WO2009026053A2

WO2009026053A2 - Inhibitors of iaspp interaction with an iaspp binding partner and methods of use

Info

Publication number: WO2009026053A2
Application number: PCT/US2008/072918
Authority: WO
Inventors: Edith Yvonne Jones; Xin Lu; Christian Siebold; Ross Alexander Robinson
Original assignee: Cancer Research Technology Limited; Ludwig Institute For Cancer Research Ltd.
Priority date: 2007-08-21
Filