure of non-binding species of HK, such as yeast HK, or trypsin- or chymotrypsin-treated HK-I (Fig. 2D) to prevent PTP opening, confirmed the specificity of the HK-I effect, and of the requirement of the HK-I N-terminal region for its interaction with VDAC.

HK-I prevented the release of cytochrome c from mitochondria (as revealed by Western blot analysis, Fig. 1C). The effects of HK-I are similar to those of the well-known inhibitor of PTP, cyclosporine A (CsA) (Figs. 1C and 2B).

Example 3 : HK-I over-expression protects against apoptotic cell death induced by over-expression of native and some VDAC1 variants, but not of E72Q- and E65Q-mVDACl Leukemic U937 cells were transformed to express green fluorescent protein (GFP) and VDAC1-GFP. While cells expressing GFP showed diffuse green fluorescence, cells expressing VDAC1-GFP showed mainly punctuated fluorescence confined to membranes (Fig. 3A). Immunoblot analysis of cell extracts using anti-VDAC or anti-GFP antibodies confirmed the expression of VDAC1-GFP (Fig. 3B). It is noteworthy that the fused VDAC1-GFP was highly protease-sensitive and was readily cleaved to VDAC1 and GFP, as reflected in the appearance of GFP in the VDAC1-GFP expressing cells (Fig. 3B). As shown, in cells transfected to stably over-express HK-I, with and without a second transfection with mVDACl-GFP or E72Q-mVDACl-GFP, the level of HK-I was about 3-4-fold higher than in HK-I non-transfected cells The effect of HK-I over-expression on apoptotic cell death of the GFP-tagged version of native and E72Q-mVDACl over-expressing cells was examined. Cells were first transformed to over-express HK-I and then transfected with native mVDACl-GFP or E72Q-mVDACl-GFP. Apoptotic cell death induced by native mVDACl over-expression was dramatically reduced in the cells over-expressing HK-I to a low level of apoptosis (< 10%), equal to the control (Fig. 3C and 3D).

The E72 amino acid is not conserved in the yeast VDAC1, known for its low ability to bind HK-I. Comparison of the amino acid sequence in the structural cytosolic β-loop 2 domain, which includes the E72 amino acid residue in human or murine VDAC1 with that of VDAC1 from Saccharomyces cerevisiae, or Neurospora crassa reveals that in addition to E72, the charged amino acids D77, K73, and E65 are not 44 conserved. To verify these residues' function in HK-I binding, they were replaced by N, L, and Q, respectively, using overlapping PCR.

Since VDACl overexpression induces cell death, U937 cells (Fig. 4A, 4B) or T-Rex™ 293 human embryonic kidney cells (Fig. 4B) were first transformed to stably over-express HK-I and then transfected with native, E65Q, E72Q, K73L, D77N, N75A, or G67A mutated mVDACl-GFP. Apoptotic cell death induced by native mVDACl and by VDACl mutated at non-charged amino acids N75A or G67A mutated-m VDACl over-expression was dramatically reduced in the cells over-expressing HK-I (Fig. 4B). On the other hand, as obtained with E72Q-expressing cells, no protective effect of HK-I against cell death was obtained in cells over-expressing E65Q-, K73L-, D77N- or E202Q-m VDACl variants (Fig. 4). These results suggest that HK-I interaction with VDACl involves charged amino acids in the cytosolic β-loop domain in which the E72 residue is located. This β-loop includes amino acid residues T64 to about N78. Fig. 3E shows that HK-I protects against apoptosis induced by STS.

Example 4 : Ectopic expression of VDACl induces apoptosis Expression of exogenous murine or rat VDACl or VDAC1-GFP, E72Q- or other mutated mVDACl variants in various cell lines resulted in cell death characterized by nuclear fragmentation. About 70 to 85% of the cells had died 70 to 78 hrs following cell transfection (Figs. 3-5; Tables 2-4 herein below; Zaid et al, 2005). Similar results were obtained upon transfection of Jurkat cells with rice or human VDAC (Godbole et al, 2003). These results indicate that the cellular expression level of VDAC may play a crucial factor in the process of mitochondria-mediated apoptosis.

The mechanism by which enhanced VDACl expression leads to cell death is not known. Without wishing to be bound to theory, it is possible that a transient increase in the VDACl levels produce a significant increase in the leak of the outer mitochondrial membranes and this, in turn, decreases cell viability. This suggestion is in line with the finding that expression of exogenous VDACl could only be observed upon exposure of the native but not VDACl variant transfected cells to RuR, a treatment that protects against cell death. (Fig. 5D). It thus seems that it is not an increase in the total amount of VDACl, but rather an increase in its functionality that is responsible for the apoptotic cell death. Similarly, cells over-expressing HK-I, which interact with VDACl and inhibit 45 cytochrome c release, were resistant to VDACl exogenous expression induced cell death (Figs. 3 and 4).

Recently, we have shown that dynamic VDAC oligomerization is involved in the release of cytochrome c from mitochondria and suggested that cytochrome c may cross the outer mitochondrial membrane via the large flexible pore formed between individual subunits of a VDACl tetramer (Zaik, et al., 2005). Without wishing to be bound to theory, VDACl over-expression may encourage VDACl oligomerization and thus allow release of pro-apoptotic proteins, such as cytochrome c, from the mitochondrial intermembrane space. The apoptosis-inducing effect of the reagent As203, was recently attributed to an induction of homodimerization of VDAC molecules (Zheng, et al, 2004). This effect could be prevented by over-expression of the anti-apoptotic protein Bcl-xL.

Figure 6 shows the transmembrane topology of the VDACl protein according to thel3 transmembrane β-strand model (modified from Colombini, M., 2004). Proposed cytoplasmic domains of VDACl, as well as the mutated amino acids are shown schematically. The gray sequences are the proposed peptides for involvement in protein-protein interactions. Mutation of the circled amino acids E65, E72, K73, D77, E202 eliminates HK-I binding to VDAC and HK-I protection against cell death (Figs.3 and 4).

Example 5 : RuR protects against apoptotic cell death induced by over-expression of native, but not of mutated mVDAC.

To further investigate the relationship between the protective effect of RuR and its interaction with VDACl, leukemic U937 cells were transfected with a plasmid encoding native, E65Q, E72Q, K73L, or D77N-mVDACl variant. Fifty six (56) hr after transfection, cells were exposed for 20 hr to RuR ( 1 μΜ) and cell viability was analyzed using acridine orange/ethidium bromide staining. In each independent experiment, approximately 250 cells were counted for each including early and late apoptotic cells. Data shown in Fig. 5 and Table 2 are the mean ± SEM (n=3). Cells transfected with native, or mutated mVDACl or mVDACl-GFP, to induce over-expression, resulted in increased apoptotic cell death (70-75%), as characterized by enhanced nuclear fragmentation indicative of apoptotic cell death (Fig. 5A). In contrast, control or GFP-transfected cells showed about 14-16% nuclear fragmentation (Fig. 5B and 5C). Pre-1 incubation of cells transfected to induce over-expression of VDACl or VDAC1-GFP with 1 uM RuR for 18 hr or with 5 μΜ RuR for 5 hr dramatically reduced their apoptotic 46 cell death (64-72 % protection). RuR had no effect or even slightly increased apoptotic death in control or plasmid-transfected cells (Fig. 5B and 5C).

Strikingly, the protective effect of RuR on recombinant VDAC1 over-expression- induced cell death was not observed in cells expressing E72Q-mVDACl, E72Q- mVDACl-GFP or E65Q-mVDACl (Fig. 5B and 5C). Given the RuR-insensitive behavior of E72Q-mVDACl, as reflected by the inability of RuR to inhibit channel activity in this mutant (Fig. 5E), it appears that RuR protection against apoptosis is exerted through its direct interaction with VDAC. Moreover, the increase (over 5 -fold) in the total level of VDAC1 protein in the transfected cells was observed only upon exposure of cells expressing native mVDACl to RuR, but not in cells expressing E72Q- mVDACl (Fig. 5D).

This indicates that the apoptotic cell death induced by exogenous VDAC1 expression leads to VDAC1 degradation that was prevented upon RuR binding to VDAC1 and protecting against apoptotic cell death.

Table 2 Example 6 : RuR induces channel closure: of native but not E72Q mVDACl variant RuR, known to interact with several Ca2+ binding proteins and to inhibit Ca2+ transport in mitochondria, inhibited channel activity of native but not of E72Q-mutated VDAC1 (Fig. 5E). Purified recombinant native and E72Q-mVDACl variant were reconstituted into a planar lipid bilayer (PLB) and the currents produced in response to voltages stepped from a holding potential of 0 mV to -20 mV were recorded. In nine of ten experiments, RuR inhibited recombinant native mVDACl channel activity and stabilized the channel in an almost completely closed state. The level of inhibition calculated from five different experiments was 89.2 ± 2.8 % (mean ± SD, n= 9). In 47 contrast, the channel activity of E72Q-mVDACl was not inhibited by RuR in all ten experiments.

Example 7 : Cell type dependent apoptosis induction by various stimuli, but not by VDAC1 overexpression Cells were incubated with curcumin (120 μΜ) or ASJOJ (20 μΜ) for 48 hrs, or with STS (1.25 uM) for 7 hrs. Cell viability was analyzed by acridine orange/ethidium Bromide staining. In each independent experiment, approximately 250 cells were counted for each treatment, in which early and late apoptotic cells were also counted. The data shown in table 3 is the mean ± S.E.M. (n=3).

Table 3 In contrast to the cell-type dependent effect of the various apoptosis stimuli (Table 3), Table 4 herein below shows that over-expression of native and E72Q variant mVDACl induces apoptotic cell death in all cell lines tested.

The following cell types T-Rex-293 (human embryonic kidney), HeLa (human cervical carcinoma), Pane (human pancreas adenocarcinoma), B-16 (mouse skin melanoma), Du-145 (human prostate carcinoma) and PC- 12 (rat adrenal pheochromocytoma) were transfected with native or mutated E72Q mVDACl (5ug) by metafectene reagent. Cell viability was analyzed 76h after transfection by acridine orange/ethidium bromide staining. In each independent experiment, approximately 250 cells including early and late apoptotic cells were counted. 48 Table 4 Example 8 : Peptide Synthesis The following VDAC1 peptides were chemically synthesized: Peptide N (SEQ ID NO:l) is a 26 amino acid peptide which comprises an amino acid sequence derived from the N-terminal cytosolic segment of VDAC1, the peptide comprising amino acids 1-26 of VDAC1: ' M A VPPT Y ADLGKS ARD VFTKG YGFGL ; encoded by Nuc N (SEQ ID NO:6): 5'ATG GCT GTG CCA CCC ACG TAT GCC GAT CTT GGC AAA TCT GCC AGG GAT GTC TTC ACC AAG GGC TAT GGA TTT GGC TTA Peptide LP1 (SEQ ID NO:2) is a 15 amino acid peptide comprising an amino acid sequence derived from the first cytosolic β-loop of VDAC1, the peptide comprising amino acids 63-78: 5' WTEYGLTFTEKWNTD; encoded by Nuc LP1 (SEQ ID NO:7): TGG ACT GAG TAC GGC CTG ACG TTT ACA GAG AAA TGG AAT ACC GAC AAT Peptide LP2 (SEQ ID NO:3) is a 20 amino acid peptide comprising an amino acid sequence derived from the second cytosolic p oop of VDAC1, the peptide comprising amino acids 100-119: 5'SSFSPNTGKKNAKIKTGYKR; encoded by Nuc LP2 (SEQ ID NO:8): TCA TCC TTC TCA CCT AAC ACT GGG AAA AAA AAT GCT AAA ATC AAG ACA GGG TAC AAG CGG GAG CAC ATT Peptide LP3 (SEQ ID NO: 4) is an 18 amino acid peptide comprising an amino acid sequence derived from an inter-membranal sequence of VDAC1, the peptide comprising amino acids 157-174: 5' ET AKSRVTQ SNF A VG YKT ; encoded by Nuc LP3 (SEQ ID NO:9): GAG ACT GCA AAA TCC CGA GTG ACC CAG AGC AAC TTT GCA GTT GGC TAC AAG ACT. LP3 serves as a negative control. 49 Peptide LP4 (SEQ ID NO:5) is a 17 amino acid peptide comprising an amino acid sequence of cytosolic β-1οορ 4 of VDACl, the peptide comprising amino acids 199-215: 5'KKLET AVNLAWTAGNSN, encoded by Nuc LP4 (SEQ ID NO:10): AAG AAG TTG GAG ACC GCT GTC AAT CTT GCC TGG ACA GCA GGA AAC AGT AAC The peptides are expressed in pRSET as His6 fusion proteins and are purified using Ni affinity chromatography and, if necessary, HPLC. The effects of these peptides on HK-I binding to isolated mitochondria and on reconstituted VDACl channel activity are tested. Similar studies are carried out for Bax, Bcl2, Bcl-xL and tBids to search for peptides that specifically interact with these anti-apoptotic proteins.

Figure 8 shows alignment of the human VDAC isoforms to the corresponding mouse and rat sequences, for reference only.

Example 9 : VDACl peptide detaches bound HK-I from brain mitochondria Five VDACl peptides were chemically synthesized (Oligonucleotide and Peptide Synthesis Unit, Weizmann Institute). Rat brain mitochondria, known to possess significant amounts of bound HK-I, were used for testing the effects of the VDACl peptide on detachment of bound HK-I. As shown in Figure 9, VDACl loop 1 peptide (LP1), containing the E72, was able to release mitochondrial bound HK-I as its activity increased in the supernatant. HK-I. The peptide induced the detachment of mitochondria-bound HK-I, suggesting that a dynamic equilibrium between free and mitochondria-bound HK-I exists.

Figures 10A- 1OB shows that peptide LP1 eliminates the HK-I prevention of PTP opening. Mitochondrial swelling was assayed in the absence (control · ) or the presence of HK-I (gray · ) and in presence HK-I pre-incubated with peptide LP1. Mitochondrial swelling was assayed as in Fig. 2 in the absence and the presence of peptide LP1 (0.1 mM or 0.3 mM), brain HK-I (0.25U/ml) or with HK-I pre-incubated for 10 min with LP1. Dark circles represents control, light circles are HK-I alone; triangles are HK-I and LP1 (0.1 mM), dark squares are HK-I and LP1 (0.3 mM).

Example 10:. Delivery of VDAC sequences Several types of delivery systems are being tested for the delivery of the VDACl specific peptides, polypeptides and nucleic acids. These delivery systems include: a) Synthetic cell type-specific cell-penetrating VDAC peptides were generated for delivery to cells; b) Non- viral system-mediated gene delivery for delivery and targeting of vectors encoding VDAC peptides and polypeptides; c) Vesicles for polypeptide, peptide and nucleic acid targeting and delivery to target cells.

Cell-penetrating VDAC peptides: The use of peptides as drugs is limited by the peptides inability to cross the cell membrane. A novel carrier system that originates from membrane shuttling proteins such as Antp, the HIV-I transcriptional factor TAT and VP22 from HSV-I has advantages for targeted delivery compared with standard translocation techniques. This "transport system" is mediated by the so-called CPPs, (Cell Penetrating Peptides), which consist of short peptide sequences that rapidly translocate large molecules into the cell interior in a seemingly energy- and receptor-independent manner. CPPs have low toxicity and a high yield of delivery.

Most small peptides are flexible in solution and do not adopt the structure that the same sequence adopts in the native protein. Some of the selected VDAC1 peptides, according to VDAC1 topological model (see Fig. 6), exist in the form of β-loops. Therefore, the peptides were designed to contain the amino acids sequence SWTWE at the N-terminus of the peptide and KWTWK at the C-terminus (together the "Tip zipper amino acids"). These sequences can induce the formation of stable β-hairpins by tryptophan-tryptophan cross-strand pairs (TRP zipper) as demonstrated for peptide LP1 in Fig. 7. The following sequences refer to the TRP zipper and or ANTP domain peptides: Peptide N-ANTP (SEQ ID NO: 11) is a 26 amino acid peptide representing the N-terminal segment of VDAC1, further comprising the ANTP domain: 'MRQIKIWFQNR MKWKKMAVPPTYADLGKSARDVFTKGYGFGL; encoded by Nuc N-ANTP as set forth in SEQ ID NO: 16: 'ATGCGTCAGATTAAAATTTGGTTTCAGAATCGTCGTATGAAATGGA AAAAAATGGCTGTGCCACCCACGTATGCCGATCTTGGCAAATCTGCCAGGGA TGTCTTCACCAAGGGCTATGGATTTGGCTTATGA Peptide LPl-trp (SEQ ID NO: 12) is a 42 amino acid peptide comprising an amino acid sequence derived from the first cytosolic loop of VDAC1, amino acids 63-78, further comprising Trp zipper amino acids and the ANTP domain: 51 'MRQIKIWFQNR MKWKKSWTWEWTEYGLTFTEKWNTDKWTWK; encoded by Nuc LPl-trp as set forth in SEQ ID NO: 17: 'ATGCGTCAGATTAAAATTTGGTTTCAGAATCGTCGTATGAAATGGA AAAAATCCTGGACCTGGGAATGGACTGAGTACGGCCTGACGTTTACAGAGA AA TGGAATACCGACAATAAATGGACCTGGAAATGA Peptide LP2-trp (SEQ ID NO: 13) is a 47 amino acid peptide comprising an amino acid sequence derived from the second cytosolic loop of VDACl, amino acids 100-1 19, further comprising Trp zipper amino acids and the ANTP domain: 'MRQIKJWFQNRRMKWKKSWTWESSFSPNTGKKNAKIKTGYKRKWTW ; encoded by Nuc LP2-trp as set forth in SEQ ID NO: 18): 'ATGCGTCAGATTAAAATTTGGTTTCAGAATCGTCGTATGAAATGGA AAAAATCCTGGACCTGGGAATCATCCTTCTCACCTAACACTGGGAAAAAAAA TGCTAAAATCAAGACAGGGTACAAGCGGGAGCACATTAAATGGACCTGGAA ATGA Peptide LP3-trp (SEQ ID NO:14) is an 45 amino acid peptide comprising an amino acid sequence derived from the inter-membranal p oop (loop 3) of VDACl, amino acids 1 7-174, further comprising Trp zipper amino acids and the ANTP domain: 'MRQD IWFQNRRMKWKKSWTWEETAKSRVTQSNFAVGYKTKWTWK; encoded by Nuc LP3-trp as set forth in SEQ ID NO: 19: 'ATGCGTCAGATTAAAATTTGGTTTCAGAATCGTCGTATGAAATGGA AAAAATCCTGGACCTGGGAAGAGACTGCAAAATCCCGAGTGACCCAGAGCA ACTTTGCAGTTGGCTACAAGACTAAATGGACCTGGAAATGA Peptide LP4-trp (SEQ DD NO: 15) is a 44 amino acid peptide comprising an amino acid sequence derived from the fourth Moop (cytosolic) of VDACl, amino acids 199-215, further comprising Trp zipper amino acids and the ANTP domain: 'MRQIKIWFQNRRMKWKKSWTWEKKLETAVNLAWTAGNSNKWTWK, encoded by Nuc LP4-trp as set forth in SEQ ID NO:20: 'ATGCGTCAGATTAAAATTTGGTTTCAGAATCGTCGTATGAAATGGA AAAAATCCTGGACCTGGGAAAAGAAGTTGGAGACCGCTGTCAATCTTGCCTG GACAGCAGGAAACAGTAACAAATGGACCTGGAAATGA 52 To facilitate the translocation of the peptides across the cell membrane, the sequence peptide was linked to the C terminus of the cell penetrating peptide (CPP) derived from the Drosophila homeobox protein Antennapedia (Antp). By using available antibodies against Antp, the expression/penetration of the peptide in the cell can be followed.

Thus, by linking VDAC peptide sequences, for example VDACl peptide sequences to a CPP, as depicted in Figure 7, the peptides are introduced into the cell. Without wishing to be bound to theory, the peptide alters the existing VDAC1-HK-I and/or VDAC1-Bcl2 interactions and thereby reduces their anti-apoptotic activity. Control cells and HK-I or Bcl2 over-expressing B- 16 cells are incubated without and with different concentrations of VDAC peptides or Antp- VDAC peptides, and apoptosis induced by staurosporine and other reagents is monitored. The peptides with the highest activity in preventing the HK-I or Bcl2 anti-apoptotic activity are further tested in cancer cell lines, including prostate, breast, lung, kidney, neuroblastoma, leukemia, glioblastoma cell lines.

Non-Viral Peptide Delivery: Another tool for delivery and targeting VDAC peptides is the use of "shielding particles". The particle surface of these delivery particles can be coated with hydrophilic polymers, e.g. polyethylene glycol (PEG), which prevent binding to, for example, plasma proteins and blood cells and also enable prolonged circulation time in the bloodstream (Oupicky et al., 2002) To provide vectors with the ability to distinguish between target and non-target tissue, cell-binding ligands that recognize target-specific cellular receptors can be added to the particle. Numerous cell-targeting ligands, including peptides, growth factors, antibodies or other proteins, are incorporated into polyplexes (Wagner, et al, 2005) after chemical conjugation to cationic polymers (Schatzlein, 2003). Thus, tailor-made synthetic vectors can be used to achieve targeting to tumor tissue. Therapeutic concepts based, for example, on suicide genes or cytokines, showed encouraging results in preclinical and also in first clinical evaluations. The aim of this proposal is to use a novel targeted gene transfer systems based on DNA/ligand-polycation complexes with tumor targeting ligands to deliver VDAC peptides and derivatives and analogs thereof to cancer cells.

Delivery Vesicles: The delivery vesicles are preferably designed to prevent binding of the VDACl peptide and derivatives and analogs thereof to plasma proteins or 53 to blood cells and will enable a prolonged circulatory time in the blood stream. To provide vectors with the ability to distinguish between target and non-target tissue, cell-binding ligands that recognize target-specific cellular receptors are introduced. For tumor specific targeting, receptor-targeted polyplexes based on PEI and using transferrin (Tf), epidermal growth factor (EGF) or other ligand for targeting. Depending on the cell type, part of the PEI polymer in the complex is replaced by ligand-PEI conjugate (for example, Tf-PEI or EGF-PEI). All formulations contain PEG-PEI conjugate for shielding purposes.

Example 1 1 : Formulation and stabilization of peptide drugs Pharmaceutical macromolecules may be damaged during bio-processing and storage; thus, analytical tools are needed for the stage of formulation development and stabilization of peptides. Strategies for solubility optimization, storage, the use of stabilizing agents and stability-indicating assays are developed for the VDAC peptides.

Moreover, certain peptides, when introduced to the cell interior, are rapidly degraded, and thus the effect of the peptide might not be as long lasting as desired. To make the peptides more effective drugs, the preparation of peptide analogs, including peptide derivatives and peptidomimetics, having increased stability, including protection against proteolysis, is required. Peptide analogs containing for example N (alpha)-methylamino acids are increasingly recognised as potentially useful therapeutics (Jarver and Langel, 2004).

In addition, the use of an analog of the VDAC effective peptides comprising some or all D-amino acids fused to a D-configured amino acid analog of the Antp are constructed and tested.

Liposomes containing lipid-linked maleinimide is generated by standard loading technique with VDAC1 -derived peptides (Derycke and Witte, 2002). Following separation of peptide loaded liposomes from free peptide by size exclusion chromatography, transferrin is modified with hetero-bifunctional cross linker introducing a free thiol group into the transferrin molecule. Covalent coupling to the liposome surface is carried out by reaction of the thiol group with maleinimide residues on the liposome surface. 54 Example 12: Pro-apoptotic activity of VDAC1 peptide fragmentsjn cancer cells The effect of VDAC1 peptides in minimizing the self-defense mechanisms of cancer cells, which involve over-expression of anti-apoptotic proteins such as HK-I, are tested in several cancer cell lines, among them recombinant HK-I over-expressing cells. The ability to increase the efficacy of chemotherapeutic drugs by VDAC-based peptides is a step toward developing practical therapies to minimize the self-defense mechanisms of cancer cells involving over-expression of anti-apoptotic proteins such as HK-I.

In a non-limiting example, peptides are introduced into cells by one of at least three ways: Cells are transfected with mammalian expression vector encoding a VDAC peptide or peptide analog.

VDAC1 polypeptides or peptides fused to penetrating peptides are added to the cells. Alternatively, VDAC1 polypeptide or peptide packed in shield particles containing targeting ligand are added to the cells.

Cell viability and apoptotic cell death of control, HK-I and peptide containing cells is followed by staining cells with acridine orange (AcOr) and ethidium bromide (EtBr) and by Flow Cytometry (FACS) analysis. The expression levels of HK-I, HK-II, Bcl2, Bcl-xL and Bax in these cells are estimated using antibodies specific to each protein. In addition, the level of VDAC peptide in the cells is analyzed using anti-Antp antibodies (anti-HK antibodies from Santa Cruz; anti-VDAC antibodies from Calbiochem; anti-ANTP antibodies from GentTex Inc.) Example 13: Pro-apoptotic activity of VDAC peptides in animal models Imaging and follow up of synthetic VDAC peptides in vivo by non-invasive measurement of apoptosis and monitoring of glucose metabolism, with positron emission tomography (PET) and fluorodeoxyglucose, respectively are essential components of a study of this nature. Thus, in vitro and in vivo studies with the most effective VDAC peptides are carried out. Stability of the peptides is evaluated in vivo in this study by utilizing scintographic imaging and HPLC serum.

First, to evaluate their uptake kinetics, the peptides or peptide analogs are labeled with 13II and bio-distribution of the labeled peptides is determined in tumor-bearing animals. Complementary animal models are performed including different subcutaneous 185500/2 tumor models, either in immune competent mice (e.g. neuroblastoma in A/J, melanoma in C57/BL6, CT26 in Balb/c) or SCID mice (hepatoma and others).

Example 14: VP AC peptides enhance sensitivity of cancer cells to chemotherapy The molecular events responsible for chemotherapy resistance of cancer cells remain largely unknown. Pro-survival members of the Bcl-2 family are expressed in many tumors and are associated with the repression of apoptosis induced by chemotherapeutic drugs in vitro. Since the anti-apoptotic Bcl-2 proteins are thought to act via interaction with VDAC, the effect of VDAC-based peptides on Bcl2 over-expressing cells to sensitivity to chemotherapy is studied.

The Bcl-2 protein is produced at high levels in many types of cancer, including about 90% of colorectal, 30-60% of prostate, 70% of breast, 20% of non-small cell lung cancers, and 65% of lymphomas. The in vivo, expression of Bcl-2 has been associated with a poor response to therapy in certain subgroups of cancer patients, including some patients with lymphoma, acute leukemia, and prostate cancer.

Example 15: Pro-apoptotic activity of VDAC peptides in apoptosis-defective and chemotherapy-resistant cancer cell models To test the effect of the VDAC-based peptides in apoptosis-defective and chemotherapy-resistant cancer cells, an apoptosis-resistance model is used. For example, the non-small cell lung carcinoma (NSCLC) cell lines exhibit an intrinsic resistance to chemo- and radiotherapy, are used to study the mechanisms of apoptosis resistance and to find ways of overcoming this resistance with the VDAC peptides and analogs thereof.

All parts of the description that are not encompassed by the claims are not part of the present invention. 56 REFERENCES Ausubel, et al (Eds), Current Protocols in Molecular Biology . John Wiley & Sons, Inc. (New York) (1987-1999) Azoulay-Zohar, H., Israelson, A., Abu-Hamad, S. and Shoshan-Barmatz, V. (2004) In self-defense: hexokinase promotes voltage-dependent anion channel closure and prevents mitochondria-mediated apoptotic cell death. Biochem. J. 377: 347-355.

Blachly-Dyson E, Zambronicz EB, Yu WH, Adams V5 McCabe ER, Adelman J, Colonibini M, Forte M . 1993. Cloning and functional expression in yeast of two human isoforms of the outer mitochondrial membrane channel, the voltage- dependent anion channel. J Biol Chem. 268(3): 1835-41 .

Colombini, M . (2004) VDAC: The channel at the interface between mitochondria and the cytosol. Molecular and Cellular Biochemistry 256/257, 107-1 15.

Cochran, AG, Skelton, NJ and Starovasnik, MA. (2001). Tryptophan zippers: Stable, monomeric-hairpins. PNAS, 98:10 pp 5578-5583.

Derycke, AS and Witte PA, (2002) "Liposomes for photodynamic therapy" Int. J. Oncology, 20, ppl81-187 Gennaro (ed.), Remington's Pharmaceutical Sciences, 18th Ed. (Mack Publishing Co., 1990) Godbole A, Varghese J, Sarin A and Mathew MK. (2003) VDAC is a conserved element of death pathways in plant and animal systems. Biochim. Biophys. Acta. 1642: 87-96. Jarver, P. and Langel, U . (2004) The use of cell-penetrating peptides as a tool for gene regulation. .Drug Discov Today 9, 395-402.

Johnson , D. and Lardy, H . (1967) Rat liver mitochondria preparation. Meth. Enzymol., 94-96 Kim R. (2005) Unknotting the roles of Bcl-2 and Bcl-xL in cell death. Biochem Biophys Res Commun. 333(2):336-43.

Li L, Thomas RM, Suzuki H, De Brabander JK, Wang X, Harran PG. (2004). A small molecule Smac mimic potentiates TRAIL- and TNF alpha-mediated cell death. Science.305(5689): 1471-4. 57 Meienhofer, J (1973). "Hormonal Proteins and Peptides," vol. 2, p. 46, Academic Press (New York).

Mendoza, FJ, Espino, PS5 Cann, KL5 Brisrow, N5 McCrea, K and Los, M. (2005) "Anti-1 tumor chemotherapy utilizing peptide based approaches-apoptotic pathways, kinases and proteasome targets". Arch Immunol Ther Exp 53:47-60.

Oupicky D., Ogris M. and Seymour L.W. (2002) Development of long-circulating polyelectrolyte complexes for systemic delivery of genes. J Drug Target. 10: 93-98 Pillai 05 Panchagnula R. (2001) Polymers in drug delivery. Curr. Opin. Chem. Biol. 5, 447-51, Sambrook, J5 Fritsch, E. F . and Maniatis, T. (1989) Molecular Cloning: A laboratory manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, USA.

Sapra, P and Allen TM (2004) "Improved outcome when B-cell lymphoma is treated with combinations of immunoliposomal anticancer drugs targeted to both the CD 19 and CD20 epitopes." Clin Cancer Res. 10(7):2530-7).

Schatzlein AG. (2003) Targeting of Synthetic Gene Delivery Systems. J Biomed Biotechnol. 2003, 149-158 Schroder, G and Lupke, K. (1965). The Peptides, vol. 15 Academic Press (New York).

Shoshan-Barmatz, V and Gincel D. (2003) The voltage-dependent anion channel: characterization, modulation, and role in mitochondrial function in cell life and death. Cell Biochem. Biophys. 39: 279-292.

Stewart, J. M. and Young, J. D. (1963), "Solid Phase Peptide Synthesis," W. H. Freeman Co. (San Francisco).

Tracy, MA. (1998) Development and scale-up of a microsphere protein delivery system. Biotechnol. Prog. 14, 108.

Wagner E5 Culmsee C, Boeckle S , (2005). "Targeting of Polyplexes: Toward Synthetic Virus Vector Systems" Adv Genet. 53PA:333-354.

Walensky LD5 Kung AL, Escher I, Malia TJ, Barbuto S, Wright RD, Wagner G, Verdine GL, Korsmeyer SJ. Activation of apoptosis in vivo by a hydrocarbon- stapled BH3 helix. (2004). Science. 305(5689): 1466-70. 58 Wilson, J. E . (1989). Rapid purification of mitochondrial hexokinase from rat brain by a single affinity chromatography step on Affi-Gel blue. Prep Biochem 19, 13-21.

Zabala M , Wang L, Hernandez-Alcoceba R, Hillen W, Qian C, Prieto J 5 Kramer MG. (2004), Optimization of the Tet-on system to regulate interleukin 12 expression in the liver for the treatment of hepatic tumors. Cancer Res. 64(8):2799-804.

Zalk R, Israelson A , Garty E , Azoulay-Zohar H and Shoshan-Barmatz V . (2005).

Oligomeric states of the voltage-dependent anion channel and cytochrome c release from mitochondria. Biochem. J. Biochem J 386, 73-83.

Zheng Y, Shi Y, Tian C, Jiang C, Jin H , Chen J, Almasan A , Tang H and Chen Q . (2004) Essential role of the voltage-dependent anion channel (VDAC) in mitochondrial permeability transition pore opening and cytochrome c release induced by arsenic trioxide. Oncogene 23: 1239-1247.

Zaid, H., Abu-Hamad, S., Israelson, A., Nathan, L and Shoshan-Barmatz, V . (2005) Cell Death Differ 12, 751-6.

Citation of any document herein is not intended as an admission that such document is pertinent prior art, or considered material to the patentability of any claim of the present application. 59 185500/2 <160> Number of SEQ ID NOS <170> Patentln version 3.3 <210> 1 <211> 26 <212> PRT <213> Artificial <220> <223> SYNTHETIC PEPTIDE <400> 1 Met Ala Val Pro Pro Thr Tyr Ala Asp Leu Gly Lys Ser Ala Arg Asp 1 5 10 15 Val Phe Thr Lys Gly Tyr Gly Phe Gly Leu 25 <210> 2 <211> 15 <212> PRT <213> Artificial <220> <223> SYNTHETIC PEPTIDE <400> Trp Thr Glu Tyr Gly Leu Thr Phe Thr Glu Lys Trp Asn Thr Asp 1 10 15 <210> 3 <211> 20 <212> PRT <213> Artificial <220> <223> SYNTHETIC PEPTIDE <400> 3 Ser Ser Phe Ser Pro Asn Thr Gly Lys Lys Asn Ala Lys lie Lys Thr 1 5 10 15 Gly Tyr Lys Arg <210> 4 <211> 18 <212> PRT <213> Artificial <220> <223> SYNTHETIC PEPTIDE <400> 60 185500/2 Glu Thr Ala Lys Ser Arg Val Thr Gin Ser Asn Phe Ala Val Gly Tyr 1 5 10 15 Lys Thr <210> 5 <211> 17 <212> PRT <213> Artificial <220> <223> SYNTHETIC PEPTIDE <400> 5 Lys Lys Leu Glu Thr Ala Val Asn Leu Ala Trp Thr Ala Gly Asn Ser 1 5 10 15 Asn <210> 6 <211> 78 <212> DNA <213> Artificial <220> <223> NUCLEOTIDE SEQUENCE CORRESPONDING TO SYNTHETIC PEPTIDE <400> 6 atggctgtgc cacccacgta tgccgatctt ggcaaatctg ccagggatgt cttcaccaag 60 ggctatggat ttggctta 78 <210> 7 <211> 48 <212> DNA <213> Artificial <220> <223> NUCLEOTIDE SEQUENCE CORRESPONDING TO SYNTHETIC PEPTIDE <400> 7 tggactgagt acggcctgac gtttacagag aaatggaata ccgacaat 48 <210> 8 <211> 69 <212> DNA <213> Artificial <220> <223> NUCLEOTIDE SEQUENCE CORRESPONDING TO SYNTHETIC PEPTIDE 61 1 <400> 8 tcatccttct cacctaacac tgggaaaaaa aatgctaaaa tcaagacagg gtacaagcgg 60 gagcacatt 69 <210> 9 <211> 54 <212> DNA <213> Artificial <220> <223> NUCLEOTIDE SEQUENCE CORRESPONDING TO SYNTHETIC PEPTIDE <400> 9 gagactgcaa aatcccgagt gacccagagc aactttgcag ttggctacaa gact 54 <210> 10 <211> 51 <212> DNA <213> Artificial <220> <223> NUCLEOTIDE SEQUENCE CORRESPONDING TO SYNTHETIC PEPTIDE <400> 10 aagaagttgg agaccgctgt caatcttgcc tggacagcag gaaacagtaa c 51 <210> 11 <211> 43 <212> PRT <213> Artificial <220> <223> SYNTHETIC PEPTIDE <400> 11 Met Arg Gin lie Lys lie Trp Phe Gin Asn Arg Arg Met Lys Trp Lys 1 5 10 15 Lys Met Ala Val Pro Pro Thr Tyr Ala Asp Leu Gly Lys Ser Ala Arg 25 30 Asp Val Phe Thr Lys Gly Tyr Gly Phe Gly Leu 40 <210> 12 <211> 42 <212> PRT <213> Artificial <220> <223> ' SYNTHETIC PEPTIDE 62 185500/2 <400> 12 Met Arg Gin lie Lys lie Trp Phe Gin Asn Arg Arg Met Lys Trp Lys 1 5 10 15 Lys Ser Trp Thr Trp Glu Trp Thr Glu Tyr Gly Leu Thr Phe Thr Glu 25 30 Lys Trp Asn Thr Asp Lys Trp Thr Trp Lys 40 <210> 13 <211> 47 <212> PRT <213> Artificial <220> <223> SYNTHETIC <400> 13 Met Arg Gin lie Lys lie Trp Phe Gin Asn Arg Arg Met Lys Trp Lys 1 5 10 15 Lys Ser Trp Thr Trp Glu Ser Ser Phe Ser Pro Asn Thr Gly Lys Lys 25 30 Asn Ala Lys lie Lys Thr Gly Tyr Lys Arg Lys Trp Thr Trp Lys 40 45 <210> 14 <211> 45 <212> PRT <213> Artificial <220> <223> SYNTHETIC <400> 14 Met Arg Gin lie Lys lie Trp Phe Gin Asn Arg Arg Met Lys Trp Lys 1 5 10 15 Lys Ser Trp Thr Trp Glu Glu Thr Ala Lys Ser Arg Val Thr Gin Ser 25 30 Asn Phe Ala Val Gly Tyr Lys Thr Lys Trp Thr Trp Lys 40 45 <210> 15 <211> 44 <212> PRT 63 <213> Artificial <220> <223> SYNTHETIC PEPTIDE <400> 15 Met Arg Gin lie Lys lie Trp Phe Gin Asn Arg Arg Met Lys Trp Lys 1 5 10 15 Lys Ser Trp Thr Trp Glu Lys Lys Leu Glu Thr Ala Val Asn Leu Ala 25 30 Trp Thr Ala Gly Asn Ser Asn Lys Trp Thr Trp Lys 40 <210> 16 <211> 132 <212> DNA <213> Artificial <220> <223> NUCLEOTIDE SEQUENCE CORRESPONDING TO SYNTHETIC PEPTIDE <400> 16 atgcgtcaga ttaaaatttg gtttcagaat cgtcgtatga aatggaaaaa aatggctgtg 60 ccacccacgt atgccgatct tggcaaatct gccagggatg tcttcaccaa gggctatgga 120 tttggcttat ga 132 <210> 17 <211> 132 <212> DNA <213> Artificial <220> <223> NUCLEOTIDE SEQUENCE CORRESPONDING TO SYNTHETIC PEPTIDE <400> 17 atgcgtcaga ttaaaatttg gtttcagaat cgtcgtatga aatggaaaaa atcctggacc 60 tgggaatgga ctgagtacgg cctgacgttt acagagaaat ggaataccga caataaatgg 120 acctggaaat ga 132 <210> 18 <211> 153 <212> DNA <213> Artificial <220> 64 <223> NUCLEOTIDE SEQUENCE CORRESPONDING TO SYNTHETIC PEPTIDE <400> 18 atgcgtcaga ttaaaatttg gtttcagaat cgtcgtatga aatggaaaaa atcctggacc 60 tgggaatcat ccttctcacc taacactggg aaaaaaaatg ctaaaatcaa gacagggtac 120 aagcgggagc acattaaatg gacctggaaa tga 153 <210> 19 <211> 138 <212> DNA <213> Artificial <220> <223> NUCLEOTIDE <400> 19 atgcgtcaga ttaaaatttg gtttcagaat cgtcgtatga aatggaaaaa atcctggacc 60 tgggaagaga ctgcaaaatc ccgagtgacc cagagcaact ttgcagttgg ctacaagact 120 aaatggacct ggaaatga 138 <210> 20 <211> 135 <212> DNA <213> Artificial <220> <223> NUCLEOTIDE <400> 20 atgcgtcaga ttaaaatttg gtttcagaat cgtcgtatga aatggaaaaa atcctggacc 60 tgggaaaaga agttggagac cgctgtcaat cttgcctgga cagcaggaaa cagtaacaaa 120 tggacctgga aatga 135 <210> 21 <211> 283 <212> PRT <213> HOMO SAPIENS <300> <308> NP_00365 <309> 2005-12-25 <313> (1) .. (283) <400> 21 65 Met Ala Val Pro Pro Thr Tyr Ala Asp Leu Gly Lys Ser Ala Arg Asp 1 5 10 15 Val Phe Thr Lys Gly Tyr Gly Phe Gly Leu lie Lys Leu Asp Leu Lys 25 30 Thr Lys Ser Glu Asn Gly Leu Glu Phe Thr Ser Ser Gly Ser Ala Asn 40 45 Thr Glu Thr Thr Lys Val Thr Gly Ser Leu Glu Thr Lys Tyr Arg Trp 50 55 60 Thr Glu Tyr Gly Leu Thr Phe Thr Glu Lys Trp Asn Thr Asp Asn Thr 65 70 75 80 Leu Gly Thr Glu lie Thr Val Glu Asp Gin Leu Ala Arg Gly Leu Lys 85 90 95 Leu Thr Phe Asp Ser Ser Phe Ser Pro Asn Thr Gly Lys Lys Asn Ala 100 105 110 Lys lie Lys Thr Gly Tyr Lys Arg Glu His lie Asn Leu Gly Cys Asp 115 120 125 Met Asp Phe Asp lie Ala Gly Pro Ser lie Arg Gly Ala Leu Val Leu 130 135 140 Gly Tyr Glu Gly Trp Leu Ala Gly Tyr Gin Met Asn Phe Glu Thr Ala 145 150 155 160 Lys Ser Arg Val Thr Gin Ser Asn Phe Ala Val Gly Tyr Lys Thr Asp 165 170 175 Glu Phe Gin Leu His Thr Asn Val Asn Asp Gly Thr Glu Phe Gly Gly 180 185 190 Ser lie Tyr Gin Lys Val Asn Lys Lys Leu Glu Thr Ala Val Asn Leu 195 200 205 Ala Trp Thr Ala Gly Asn Ser Asn Thr Arg Phe Gly lie Ala Ala Lys 210 215 220 Tyr Gin lie Asp Pro Asp Ala Cys Phe Ser Ala Lys Val Asn Asn Ser 225 230 235 240 66 Ser Leu He Gly Leu Gly Tyr Thr Gin Thr Leu Lys Pro Gly He Lys 245 250 255 Leu Thr Leu Ser Ala Leu Leu Asp Gly Lys Asn Val Asn Ala Gly Gly 260 265 270 His Lys Leu Gly Leu Gly Leu Glu Phe Gin Ala 275 280 <210> 22 <211> 294 <212> PRT <213> HOMO SAPIENS <300> <308 > NP 00366 <309> 2005-10- 18 <313> ( 1 ) . . ( 294 ) <400> 22 Met Ala Thr His Gly Gin Thr Cys Ala Arg Pro Met Cys He Pro Pro 1 5 10 15 Ser Tyr Ala Asp Leu Gly Lys Ala Ala Arg Asp He Phe Asn Lys Gly 25 30 Phe Gly Phe Gly Leu Val Lys Leu Asp Val Lys Thr Lys Ser Cys Ser 40 45 Gly Val Glu Phe Ser Thr Ser Gly Ser Ser Asn Thr Asp Thr Gly Lys 50 55 60 Val Thr Gly Thr Leu Glu Thr Lys Tyr Lys Trp Cys Glu Tyr Gly Leu 65 70 75 80 Thr Phe Thr Glu Lys Trp Asn Thr Asp Asn Thr Leu Gly Thr Glu He 85 90 95 Ala He Glu Asp Gin He Cys Gin Gly Leu Lys Leu Thr Phe Asp Thr 100 105 110 Thr Phe Ser Pro Asn Thr Gly Lys Lys Ser Gly Lys He Lys Ser Ser 115 120 125 Tyr Lys Arg Glu Cys He Asn Leu Gly Cys Asp Val Asp Phe Asp Phe 130 135 140 Ala Gly Pro Ala He His Gly Ser Ala Val Phe Gly Tyr Glu Gly Trp 67 145 150 155 160 Leu Ala Gly Tyr Gin Met Thr Phe Asp Ser Ala Lys Ser Lys Leu Thr 165 170 175 Arg Asn Asn Phe Ala Val Gly Tyr Arg Thr Gly Asp Phe Gin Leu His 180 185 190 Thr Asn Val Asn Asp Gly Thr Glu Phe Gly Gly Ser lie Tyr Gin Lys 195 200 205 Val Cys Glu Asp Leu Asp Thr Ser Val Asn Leu Ala Trp Thr Ser Gly 210 215 220 Thr Asn Cys Thr Arg Phe Gly He Ala Ala Lys Tyr Gin Leu Asp Pro 225 230 235 240 Thr Ala Ser He Ser Ala Lys Val Asn Asn Ser Ser Leu He Gly Val 245 250 255 Gly Tyr Thr Gin Thr Leu Arg Pro Gly Val Lys Leu Thr Leu Ser Ala 260 265 270 Leu Val Asp Gly Lys Ser He Asn Ala Gly Gly His Lys Val Gly Leu 275 280 285 Ala Leu Glu Leu Glu Ala 290 <210> 23 <211> 283 <212> PRT <213> HOMO SAPIENS <300> <308> NP 005653 <309> 2005-10-17 <313> (1) .. (283) <400> 23 Met Cys Asn Thr Pro Thr Tyr Cys Asp Leu Gly Lys Ala Ala Lys Asp 1 5 10 15 Val Phe Asn Lys Gly Tyr Gly Phe Gly Met Val Lys He Asp Leu Lys 25 30 Thr Lys Ser Cys Ser Gly Val Glu Phe Ser Thr Ser Gly His Ala Tyr 40 45 68 Thr Asp Thr Gly Lys Ala Ser Gly Asn Leu Glu Thr Lys Tyr Lys Val 50 55 60 Cys Asn Tyr Gly Leu Thr Phe Thr Gin Lys Trp Asn Thr Asp Asn Thr 65 70 75 80 Leu Gly Thr Glu He Ser Trp Glu Asn Lys Leu Ala Glu Gly Leu Lys 85 90 95 Leu Thr Leu Asp Thr He Phe Val Pro Asn Thr Gly Lys Lys Ser Gly 100 105 110 Lys Leu Lys Ala Ser Tyr Lys Arg Asp' Cys Phe Ser Val Gly Ser Asn 115 120 125 Val Asp He Asp Phe Ser Gly Pro Thr He Tyr Gly Trp Ala Val Leu 130 135 140 Ala Phe Glu Gly Trp Leu Ala Gly Tyr Gin Met Ser Phe Asp Thr Ala 145 150 155 160 Lys Ser Lys Leu Ser Gin Asn Asn Phe Ala Leu Gly Tyr Lys Ala Ala 165 170 175 Asp Phe Gin Leu His Thr His Val Asn Asp Gly Thr Glu Phe Gly Gly 180 185 190 Ser He Tyr Gin Lys Val Asn Glu Lys He Glu Thr Ser He Asn Leu 195 200 205 Ala Trp Thr Ala Gly Ser Asn Asn Thr Arg Phe Gly He Ala Ala Lys 210 215 220 Tyr Met Leu Asp Cys Arg Thr Ser Leu Ser Ala Lys Val Asn Asn Ala 225 230 235 240 Ser Leu He Gly Leu Gly Tyr Thr Gin Thr Leu Arg Pro Gly Val Lys 245 250 255 Leu Thr Leu Ser Ala Leu He Asp Gly Lys Asn Phe Ser Ala Gly Gly 260 265 270 His Lys Val Gly Leu Gly Phe Glu Leu Glu Ala 275 280 69 <210> 24 <211> 283 <212> PRT <213> MUS MUSCULUS <300> <308> ΝΡ 035824 <309> 2005-12-11 <313> (1) .. (283) <400> 24 Met Ala Val Pro Pro Thr Tyr Ala Asp Leu Gly Lys Ser Ala Arg Asp 1 5 10 15 Val Phe Thr Lys Gly Tyr Gly Phe Gly Leu lie Lys Leu Asp Leu Lys 25 30 Thr Lys Ser Glu Asn Gly Leu Glu Phe Thr Ser Ser Gly Ser Ala Asn 40 45 Thr Glu Thr Thr Lys Val Asn Gly Ser Leu Glu Thr Lys Tyr Arg Trp 50 55 60 Thr Glu Tyr Gly Leu Thr Phe Thr Glu Lys Trp Asn Thr Asp Asn Thr 65 70 75 80 Leu Gly Thr Glu lie Thr Val Glu Asp Gin Leu Ala Arg Gly Leu Lys 85 90 95 Leu Thr Phe Asp Ser Ser Phe Ser Pro Asn Thr Gly Lys Lys Asn Ala 100 105 110 Lys lie Lys Thr Gly Tyr Lys Arg Glu His lie Asn Leu Gly Cys Asp 115 120 125 Val Asp Phe Asp lie Ala Gly Pro Ser lie Arg Gly Ala Leu Val Leu 130 135 140 Gly Tyr Glu Gly Trp Leu Ala Gly Tyr Gin Met Asn Phe Glu Thr Ser 145 150 155 160 Lys Ser Arg Val Thr Gin Ser Asn Phe Ala Val Gly Tyr Lys Thr Asp 165 170 175 Glu Phe Gin Leu His Thr Asn Val Asn Asp Gly Thr Glu Phe Gly Gly 180 185 190 70 Ser He Tyr Gin Lys Val Asn Lys Lys Leu Glu Thr Ala Val Asn Leu 195 200 205 Ala Trp Thr Ala Gly Asn Ser Asn Thr Arg Phe Gly He Ala Ala Lys 210 215 220 Tyr Gin Val Asp Pro Asp Ala Cys Phe Ser Ala Lys Val Asn Asn Ser 225 230 235 240 Ser Leu He Gly Leu Gly Tyr Thr Gin Thr Leu Lys Pro Gly He Lys 245 250 255 Leu Thr Leu Ser Ala Leu Leu Asp Gly Lys Asn Val Asn Ala Gly Gly 260 265 270 His Lys Leu Gly Leu Gly Leu Glu Phe Gin Ala 275 280 <210> 25 <211> 295 <212> PRT <213> MUS MUSCULUS <300> <308> NP 035825 <309> 2005-12-11 <313> (1) · · (295) <400> 25 Met Ala . Glu Cys Cys 1 5 10 15 Pro Tyr Ala Asp Leu Gly Lys Ala Ala Arg Asp He Phe Asn Lys 25 30 Gly Phe Gly Phe Gly Leu Val Lys Leu Asp Val Lys Thr Lys Ser Cys 40 45 Ser Gly Val Glu Phe Ser Thr Ser Gly Ser Ser Asn Thr Asp Thr Gly 50 55 60 Lys Val Ser Gly Thr Leu Glu Thr Lys Tyr Lys Trp Cys Glu Tyr Gly 65 70 75 80 Leu Thr Phe Thr Glu Lys Trp Asn Thr Asp Asn Thr Leu Gly Thr Glu 85 90 95 71 He Ala He Glu Asp Gin He Cys Gin Gly Leu Lys Leu Thr Phe Asp 100 105 110 Thr Thr Phe Ser Pro Asn Thr Gly Lys Lys Ser Gly Lys He Lys Ser 115 120 125 Ala Tyr Lys Arg Glu Cys He Asn Leu Gly Cys Asp Val Asp Phe Asp 130 135 140 Phe Ala Gly Pro Ala He His Gly Ser Ala Val Phe Gly Tyr Glu Gly 145 150 155 160 Trp Leu Ala Gly Tyr Gin Met Thr Phe Asp Ser Ala Lys Ser Lys Leu 165 170 175 Thr Arg Ser Asn Phe Ala Val Gly Tyr Arg Thr Gly Asp Phe Gin Leu 180 185 190 His Thr Asn Val Asn Asn Gly Thr Glu Phe Gly Gly Ser He Tyr Gin 195 200 205 Lys Val Cys Glu Asp Phe Asp Thr Ser Val Asn Leu Ala Trp Thr Ser 210 215 220 Gly Thr Asn Cys Thr Arg Phe Gly He Ala Ala Lys Tyr Gin Leu Asp 225 230 235 240 Pro Thr Ala Ser He Ser Ala Lys Val Asn Asn Ser Ser Leu He Gly 245 250 255 Val Gly Tyr Thr Gin Thr Leu Arg Pro Gly Val Lys Leu Thr Leu Ser 260 265 270 Ala Leu Val Asp Gly Lys Ser Phe Asn Ala Gly Gly His Lys Leu Gly 275 280 285 Leu Ala Leu Glu Leu Glu Ala 290 295 <210> 26 <211> 283 <212> PRT <213> MUS MUSCULUS <300> <308> NP 035826 <309> 2005-12-11 <313> (1) · · (283) 72 <400> 26 Met Cys Asn Thr Pro Thr Tyr Cys Asp Leu Gly Lys Ala Ala Lys Asp 1 5 10 15 Val Phe Asn Lys Gly Tyr Gly Phe Gly Met Val Lys He Asp Leu Lys 25 30 Thr Lys Ser Cys Ser Gly Val Glu Phe Ser Thr Ser Gly His Ala Tyr 40 45 Thr Asp Thr Gly Lys Ala Ser Gly Asn Leu Glu Thr Lys Tyr Lys Val 50 55 60 Cys Asn Tyr Gly Leu Thr Phe Thr Gin Lys Trp Asn Thr Asp Asn Thr 65 70 75 80 Leu Gly Thr Glu He Ser Trp Glu Asn Lys Leu Ala Glu Gly Leu Lys 85 90 95 Leu Thr Leu Asp Thr He Phe Val Pro Asn Thr Gly Lys Lys Ser Gly 100 105 110 Lys Leu Lys Ala Ser Tyr Arg Arg Asp Cys Phe Ser Leu Gly Ser Asn 115 120 125 Val Asp He Asp Phe Ser Gly Pro Thr He Tyr Gly Trp Ala Val Leu 130 135 140 Ala Phe Glu Gly Trp Leu Ala Gly Tyr Gin Met Ser Phe Asp Thr Ala 145 150 155 160 Lys Ser Lys Leu Ser Gin Asn Asn Phe Ala Leu Gly Tyr Lys Ala Ala 165 170 175 Asp Phe Gin Leu His Thr His Val Asn Asp Gly Thr Glu Phe Gly Gly 180 185 190 Ser He Tyr Gin Lys Val Asn Glu Arg He Glu Thr Ser He Asn Leu 195 200 205 Ala Trp Thr Ala Gly Ser Asn Asn Thr Arg Phe Gly He Ala Ala Lys 210 215 220 Tyr Lys Leu Asp Cys Arg Thr Ser Leu Ser Ala Lys Val Asn Asn Ala 73 225 230 235 240 Ser Leu He Gly Leu Gly Tyr Thr Gin Thr Leu Arg Pro Gly Val Lys 245 250 255 Leu Thr Leu Ser Ala Leu He Asp Gly Lys Asn Phe Asn Ala Gly Gly 260 265 270 His Lys Val Gly Leu Gly Phe Glu Leu Glu Ala 275 280 <210> 27 <211> 283 <212> PRT <213> RATTUS NORVEGICUS <300> <308> NP 112643 <309> 2005-10-17 <313> (1) · · (283) <400> 27 Met Ala Val Pro Pro Thr Tyr Ala Asp Leu Gly Lys Ser Ala Arg Asp 1 5 10 15 Val Phe Thr Lys Gly Tyr Gly Phe Gly Leu He Lys Leu Asp Leu Lys 25 30 Thr Lys Ser Glu Asn Gly Leu Glu Phe Thr Ser Ser Gly Ser Ala Asn 40 45 Thr Glu Thr Thr Lys Val Asn Gly Ser Leu Glu Thr Lys Tyr Arg Trp 50 55 60 Thr Glu Tyr Gly Leu Thr Phe Thr Glu Lys Trp Asn Thr Asp Asn Thr 65 70 75 80 Leu Gly Thr Glu He Thr Val Glu Asp Gin Leu Ala Arg Gly Leu Lys 85 90 95 Leu Thr Phe Asp Ser Ser Phe Ser Pro Asn Thr Gly Lys Lys Asn Ala 100 105 110 Lys He Lys Thr Gly Tyr Lys Arg Glu His He Asn Leu Gly Cys Asp 115 120 125 Val Asp Phe Asp He Ala Gly Pro Ser He Arg Gly Ala Leu Val Leu 130 135 140 74 Gly Tyr Glu Gly Trp Leu Ala Gly Tyr Gin Met Asn Phe Glu Thr Ser 145 150 155 160 Lys Ser Arg Val Thr Gin Ser Asn Phe Ala Val Gly Tyr Lys Thr Asp 165 170 175 Glu Phe Gin Leu His Thr Asn Val Asn Asp Gly Thr Glu Phe Gly Gly 180 185 190 Ser lie Tyr Gin Lys Val Asn Lys Lys Leu Glu Thr Ala Val Asn Leu 195 200 205 Ala Trp Thr Ala Gly Asn Ser Asn Thr Arg Phe Gly lie Ala Ala Lys 210 215 220 Tyr Gin Val Asp Pro Asp Ala Cys Phe Ser Ala Lys Val Asn Asn Ser 225 230 235 240 Ser Leu lie Gly Leu Gly Tyr Thr Gin Thr Leu Lys Pro Gly lie Lys 245 250 255 Leu Thr Leu Ser Ala Leu Leu Asp Gly Lys Asn Val Asn Ala Gly Gly 260 265 270 His Lys Leu Gly Leu Gly Leu Glu Phe Gin 275 280 <210> 28 <211> 295 <212> PRT <213> RATTUS NORVEGICUS <300> <308> NP 112644 <309> 2005-04-20 <313> (1) .. (295) <400> 28 Met Ala Glu Cys Cys Val Pro Val Cys Gin 1 5 10 15 Pro Pro Tyr Ala Asp Leu Gly Lys Ala Ala 25 30 Gly Phe Gly Phe Gly Leu Val Lys Leu Asp Val Lys Thr Lys Ser Cys 40 45 75 Ser Gly Val Glu Phe Ser Thr Ser Gly Ser Ser Asn Thr Asp Thr Gly 50 55 60 Lys Val Ser Gly Thr Leu Glu Thr Lys Tyr Lys Trp Cys Glu Tyr Gly 65 70 75 80 Leu Thr Phe Thr Glu Lys Trp Asn Thr Asp Asn Thr Leu Gly Thr Gl 85 90 95 He Ala He Glu Asp Gin He Cys Gin Gly Leu Lys Leu Thr Phe Asp 100 105 110 Thr Thr Phe Ser Pro Asn Thr Gly Lys Lys Ser Gly Lys He Lys Ser 115 120 125 Ala Tyr Lys Arg Glu Cys He Asn Leu Gly Cys Asp Val Asp Phe Asp 130 135 140 Phe Ala Gly Pro Ala He His Gly Ser Ala Val Phe Gly Tyr Glu Gly 145 150 155 160 Trp Leu Ala Gly Tyr Gin Met Thr Phe Asp Ser Ala Lys Ser Lys Leu 165 170 175 Thr Arg Ser Asn Phe Ala Val Gly Tyr Arg Thr Gly Asp Phe Gin Leu 180 185 190 His Thr Asn Val Asn Asn Gly Thr Glu Phe Gly Gly Ser He Tyr Gin 195 200 205 Lys Val Cys Glu Asp Phe Asp Thr Ser Val Asn Leu Ala Trp Thr 210 215 220 Gly Thr Asn Cys Thr Arg Phe Gly He Ala Ala Lys Tyr Gin Leu Asp 225 230 235 240 Pro Thr Ala Ser He Ser Ala Lys Val Asn Asn Ser Ser Leu He Gly 245 250 255 Val Gly Tyr Thr Gin Thr Leu Arg Pro Gly Val Lys Leu Thr Leu 260 265 270 Ala Leu Val Asp Gly Lys Ser Phe Asn Ala Gly Gly His Lys Leu Gly 275 280 285 76 Leu Ala Leu Glu Leu Glu Ala 290 295 <210> 29 <211> 283 <212> PRT <213> RATTUS NORVEGICUS <300> <308> NP 112645 <309> 2005-04-20 <313> (1) .. (283) <400> 29 Met Cys Ser Thr Pro Thr 1 1 5 10 15 Val Phe Asn Lys Gly Tyr Gly Phe Gly Met Val Lys He Asp Leu Lys 25 30 Thr Lys Ser Cys Ser Gly Val Glu Phe Ser Thr Ser Gly His Ala Tyr 40 45 Thr Asp Thr Gly Lys Ala Ser Gly Asn Leu Glu Thr Lys Tyr Lys Val 50 55 60 Cys Asn Tyr Gly Leu He Phe Thr Gin Lys Trp Asn Thr Asp Asn Thr 65 70 75 80 Leu Gly Thr Glu He Ser Trp Glu Asn Lys Leu Ala Glu Gly Leu Lys 85 90 95 Leu Thr Val Asp Thr He Phe Val Pro Asn Thr Gly Lys Lys Ser Gly 100 105 110 Lys Leu Lys Ala Ser Tyr Arg Arg Asp Cys Phe Ser Val Gly Ser Lys 115 120 125 Val Asp He Asp Phe Ser Gly Pro Thr He Tyr Gly Trp Ala Val Leu 130 135 140 Ala Phe Glu Gly Trp Leu Ala Gly Tyr Gin Met Ser Phe Asp Thr Ala 145 150 155 160 Lys Ser Lys Leu Cys Gin Asn Asn Phe Ala Leu Gly Tyr Lys Ala Glu 165 170 175 77 Asp Phe Gin Leu His Thr His Val Asn Asp Gly Thr Glu Phe Gly Gly 180 185 190 Ser He Tyr Gin Arg Val Asn Glu Lys He Glu Thr Ser He Asn Leu 195 200 205 Ala Trp Thr Ala Gly Ser Asn Asn Thr Arg Phe Gly He Ala Ala Lys 210 215 220 Tyr Arg Leu Asp Cys Arg Thr Ser Leu Ser Ala Lys Val Asn Asn Ala 225 230 235 240 Ser Leu He Gly Leu Gly Tyr Thr Gin Ser Leu Arg Pro Gly Val Lys 245 250 255 Leu Thr Leu Ser Ala Leu Val Asp Gly Lys Asn Phe Asn Ala Gly Gly 260 265 270 His Lys Val Gly Leu Gly Phe Glu Leu Glu Ala 275 280 <210> 30 <211> 852 <212> DNA <213> Homo sapiens <300> <308> NM_003374 <309> 2005-12-24 <313> (1) .. (852) <400> 30 atggctgtgc cacccacgta tgccgatctt ggcaaatctg ccagggatgt cttcaccaag 60 ggctatggat ttggcttaat aaagcttgat ttgaaaacaa aatctgagaa tggattggaa 120 tttacaagct caggctcagc caacactgag accaccaaag tgacgggcag tctggaaacc 180 aagtacagat ggactgagta cggcctgacg tttacagaga aatggaatac cgacaataca 240 ctaggcaccg agattactgt ggaagatcag cttgcacgtg gactgaagct gaccttcgat 300 tcatccttct cacctaacac tgggaaaaaa aatgctaaaa tcaagacagg gtacaagcgg 360 gagcacatta acctgggctg cgacatggat ttcgacattg ctgggccttc catccggggt 420 78 gctctggtgc taggttacga gggctggctg gccggctacc agatgaattt tgagactgca 480 aaatcccgag tgacccagag caactttgca gttggctaca agactgatga attccagctt 540 cacactaatg tgaatgacgg gacagagttt ggcggctcca tttaccagaa agtgaacaag 600 aagttggaga ccgctgtcaa tcttgcctgg acagcaggaa acagtaacac gcgcttcgga 660 atagcagcca agtatcagat tgaccctgac gcctgcttct cggctaaagt gaacaactcc 720 agcctgatag gtttaggata cactcagact ctaaagccag gtattaaact gacactgtca 780 gctcttctgg atggcaagaa cgtcaatgct ggtggccaca agcttggtct aggactggaa 840 tttcaagcat aa 852 <210> 31 <211> 885 <212> DNA <213> Homo sapiens <300> <308> NM_003375 18-OCT-2005 <309> 2005-10-18 <313> (1) .. (885) <400> 31 atggcgaccc acggacagac ttgcgcgcgt ccaatgtgta ttcctccatc atatgctgac 60 cttggcaaag ctgccagaga tattttcaac aaaggatttg gttttgggtt ggtgaaactg 120 gatgtgaaaa caaagtcttg cagtggcgtg gaattttcaa cgtccggttc atctaataca 180 gacactggta aagttactgg gaccttggag accaaataca agtggtgtga gtatggtctg 240 actttcacag aaaagtggaa cactgataac actctgggaa cagaaatcgc aattgaagac 300 cagatttgtc aaggtttgaa actgacattt gatactacct tctcaccaaa cacaggaaag 360 aaaagtggta aaatcaagtc ttcttacaag agggagtgta taaaccttgg ttgtgatgtt 420 gactttgatt ttgctggacc tgcaatccat ggttcagctg tctttggtta tgagggctgg 480 79 cttgctggct accagatgac ctttgacagt gccaaatcaa agctgacaag gaataacttt 540 gcagtgggct acaggactgg ggacttccag ctacacacta atgtcaacga tgggacagaa 600 tttggaggat caatttatca gaaagtttgt gaagatcttg acacttcagt aaaccttgct 660 tggacatcag gtaccaactg cactcgtttt ggcattgcag ctaaatatca gttggatccc 720 actgcttcca tttctgcaaa agtcaacaac tctagcttaa ttggagtagg ctatactcag 780 actctgaggc ctggtgtgaa gcttacactc tctgctctgg tagatgggaa gagcattaat 840 gctggaggcc acaaggttgg gctcgccctg gagttggagg cttaa 885 <210> 32 <211> 852 <212> DNA <213> Homo sapiens <300> <308> NM_005662 <309> 2005-10-17 <313> (1) .. (852) <400> 32 atgtgtaaca caccaacgta ctgtgaccta ggaaaggctg ctaaggatgt cttcaacaaa 60 ggatatggct ttggcatggt caagatagac ctgaaaacca agtcttgtag tggagtggaa 120 ttttctactt ctggtcatgc ttacactgat acagggaaag catcaggcaa cctagaaacc 180 aaatataagg tctgtaacta tggacttacc ttcacccaga aatggaacac agacaatact 240 ctagggacag aaatctcttg ggagaataag ttggctgaag ggttgaaact gactcttgat 300 accatatttg taccgaacac aggaaagaag agtgggaaat tgaaggcctc ctataaacgg 360 gattgtttta gtgttggcag taatgttgat atagattttt ctggaccaac catctatggc 420 tgggctgtgt tggccttcga agggtggctt gctggctatc agatgagttt tgacacagcc 480 aaatccaaac tgtcacagaa taatttcgcc ctgggttaca aggctgcgga cttccagctg 540 80 cacacacatg tgaacgatgg cactgaattt ggaggttcta tctaccagaa ggtgaatgag 600 aagattgaaa catccataaa ccttgcttgg acagctggga gtaacaacac ccgttttggc 660 attgctgcta agtacatgct ggattgtaga acttctctct ctgctaaagt aaataatgcc 720 agcctgattg gactgggtta tactcagacc cttcgaccag gagtcaaatt gactttatca 780 gctttaatcg atgggaagaa cttcagtgca ggaggtcaca aggttggctt gggatttgaa 840 ctggaagctt aa 852 <210> 33 <211> 852 <212> DNA <213> MUS USCULUS <300> <308> NM_011694 <309> 2005-12-11 <313> (1).. (852) <400> 33 atggccgtgc ctcccacata cgccgatctt ggcaagtccg ccagggatgt cttcaccaag 60 ggctacggct ttggcttaat aaaacttgat ttgaaaacga agtcagagaa tggattggaa 120 tttaccagct caggctctgc caacacggaa accaccaaag tgaacggcag cctggaaacc 180 aagtacagat ggactgagta tgggctgacg tttacagaga agtggaacac agacaacacc 240 ctgggcactg agatcactgt ggaagaccag cttgctcgtg gactgaagct cacctttgat 300 tcgtcattct cgccgaacac tgggaaaaaa aatgctaaaa tcaagacagg gtacaagagg 360 gagcacatca acctcggctg tgacgtggac tttgacatcg ctgggccctc gatccgggg 420 gctctggtgc ttggctatga gggttggctg gctggctacc agatgaattt tgagacctc 480 aagtcccgag tgacccagag caacttcgca gttggctata agacggatga attccagctt 540 catactaatg tgaatgacgg gacagagttt ggtggctcca tttaccagaa ggtgaacaag 600 81 aagttggaga ctgctgtcaa tctcgcctgg actgcaggaa acagtaacac tcgcttcgga 660 atagcagcca agtatcaggt cgaccctgat gcctgctttt cggccaaagt gaacaactct 720 agcctgattg gcttagggta cactcagacc ctaaaaccag gtatcaaact gacgttgtca 780 gccctgctcg atggcaagaa cgtcaatgcg ggtggccaca agcttggcct aggactggaa 840 tttcaagcat aa 852 <210> 34 <211> 888 <212> DNA <213> MUS MUSCULUS <300> <308> NM_011695 ll-DEC-2005 <309> 2005-12-11 <313> (1) .. (888) <400> 34 atggctgagt gctgtgtacc ggtatgccca cggccgatgt gtatccctcc accctatgct 60 gacctcggca aagctgccag agacattttc aacaaaggat ttggctttgg gctggtgaag 120 ctggatgtga agacgaagtc atgcagcggt gtggaatttt caacatctgg ctcatctaat 180 acagacactg gtaaagttag cgggaccttg gagaccaagt acaaatggtg tgagtatggt 240 ctgactttca cagagaagtg gaacaccgat aacactctgg ggacagagat tgcaattgaa 300 gaccagattt gtcaaggttt gaaactgact tttgacacca ccttttcacc gaacacagga 360 aagaaaagtg gtaaaatcaa gtctgcttac aagagggagt gtataaacct cggctgtgat 420 gttgactttg attttgctgg acctgccatc catgggtcag ctgtctttgg ttacgagggc 480 tggcttgctg ggtaccaaat gacctttgac agtgccaagt caaagctgac aaggagtaac 540 tttgcagtcg gctacaggac tggggacttc cagctacaca caaatgtaaa taatgggaca 600 gaatttggag gatcaattta tcagaaagta tgtgaagatt ttgacacttc agtaaacctc 660 82 gcttggacat caggtaccaa ctgcactcgt tttggcattg cagctaaata ccagttggat 720 cctactgctt ctatctctgc aaaggtcaac aactctagtt taattggagt gggctatact 780 cagactctga ggcctggtgt gaagcttaca ctgtctgctc tggtagacgg gaagagcttt 840 aatgctggag gccacaaact tgggcttgcc ttggaattgg aggcttaa 888 <210> 35 <211> 852 <212> DNA <213> MUS MUSCULUS <400> 35 atgtgtaaca caccaactta ttgcgaccta ggaaaggctg ccaaggatgt ctttaacaaa 60 gggtatgggt ttggcatggt caagatagat ctgaaaacca agtcttgtag tggagtggaa 120 ttttctactt caggtcatgc ttatactgat acagggaaag catcaggcaa cctagagacc 180 aaatataagg tctgcaacta tgggctcacc ttcacccaaa agtggaatac agacaatact 240 cttgggacag aaatctcttg ggagaataag ttggctgaag ggttgaaact gactcttgat 300 accatatttg taccaaacac aggaaagaag agtgggaaat taaaggcctc ctatagacgg 360 gattgtttta gtctcggcag taatgttgat atagattttt ctggaccgac catctatggc 420 tgggctgtgt tggcctttga aggttggctt gctggctatc agatgagttt tgacacagcc 480 aaatccaaac tgtctcagaa taatttcgct cttggttaca aggctgcaga cttccagctg 540 catactcacg tgaatgatgg cactgagttt ggaggctcaa tctaccagaa agttaacgag 600 aggattgaaa cgtcaataaa cctggcatgg acagctggca gcaacaacac tcgttttggc 660 atcgctgcta aatataagct ggattgtaga acttctctat ctgccaaagt aaacaatgcc 720 agtttaattg gactgggtta tacgcagacc ctccgaccag gagtcaaact gaccctgtca 780 gctttaatag atggaaagaa cttcaatgca ggaggccaca aggttggatt gggatttgaa 840 83 ctggaggctt ag 852 <210> 36 <211> 852 <212> DNA <213> RATIOS NORVEGICUS <300> <308> NM_031353 <309> 2005-10-17 <313> (1) .. (852) <400> 36 atggctgtgc ctcccacata tgctgatctt ggcaagtccg ccagggatgt cttcaccaag 60 ggctacggct ttggcttaat aaaacttgat ttgaaaacga agtccgagaa tggattggaa 120 tttactagct caggttctgc caacacggag accaccaaag tgaacggcag tctggaaacc 180 aagtacagat ggaccgagta tgggctgacg tttactgaga agtggaacac agacaacacc 240 ctgggcactg agatcaccgt ggaagaccag cttgctcgtg gactgaagct gacctttgat 300 tcatctttct cgcctaacac tgggaaaaaa aatgctaaaa tcaagacagg gtacaagagg 360 gagcatatca acctgggctg tgatgtggac tttgacatcg ctgggccctc aatccggggc 420 gctctggtgc ttggctatga gggttggctg gctggctacc agatgaattt tgagacctcg 480 aagtcccgag tgacccagag caactttgca gttggctaca agacggacga attccagctt 540 catactaatg tgaatgatgg gacggagttt ggtggctcca tttaccagaa ggtgaacaag 600 aagttggaga ctgctgtcaa tctcgcctgg accgcaggaa acagtaacac tcgctttgga 660 atagcagcca agtatcaggt cgaccctgat gcctgctttt cggccaaagt gaacaactcc 720 agtctaattg gcttagggta cactcagacc ctaaaaccag gtatcaaact gacactgtca 780 gccctgctgg atggcaagaa cgtcaatgcg ggtggccaca agcttggttt aggactggaa 840 tttcaagcat aa 852 84 <210> 37 <211> 888 <212> DNA <213> RATTUS NORVEGICUS <300> <308> NM_031354 <309> 2005-04-20 <313> (1) .. (888) <400> 37 atggctgaat gttgtgtacc ggtatgccaa cggccaattt gtatccctcc accctatgct 60 gaccttggca aagctgccag agatattttc aacaaaggat ttggttttgg gttggtaaag 120 ctggatgtga aaacgaagtc atgcagtggt gtggaatttt caacatctgg ctcatctaat 180 acagacactg gtaaagtcag tgggaccttg gagaccaagt acaaatggtg tgagtatggt 240 ctgactttca cagagaaatg gaacactgac aacactctgg ggacggagat tgcaattgaa 300 gaccagattt gtcaaggttt gaaactgacc tttgacacca cgttttcacc aaacacagga 360 aagaaaagtg gtaaaatcaa gtctgcttac aagagggaat gtataaacct tggctgtgat 420 gttgattttg attttgctgg acctgccatc catgggtcag ccgtctttgg ttacgagggc 480 tggcttgctg ggtaccagat gacctttgac agtgccaagt caaagctgac aaggagtaac 540 ttcgcagttg gctacaggac tggggacttc cagctacaca caaatgtaaa taatgggaca 600 gaatttggag gatcaattta tcagaaagta tgtgaagatt ttgacacttc agtaaacctt 660 gcttggacat caggtaccaa ctgcactcgt tttggcattg cagctaaata ccagttggac 720 cccactgctt ctatttctgc aaaggtcaac aactctagtt taattggagt gggctatact 780 cagactctga ggcctggtgt gaagcttaca ctgtctgctc tggtagacgg gaagagcttt 840 aatgctggag gccacaaact tgggcttgcc ttggaattgg aggcttaa 888 <210> 38 <211> 852 <212> DNA 85 <213> RATTUS NORVEGICUS <300> <308> NM_031355 <309> 2005-04-20 <313> (1) .. (852) <400> 38 atgtgtagca caccaactta ctgcgaccta ggaaaggctg ccaaggatgt ctttaacaaa 60 gggtatgggt ttggcatggt caaaatagat ctgaaaacca agtcttgtag tggagtggaa 120 ttttctactt ctggtcatgc ttatactgat acagggaaag catcaggcaa cctagagacc 180 aaatataagg tctgtaacta cgggctcatc ttcacccaaa agtggaatac agacaatact 240 cttgggacag aaatctcttg ggagaataag ttggctgaag ggttgaaact gacggttgat 300 accatatttg taccaaacac agggaagaag agtgggaaat taaaggcctc ctatagacgg 360 gattgtttta gtgtgggcag taaggttgac atagattttt ctggaccgac catctatggc 420 tgggccgtgt tggcctttga aggttggctt gctggctacc agatgagttt tgacacagcc 480 aaatccaaac tgtgtcagaa taattttgct cttggttaca aggctgaaga cttccaactg 540 catactcatg taaacgatgg cactgaattt ggaggctcca tctaccagag agttaatgag 600 aagatcgaaa catcaataaa cctggcatgg acagctggca gcaacaacac tcgttttggc 660 atcgctgcta agtataggct ggattgtaga acttctctgt ctgccaaagt aaacaatgcc 720 agtttaattg gactgggtta tacgcagagt ctccgaccgg gagtcaaact gaccctgtca 780 gctttagtgg atggaaagaa cttcaatgca ggaggccaca aggttggctt gggatttgaa 840 ctggaagctt aa 852 <210> 39 <211> 17 <212> PRT <213> Artificial <220> <223> SYNTHETIC PEPTIDE 86 <400> 39 Met Arg Gin lie Lys lie Trp Phe Gin Asn Arg Arg Met Lys Trp Lys 1 5 10 15 Lys <210> 40 <211> 51 <212> DNA <213> Artificial <220> <223> NUCLEOTIDE SEQUENCE CORRESPONDING TO SYNTHETIC PEPTIDE <400> 40 atgcgtcaga ttaaaatttg gtttcagaat cgtcgtatga aatggaaaaa a 51 <210> 41 <211> 5 <212> PRT <213> Artificial <220> <223> SYNTHETIC PEPTIDE <400> 41 Ser Trp Thr Trp Glu <210> 42 <211> 5 <212> PRT <213> Artificial <220> <223> SYNTHETIC PEPTIDE <400> 42 Lys Trp Thr Trp Lys <210> 43 <211> 283 <212> PRT <213> homo sapiens <220> <221> VARIANT <222> (72) .. (72) <223> E72Q SUBSTITUTION <400> 43 87 Met Ala Val Pro Pro Thr Tyr Ala Asp Leu Gly Lys Ser Ala Arg Asp 1 5 10 15 Val Phe Thr Lys Gly Tyr Gly Phe Gly Leu lie Lys Leu Asp Leu Lys 25 30 Thr Lys Ser Glu Asn Gly Leu Glu Phe Thr Ser Ser Gly Ser Ala Asn 40 45 Thr Glu Thr Thr Lys Val Thr Gly Ser Leu Glu Thr Lys Tyr Arg Trp 50 55 60 Thr Glu Tyr Gly Leu Thr Phe Thr Gin Lys Trp Asn Thr Asp Asn Thr 65 70 75 80 Leu Gly Thr Glu He Thr Val Glu Asp Gin Leu Ala Arg Gly Leu Lys 85 90 95 Leu Thr Phe Asp Ser Ser Phe Ser Pro Asn Thr Gly Lys Lys Asn Ala 100 105 110 Lys He Lys Thr Gly Tyr Lys Arg Glu His He Asn Leu Gly Cys Asp 115 120 125 Met Asp Phe Asp He Ala Gly Pro Ser He Arg Gly Ala Leu Val Leu 130 135 140 Gly Tyr Glu Gly Trp Leu Ala Gly Tyr Gin Met Asn Phe Glu Thr Ala 145 150 155 160 Lys Ser Arg Val Thr Gin Ser Asn Phe Ala Val Gly Tyr Lys Thr Asp 165 170 175 Glu Phe Gin Leu His Thr Asn Val Asn Asp Gly Thr Glu Phe Gly Gly 180 185 190 Ser He Tyr Gin Lys Val Asn Lys Lys Leu Glu Thr Ala Val Asn Leu 195 200 205 Ala Trp Thr Ala Gly Asn Ser Asn Thr Arg Phe Gly He Ala Ala Lys 210 215 220 Tyr Gin He Asp Pro Asp Ala Cys Phe Ser Ala Lys Val Asn Asn Ser 225 230 235 240 88 Ser Leu He Gly Leu Gly Tyr Thr Gin Thr Leu Lys Pro Gly He Lys 245 250 255 Leu Thr Leu Ser Ala Leu Leu Asp Gly Lys Asn Val Asn Ala Gly Gly 260 265 270 His Lys Leu Gly Leu Gly Leu Glu Phe Gin Ala 275 280 <210> 44 <211> 283 <212> PRT <213> homo sapiens <220> <221> VARIANT <222> (87) . . · (87) <223> E87Q SUBSTITUTION <220> <221> VARIANT <222> (88) . .. (88) <223> D88N SUBSTITUTION <400> 44 Met Ala Val Pro Pro Thr 1 1 5 10 15 Val Phe Thr Lys Gly Tyr i 2 200 25 30 Thr Lys Ser Glu Asn Gly : 40 45 Thr Glu Thr Thr Lys Val Thr Gly Ser Leu Glu Thr Lys Tyr Arg Trp 50 55 60 Thr Glu Tyr Gly Leu Thr Phe Thr Glu Lys Trp Asn Thr Asp Asn Thr 65 70 75 80 Leu Gly Thr Glu He Thr Val Gin Asn Gin Leu Ala Arg Gly Leu Lys 85 90 95 Leu Thr Phe Asp Ser Ser Phe Ser Pro Asn Thr Gly Lys Lys Asn Ala 100 105 110 Lys He Lys Thr Gly Tyr Lys Arg Glu His He Asn Leu Gly Cys Asp 115 120 125 89 Met Asp Phe Asp lie Ala Gly Pro Ser lie Arg Gly Ala Leu Val Leu 130 135 140 Gly Tyr Glu Gly Trp Leu Ala Gly Tyr Gin Met Asn Phe Glu Thr Ala 145 150 155 160 Lys Ser Arg Val Thr Gin Ser Asn Phe Ala Val Gly Tyr Lys Thr Asp 165 170 175 Glu Phe Gin Leu His Thr Asn Val Asn Asp Gly Thr Glu Phe Gly Gly 180 185 190 Ser lie Tyr Gin Lys Val Asn Lys Lys Leu Glu Thr Ala Val Asn Leu 195 200 205 Ala Trp Thr Ala Gly Asn Ser Asn Thr Arg Phe Gly lie Ala Ala Lys 210 215 220 Tyr Gin lie Asp Pro Asp Ala Cys Phe Ser Ala Lys Val Asn Asn Ser 225 230 235 240 Ser Leu lie Gly Leu Gly Tyr Thr Gin Thr Leu Lys Pro Gly He Lys 245 250 255 Leu Thr Leu Ser Ala Leu Leu Asp Gly Lys Asn Val Asn Ala Gly Gly 260 265 270 His Lys Leu Gly Leu Gly Leu Glu Phe Gin Ala 275 280 <210> 45 <211> 283 <212> PRT <213> homo sapiens <220> <221> VARIANT <222> (65) .. (65) <223> E65Q SUBSTITUTION <400> 45 Met Ala Val Pro Pro Thr 1 1 5 10 15 Val Phe Thr Lys Gly Tyr Gly Phe Gly Leu He Lys Leu Asp Leu Lys 25 30 90 Thr Lys Ser Glu Asn Gly Leu Glu Phe Thr Ser Ser Gly Ser Ala Asn 35 40 45 Thr Glu Thr Thr Lys Val Thr Gly Ser Leu Glu Thr Lys Tyr Arg Trp 50 55 60 Thr Gin Tyr Gly Leu Thr Phe Thr Glu Lys Trp Asn Thr Asp Asn Thr 65 70 75 80 Leu Gly Thr Glu He Thr Val Glu Asp Gin Leu Ala Arg Gly Leu Lys 85 90 95 Leu Thr Phe Asp Ser Ser Phe Ser Pro Asn Thr Gly Lys Lys Asn Ala 100 105 110 Lys He Lys Thr Gly Tyr Lys Arg Glu His He Asn Leu Gly Cys Asp 115 120 125 Met Asp Phe Asp He Ala Gly Pro Ser He Arg Gly Ala Leu Val Leu 130 135 140 Gly Tyr Glu Gly Trp Leu Ala Gly Tyr Gin Met Asn Phe Glu Thr Ala 145 150 155 160 Lys Ser Arg Val Thr Gin Ser Asn Phe Ala Val Gly Tyr Lys Thr Asp 165 170 175 Glu Phe Gin Leu His Thr Asn Val Asn Asp Gly Thr Glu Phe Gly Gly 180 185 190 Ser He Tyr Gin Lys Val Asn Lys Lys Leu Glu Thr Ala Val Asn Leu 195 200 205 Ala Trp Thr Ala Gly Asn Ser Asn Thr Arg Phe Gly He Ala Ala Lys 210 215 220 Tyr Gin He Asp Pro Asp Ala Cys Phe Ser Ala Lys Val Asn Asn Ser 225 230 235 240 Ser Leu He Gly Leu Gly Tyr Thr Gin Thr Leu Lys Pro Gly He Lys 245 250 255 Leu Thr Leu Ser Ala Leu Leu Asp Gly Lys Asn Val Asn Ala Gly Gly 260 265 270 91 His Lys Leu Gly Leu Gly Leu Glu Phe Gin Ala 275 280 <210> 46 <211> 283 <212> PRT <213> homo sapiens <220> <221> VARIANT <222> (73) .. (73) <223> K73L SUBSTITUTION <400> 46 Met Ala Val Pro Pro Thr Tyr Ala Asp Leu Gly Lys Ser Ala Arg Asp 1 5 10 15 Val Phe Thr Lys Gly Tyr Gly Phe Gly Leu lie Lys Leu Asp Leu Lys 25 30 Thr Lys Ser Glu Asn Gly Leu Glu Phe Thr Ser Ser Gly Ser Ala Asn 40 45 Thr Glu Thr Thr Lys Val Thr Gly Ser Leu Glu Thr Lys Tyr Arg Trp 50 55 60 Thr Glu Tyr Gly Leu Thr Phe Thr Glu Leu Trp Asn Thr Asp Asn Thr 65 70 75 80 Leu Gly Thr Glu lie Thr Val Glu Asp Gin Leu Ala Arg Gly Leu Lys 85 90 95 Leu Thr Phe Asp Ser Ser Phe Ser Pro Asn Thr Gly Lys Lys Asn Ala 100 105 110 Lys lie Lys Thr Gly Tyr Lys Arg Glu His lie Asn Leu Gly Cys Asp 115 120 125 Met Asp Phe Asp lie Ala Gly Pro Ser lie Arg Gly Ala Leu Val Leu 130 135 140 Gly Tyr Glu Gly Trp Leu Ala Gly Tyr Gin Met Asn Phe Glu Thr Ala 145 150 155 160 Lys Ser Arg Val Thr Gin Ser Asn Phe Ala Val Gly Tyr Lys Thr Asp 165 170 175 Glu Phe Gin Leu His Thr Asn Val Asn Asp Gly Thr Glu Phe Gly Gly 92 180 185 190 Ser lie Tyr Gin Lys Val Asn Lys Lys Leu Glu Thr Ala Val Asn Leu 195 200 205 Ala Trp Thr Ala Gly Asn 210 215 220 Tyr Gin He Asp Pro Asp . 225 230 235 240 Ser Leu He Gly Leu Gly 245 250 255 Leu Thr Leu Ser Ala Leu 260 265 270 His Lys Leu Gly Leu Gly 275 280 <210> 47 <211> 283 <212> PRT <213> homo sapiens <220> <221> VARIANT <222> (77) . .. (77) <223> D77N SUBSTITUTION <400> 47 Met Ala Val Pro Pro Thr 1 1 5 10 15 Val Phe Thr Lys Gly Tyr I 25 30 Thr Lys Ser Glu Asn Gly : 40 45 Thr Glu Thr Thr Lys Val Thr Gly Ser Leu Glu Thr Lys Tyr Arg Trp 50 55 60 Thr Glu Tyr Gly Leu Thr Phe Thr Glu Lys Trp Asn Thr Asn Asn Thr 65 70 75 80 Leu Gly Thr Glu He Thr Val Glu Asp Gin Leu Ala Arg Gly Leu Lys 85 90 95 93 Leu Thr Phe Asp Ser Ser Phe Ser Pro Asn Thr Gly Lys Lys Asn Ala 100 105 110 Lys He Lys Thr Gly Tyr Lys Arg Glu His He Asn Leu Gly Cys Asp 115 120 125 Met Asp Phe Asp He Ala Gly Pro Ser He Arg Gly Ala Leu Val Leu 130 135 140 Gly Tyr Glu Gly Trp Leu Ala Gly Tyr Gin Met Asn Phe Glu Thr Ala 145 150 155 160 Lys Ser Arg Val Thr Gin Ser Asn Phe Ala Val Gly Tyr Lys Thr Asp 165 170 175 Glu Phe Gin Leu His Thr Asn Val Asn Asp Gly Thr Glu Phe Gly Gly 180 185 190 Ser He Tyr Gin Lys Val Asn Lys Lys Leu Glu Thr Ala Val Asn Leu 195 200 205 Ala Trp Thr Ala Gly Asn Ser Asn Thr Arg Phe Gly He Ala Ala Lys 210 215 220 Tyr Gin He Asp Pro Asp Ala Cys Phe Ser Ala Lys Val Asn Asn Ser 225 230 235 240 Ser Leu He Gly Leu Gly Tyr Thr Gin Thr Leu Lys Pro Gly He Lys 245 250 255 Leu Thr Leu Ser Ala Leu Leu Asp Gly Lys Asn Val Asn Ala Gly Gly 260 265 270 His Lys Leu Gly Leu Gly Leu Glu Phe Gin Ala 275 280 <210> 48 <211> 283 <212> PRT <213> homo sapiens <220> <221> VARIANT <222> (202) .. (202) <223> E202Q SUBSTITUTION <400> 48 94 Met Ala Val Pro Pro Thr Tyr Ala Asp Leu Gly Lys Ser Ala Arg Asp 1 5 10 15 Val Phe Thr Lys Gly Tyr Gly Phe Gly Leu lie Lys Leu Asp Leu Lys 25 30 Thr Lys Ser Glu Asn Gly Leu Glu Phe Thr Ser Ser Gly Ser Ala Asn 40 45 Thr Glu Thr Thr Lys Val Thr Gly Ser Leu Glu Thr Lys Tyr Arg Trp 50 55 60 Thr Glu Tyr Gly Leu Thr Phe Thr Glu Lys Trp Asn Thr Asp Asn Thr 65 70 75 80 Leu Gly Thr Glu lie Thr Val Glu Asp Gin Leu Ala Arg Gly Leu Lys 85 90 95 Leu Thr Phe Asp Ser Ser Phe Ser Pro Asn Thr Gly Lys Lys Asn Ala 100 105 110 Lys lie Lys Thr Gly Tyr Lys Arg Glu His lie Asn Leu Gly Cys Asp 115 120 125 Met Asp Phe Asp lie Ala Gly Pro Ser He Arg Gly Ala Leu Val Leu 130 135 140 Gly Tyr Glu Gly Trp Leu Ala Gly Tyr Gin Met Asn Phe Glu Thr Ala 145 150 155 160 Lys Ser Arg Val Thr Gin Ser Asn Phe Ala Val Gly Tyr Lys Thr Asp 165 170 175 Glu Phe Gin Leu His Thr Asn Val Asn Asp Gly Thr Glu Phe Gly Gly 180 185 190 Ser He Tyr Gin Lys Val Asn Lys Lys Leu Gin Thr Ala Val Asn Leu 195 200 205 Ala Trp Thr Ala Gly Asn Ser Asn Thr Arg Phe Gly He Ala Ala Lys 210 215 220 Tyr Gin He Asp Pro Asp Ala Cys Phe Ser Ala Lys Val Asn Asn Ser 225 230 235 240 95 Ser Leu lie Gly Leu Gly Tyr Thr Gin Thr Leu Lys Pro Gly lie Lys 245 250 255 Leu Thr Leu Ser Ala Leu Leu Asp Gly Lys Asn Val Asn Ala Gly Gly 260 265 270 His Lys Leu Gly Leu Gly Leu Glu Phe Gin Ala 275 280 <210> 49 <211> 283 <212> PRT <213> homo sapiens <220> <221> VARIANT <222> (75) .. (75) <223> N75A SUBSTITUTION <400> 49 Met Ala Val Pro Pro Thr Tyr Ala Asp Leu Gly Lys Ser Ala Arg Asp 1 5 10 15 Val Phe Thr Lys Gly Tyr Gly Phe Gly Leu lie Lys Leu Asp Leu Lys 25 30 Thr Lys Ser Glu Asn Gly Leu Glu Phe Thr Ser Ser Gly Ser Ala Asn 40 45 Thr Glu Thr Thr Lys Val Thr Gly Ser Leu Glu Thr Lys Tyr Arg Trp 50 55 60 Thr Glu Tyr Gly Leu Thr Phe Thr Glu Lys Trp Ala Thr Asp Asn Thr 65 70 75 80 Leu Gly Thr Glu He Thr Val Glu Asp Gin Leu Ala Arg Gly Leu Lys 85 90 95 Leu Thr Phe Asp Ser Ser Phe Ser Pro Asn Thr Gly Lys Lys Asn Ala 100 105 110 Lys He Lys Thr Gly Tyr Lys Arg Glu His He Asn Leu Gly Cys Asp 115 120 125 Met Asp Phe Asp He Ala Gly Pro Ser He Arg Gly Ala Leu Val Leu 130 135 140 96 Gly Tyr Glu Gly Trp Leu Ala Gly Tyr Gin Met Asn Phe Glu Thr Ala 145 150 155 160 Lys Ser Arg Val Thr Gin Ser Asn Phe Ala Val Gly Tyr Lys Thr Asp 165 170 175 Glu Phe Gin Leu His Thr Asn Val Asn Asp Gly Thr Glu Phe Gly Gly 180 185 190 Ser lie Tyr Gin Lys Val Asn Lys Lys Leu Glu Thr Ala Val Asn Leu 195 200 205 Ala Trp Thr Ala Gly Asn Ser Asn Thr Arg Phe Gly lie Ala Ala Lys 210 215 220 Tyr Gin lie Asp Pro Asp Ala Cys Phe Ser Ala Lys Val Asn Asn Ser 225 230 235 240 Ser Leu lie Gly Leu Gly Tyr Thr Gin Thr Leu Lys Pro Gly lie Lys 245 250 255 Leu Thr Leu Ser Ala Leu Leu Asp Gly Lys Asn Val Asn Ala Gly Gly 2 26600 265 270 His Lys Leu Gly Leu Gly 275 280 <210> 50 <211> 283 <212> PRT <213> homo sapiens <220> <221> VARIANT <222> (67) . . (67) <223> G67A SUBSTITUTION <400> 50 Met Ala Val Pro Pro Thr 1 1 5 10 15 Val Phe Thr Lys Gly Tyr Gly Phe Gly Leu lie Lys Leu Asp Leu Lys 25 30 Thr Lys Ser Glu Asn Gly Leu Glu Phe Thr Ser Ser Gly Ser Ala Asn 40 45 Thr Glu Thr Thr Lys Val Thr Gly Ser Leu Glu Thr Lys Tyr Arg Trp 97 50 55 60 Thr Glu Tyr Ala Leu Thr Phe Thr Glu Lys Trp Asn Thr Asp Asn Thr 65 70 75 80 Leu Gly Thr Glu lie Thr Val Glu Asp Gin Leu Ala Arg Gly Leu Lys 85 90 95 Leu Thr Phe Asp Ser Ser Phe Ser Pro Asn Thr Gly Lys Lys Asn Ala 100 105 110 lie Lys Thr Gly Tyr Lys Arg Glu His lie Asn Leu Gly Cys Asp 115 120 125 Met Asp Phe Asp lie Ala Gly Pro Ser lie Arg Gly Ala Leu Val Leu 130 135 140 Gly Tyr Glu Gly Trp Leu Ala Gly Tyr Gin Met Asn Phe Glu Thr Ala 145 150 155 160 Lys Ser Arg Val Thr Gin Ser Asn Phe Ala Val Gly Tyr Lys Thr Asp 165 170 175 Glu Phe Gin Leu His Thr Asn Val Asn Asp Gly Thr Glu Phe Gly Gly 180 185 190 Ser lie Tyr Gin Lys Val Asn Lys Lys Leu Glu Thr Ala Val Asn Leu 195 200 205 Ala Trp Thr Ala Gly Asn Ser Asn Thr Arg Phe Gly He Ala Ala Lys 210 215 220 Tyr Gin He Asp Pro Asp Ala Cys Phe Ser Ala Lys Val Asn Asn Ser 225 230 235 240 Ser Leu He Gly Leu Gly Tyr Thr Gin Thr Leu Lys Pro Gly He Lys 245 250 255 Leu Thr Leu Ser Ala Leu Leu Asp Gly Lys Asn Val Asn Ala Gly Gly 260 265 270 His Lys Leu Gly Leu Gly Leu Glu Phe Gin Ala 275 280 <210> 51 98 <211> 852 <212> DNA <213> homo sapiens <220> <221> mutation <222> (215) .. (215) <223> G>C <400> 51 atggctgtgc cacccacgta tgccgatctt ggcaaatctg ccagggatgt cttcaccaag 60 ggctatggat ttggcttaat aaagcttgat ttgaaaacaa aatctgagaa tggattggaa 120 tttacaagct caggctcagc caacactgag accaccaaag tgacgggcag tctggaaacc 180 aagtacagat ggactgagta cggcctgacg tttacagaga aatggaatac cgacaataca 240 ctaggcaccg agattactgt ggaagatcag cttgcacgtg gactgaagct gaccttcgat 300 tcatccttct cacctaacac tgggaaaaaa aatgctaaaa tcaagacagg gtacaagcgg 360 gagcacatta acctgggctg cgacatggat ttcgacattg ctgggccttc catccggggt 420 gctctggtgc taggttacga gggctggctg gccggctacc agatgaattt tgagactgca 480 aaatcccgag tgacccagag caactttgca gttggctaca agactgatga attccagctt 540 cacactaatg tgaatgacgg gacagagttt ggcggctcca tttaccagaa agtgaacaag 600 aagttggaga ccgctgtcaa tcttgcctgg acagcaggaa acagtaacac gcgcttcgga 660 atagcagcca agtatcagat tgaccctgac gcctgcttct cggctaaagt gaacaactcc 720 agcctgatag gtttaggata cactcagact ctaaagccag gtattaaact gacactgtca 780 gctcttctgg atggcaagaa cgtcaatgct ggtggccaca agcttggtct aggactggaa 840 tttcaagcat aa 852 <210> 52 <211> 852 <212> DNA <213> homo sapiens <220> 99 <221> mutation <222> (261) .. (261) <223> G>C <220> <221> mutation <222> (264) .. (264) <223> G>A <400> 52 atggctgtgc cacccacgta tgccgatctt ggcaaatctg ccagggatgt cttcaccaag 60 ggctatggat ttggcttaat aaagcttgat ttgaaaacaa aatctgagaa tggattggaa 120 tttacaagct caggctcagc caacactgag accaccaaag tgacgggcag tctggaaacc 180 aagtacagat ggactgagta cggcctgacg tttacacaga aatggaatac cgacaataca 240 ctaggcaccg agattactgt gcaaaatcag cttgcacgtg gactgaagct gaccttcgat 300 tcatccttct cacctaacac tgggaaaaaa aatgctaaaa tcaagacagg gtacaagcgg 360 gagcacatta acctgggctg cgacatggat ttcgacattg ctgggccttc catccggggt 420 gctctggtgc taggttacga gggctggctg gccggetacc agatgaattt tgagactgca 480 aaatcccgag tgacccagag caactttgca gttggctaca agactgatga attccagctt 540 cacactaatg tgaatgacgg gacagagttt ggcggctcca tttaccagaa agtgaacaag 600 aagttggaga ccgctgtcaa tcttgcctgg acagcaggaa acagtaacac gcgcttcgga 660 atagcagcca agtatcagat tgaccctgac gcctgcttct cggctaaagt gaacaactcc 720 agcctgatag gtttaggata cactcagact ctaaagccag gtattaaact gacactgtca 780 gctcttctgg atggcaagaa cgtcaatgct ggtggccaca agcttggtct aggactggaa 840 tttcaagcat aa 852 <210> 53 <211> 852 <212> DNA <213> homo sapiens 100 <220> <221> mutation <222> (196) .. (196) <223> G>C <400> 53 atggctgtgc cacccacgta tgccgatctt ggcaaatctg ccagggatgt cttcaccaag 60 ggctatggat ttggcttaat aaagcttgat ttgaaaacaa aatctgagaa tggattggaa 120 tttacaagct caggctcagc caacactgag accaccaaag tgacgggcag tctggaaacc 180 aagtacagat ggactcagta cggcctgacg tttacagaga aatggaatac cgacaataca 240 ctaggcaccg agattactgt ggaagatcag cttgcacgtg gactgaagct gaccttcgat 300 tcatccttct cacctaacac tgggaaaaaa aatgctaaaa tcaagacagg gtacaagcgg 360 gagcacatta acctgggctg cgacatggat ttcgacattg ctgggccttc catccggggt 420 gctctggtgc taggttacga gggctggctg gccggctacc agatgaattt tgagactgca 480 aaatcccgag tgacccagag caactttgca gttggctaca agactgatga attccagctt 540 cacactaatg tgaatgacgg gacagagttt ggcggctcca tttaccagaa agtgaacaag 600 aagttggaga ccgctgtcaa tcttgcctgg acagcaggaa acagtaacac gcgcttcgga 660 atagcagcca agtatcagat tgaccctgac gcctgcttct cggctaaagt gaacaactcc 720 agcctgatag gtttaggata cactcagact ctaaagccag gtattaaact gacactgtca 780 gctcttctgg atggcaagaa cgtcaatgct ggtggccaca agcttggtct aggactggaa 840 tttcaagcat aa 852 <210> 54 <211> 852 <212> DNA <213> homo sapiens <220> <221> mutation <222> (219) .. (220) <223> AA>TT 101 <400> 54 atggctgtgc cacccacgta tgccgatctt ggcaaatctg ccagggatgt cttcaccaag 60 ggctatggat ttggcttaat aaagcttgat ttgaaaacaa aatctgagaa tggattggaa 120 tttacaagct caggctcagc caacactgag accaccaaag tgacgggcag tctggaaacc 180 aagtacagat ggactgagta cggcctgacg tttacagagt tatggaatac cgacaataca 240 ctaggcaccg agattactgt ggaagatcag cttgcacgtg gactgaagct gaccttcgat 300 tcatccttct cacctaacac tgggaaaaaa aatgctaaaa tcaagacagg gtacaagcgg 360 gagcacatta acctgggctg cgacatggat ttcgacattg ctgggccttc catccggggt 420 gctctggtgc taggttacga gggctggctg gccggctacc agatgaattt tgagactgca 480 aaatcccgag tgacccagag caactttgca gttggctaca agactgatga attccagctt 540 cacactaatg tgaatgacgg gacagagttt ggcggctcca tttaccagaa agtgaacaag 600 aagttggaga ccgctgtcaa tcttgcctgg acagcaggaa acagtaacac gcgcttcgga 660 atagcagcca agtatcagat tgaccctgac gcctgcttct cggctaaagt gaacaactcc 720 agcctgatag gtttaggata cactcagact ctaaagccag gtattaaact gacactgtca 780 gctcttctgg atggcaagaa cgtcaatgct ggtggccaca agcttggtct aggactggaa 840 tttcaagcat aa 852 <210> 55 <211> 852 <212> DNA <213> homo sapiens <220> <221> mutation <222> (232) .. (232) <223> OA <400> 55 atggctgtgc cacccacgta tgccgatctt ggcaaatctg ccagggatgt cttcaccaag 60 102 ggctatggat ttggcttaat aaagcttgat ttgaaaacaa aatctgagaa tggattggaa 120 tttacaagct caggctcagc caacactgag accaccaaag tgacgggcag tctggaaacc 180 aagtacagat ggactgagta cggcctgacg tttacagaga aatggaatac caacaataca 240 ctaggcaccg agattactgt ggaagatcag cttgcacgtg gactgaagct gaccttcgat 300 tcatccttct cacctaacac tgggaaaaaa aatgctaaaa tcaagacagg gtacaagcgg 360 gagcacatta acctgggctg cgacatggat ttcgacattg ctgggccttc catccggggt 420 gctctggtgc taggttacga gggctggctg gccggctacc agatgaattt tgagactgca 480 aaatcccgag tgacccagag caactttgca gttggctaca agactgatga attccagctt 540 cacactaatg tgaatgacgg gacagagttt ggcggctcca tttaccagaa agtgaacaag 600 aagttggaga ccgctgtcaa tcttgcctgg acagcaggaa acagtaacac gcgcttcgga 660 atagcagcca agtatcagat tgaccctgac gcctgcttct cggctaaagt gaacaactcc 720 agcctgatag gtttaggata cactcagact ctaaagccag gtattaaact gacactgtca 780 gctcttctgg atggcaagaa cgtcaatgct ggtggccaca agcttggtct aggactggaa 840 tttcaagcat aa 852 <210> 56 <211> 852 <212> DNA <213> homo sapiens <220> <221> mutation <222> (605) .. (605) <223> G>C <400> 56 atggctgtgc cacccacgta tgccgatctt ggcaaatctg ccagggatgt cttcaccaag 60 ggctatggat ttggcttaat aaagcttgat ttgaaaacaa aatctgagaa tggattggaa 120 103 tttacaagct caggctcagc caacactgag accaccaaag tgacgggcag tctggaaacc 180 aagtacagat ggactgagta cggcctgacg tttacagaga aatggaatac cgacaataca 240 ctaggcaccg agattactgt ggaagatcag cttgcacgtg gactgaagct gaccttcgat 300 tcatccttct cacctaacac tgggaaaaaa aatgctaaaa tcaagacagg gtacaagcgg 360 gagcacatta acctgggctg cgacatggat ttcgacattg ctgggccttc catccggggt 420 gctctggtgc taggttacga gggctggctg gccggctacc agatgaattt tgagactgca 480 aaatcccgag tgacccagag caactttgca gttggctaca agactgatga attccagctt 540 cacactaatg tgaatgacgg gacagagttt ggcggctcca tttaccagaa agtgaacaag 600 aagttgcaga ccgctgtcaa tcttgcctgg acagcaggaa acagtaacac gcgcttcgga 660 atagcagcca agtatcagat tgaccctgac gcctgcttct cggctaaagt gaacaactcc 720 agcctgatag gtttaggata cactcagact ctaaagccag gtattaaact gacactgtca 780 gctcttctgg atggcaagaa cgtcaatgct ggtggccaca agcttggtct aggactggaa 840 tttcaagcat aa 852 <210> 57 <211> 852 <212> DNA <213> homo sapiens <220> <221> mutation <222> (176) .. (177) <223> AA>GC <400> 57 atggctgtgc cacccacgta tgccgatctt ggcaaatctg ccagggatgt cttcaccaag 60 ggctatggat ttggcttaat aaagcttgat ttgaaaacaa aatctgagaa tggattggaa 120 tttacaagct caggctcagc caacactgag accaccaaag tgacgggcag tctgggcacc 180 104 aagtacagat ggactgagta cggcctgacg tttacagaga aatggaatac cgacaataca 240 ctaggcaccg agattactgt ggaagatcag cttgcacgtg gactgaagct gaccttcgat 300 tcatccttct cacctaacac tgggaaaaaa aatgctaaaa tcaagacagg gtacaagcgg 360 gagcacatta acctgggctg cgacatggat ttcgacattg ctgggccttc catccggggt 420 gctctggtgc taggttacga gggctggctg gccggctacc agatgaattt tgagactgca 480 aaatcccgag tgacccagag caactttgca gttggctaca agactgatga attccagctt 540 cacactaatg tgaatgacgg gacagagttt ggcggctcca tttaccagaa agtgaacaag 600 aagttggaga ccgctgtcaa tcttgcctgg acagcaggaa acagtaacac gcgcttcgga 660 atagcagcca agtatcagat tgaccctgac gcctgcttct cggctaaagt gaacaactcc 720 agcctgatag gtttaggata cactcagact ctaaagccag gtattaaact gacactgtca 780 gctcttctgg atggcaagaa cgtcaatgct ggtggccaca agcttggtct aggactggaa 840 tttcaagcat 852 <210> 58 <211> 852 <212> DNA <213> homo sapiens <220> <221> mutation <222> (203) .. (203) <223> G>C <400> 58 atggctgtgc cacccacgta tgccgatctt ggcaaatctg ccagggatgt cttcaccaag 60 ggctatggat ttggcttaat aaagcttgat ttgaaaacaa aatctgagaa tggattggaa 120 tttacaagct caggctcagc caacactgag accaccaaag tgacgggcag tctggaaacc 180 aagtacagat ggactgagta cgccctgacg tttacagaga aatggaatac cgacaataca 240 105 ctaggcaccg agattactgt ggaagatcag cttgcacgtg gactgaagct gaccttcgat 300 tcatccttct cacctaacac tgggaaaaaa aatgctaaaa tcaagacagg gtacaagcgg 360 gagcacatta acctgggctg cgacatggat ttcgacattg ctgggccttc catccggggt 420 gctctggtgc taggttacga gggctggctg gccggctacc agatgaattt tgagactgca 480 aaatcccgag tgacccagag caactttgca gttggctaca agactgatga attccagctt 540 cacactaatg tgaatgacgg gacagagttt ggcggctcca tttaccagaa agtgaacaag 600 aagttggaga ccgctgtcaa tcttgcctgg acagcaggaa acagtaacac gcgcttcgga 660 atagcagcca agtatcagat tgaccctgac gcctgcttct cggctaaagt gaacaactcc 720 agcctgatag gtttaggata cactcagact ctaaagccag gtattaaact gacactgtca 780 gctcttctgg atggcaagaa cgtcaatgct ggtggccaca agcttggtct aggactggaa 840 tttcaagcat aa 852 <210> 59 <211> 22 <212> DNA <213> ARTIFICIAL <220> <223> PRIMER <400> 59 gacgtttaca cagaagtgga 22 <210> 60 <211> 22 <212> DNA <213> ARTIFICIAL <220> <223> PRIMER <400> 60 gttccacttc tgtgtaaacg 22 <210> 61 106 <211> 21 <212> DNA <213> ARTIFICIAL <220> <223> PRIMER <400> 61 tggactcagt atgggctga 21 <210> 62 <211> 20 <212> DNA <213> ARTIFICIAL <220> <223> PRIMER <400> 62 gcccatactg agtccatctg <210> 63 <211> 21 <212> DNA <213> ARTIFICIAL <220> <223> PRIMER <400> 63 acagagctgt ggaacacaga c 21 <210> 64 <211> 22 <212> DNA <213> ARTIFICIAL <220> <223> PRIMER <400> 64 gttccacagc tctgtaaacg 22 <210> 65 <211> 21 <212> DNA <213> ARTIFICIAL <220> <223> PRIMER <400> 65 gaacacacag aacaccctgg g 21 107 <210> 66 <211> 23 <212> DNA <213> ARTIFICIAL <220> <223> PRIMER <400> 66 gtgttctgtg tgttccactt etc 23 <210> 67 <211> 25 <212> DNA <213> ARTIFICIAL <220> <223> PRIMER <400> 67 gaagttgcag actgctgtca atctc <210> 68 <211> 24 <212> DNA <213> ARTIFICIAL <220> <223> PRIMER <400> 68 gcgagattga cagcagtctg 24 <210> 69 <211> 17 <212> DNA <213> ARTIFICIAL <220> <223> PRIMER <400> 69 gaacacagac gccaccc 17 <210> 70 <211> 16 <212> DNA <213> ARTIFICIAL <220> <223> PRIMER <400> 70 gtggcgtctg tgttcc 16 108 <210> 71 <211> 23 <212> DNA <213> ARTIFICIAL <220> <223> PRIMER <400> 71 gatggactga gtatgccctg acg 23 <210> 72 <211> 24 <212> DNA <213> ARTIFICIAL <220> <223> PRIMER <400> 72 gtcagggcat actcagtcca tctg 24 109

Claims (22)

1. An isolated VDACl peptide fragment, analog, chemical derivative and a salt thereof capable of inducing apoptosis in a cell, wherein the peptide fragment, analog, chemical derivative and a salt thereof is derived from a VDACl cytosolic domain or partial sequence thereof.

2. The VDACl peptide fragment, analog, chemical derivative and a salt thereof according to claim 1 wherein said peptide induces apoptosis by enhancing sensitivity to an apoptosis-inducing reagent.

3. The VDACl peptide fragment, analog, chemical derivative and a salt thereof according to claim 1 said peptide fragment comprising up to 70 amino acid residues.

4. The VDACl peptide fragment, analog, chemical derivative and a salt thereof according to claim 3 said peptide fragment comprising 15 to 30 amino acid residues.

5. The VDACl peptide according to claim 3 said peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:5.

6. The VDACl peptide according to claim 5 said peptide comprising amino acid sequence SEQ ID NO:2.

7. The VDACl peptide according to claim 3 selected from the group consisting of SEQ ID NO: 1 1 , SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 15.

8. An isolated nucleotide sequence encoding a VDACl peptide fragment, analog, chemical derivative and a salt thereof according to any one of claims 1-7.

9. The nucleotide sequence according to claim 8 selected from the group consisting of SEQ ID NO:6-SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 16-SEQ ID NO: 18 and SEQ ID NO:20.

10. A polynucleotide construct comprising an isolated nucleotide sequence according to claim 9.

11. 1 1. The polynucleotide construct according to claim 10, wherein the construct comprises an expression vector.

12. A host cell comprising a construct according to any one of claims 10 and 1 1. 1 10

13. A pharmaceutical composition comprising an isolated VDACl peptide fragment, analog, chemical derivative and a salt thereof according to claim 1 and a pharmaceutically acceptable carrier or excipient.

14. The pharmaceutical composition according to claim 13 wherein the VDACl peptide fragment is selected from SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:5.

15. The pharmaceutical composition according to claim 14 comprising SEQ ID NO:2.

16. A pharmaceutical composition comprising a nucleotide sequence according to claim 8 and a pharmaceutically acceptable carrier or excipient.

17. The pharmaceutical composition according to claim 16 comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:6 -SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 16-SEQ ID NO: 18 and SEQ ID NO:20 .

18. The pharmaceutical composition according to claim 17 comprising a polynucleotide construct comprising nucleotide sequence selected from the group consisting of SEQ ID NO:6 -SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 16-SEQ ID NO: 18 and SEQ ID NO:20.

19. The pharmaceutical composition according to claim 18, wherein the polynucleotide construct comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:7 and SEQ ID NO: 17.

20. Use of an isolated VDACl peptide fragment, analog, chemical derivative and a salt thereof capable of inducing apoptosis in a cell, according to any one of claims 1-7, for the preparation of a medicament for the treatment of a disorder associated with aberrant apoptosis selected from the group consisting of proliferative disease, drug resistant cancer, radiation resistant cancer, viral diseases and lymphoproliferative disease

21. Use of the isolated polynucleotide according to claim 8 for the preparation of a medicament for the treatment of a disorder associated with aberrant apoptosis apoptosis selected from the group consisting of proliferative disease, drug resistant cancer, radiation resistant cancer, viral diseases and lymphoproliferative disease .

22. The use according to claim 21 wherein the polynucleotide comprises a nucleic acid 1 1 1 sequence selected from the group consisting of SEQ ID NO:6-SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 16-SEQ ID NO: 18 and SEQ ID NO:20. For the Applicant Hedva Schickler Patent Attorney Webb & Co. 1 12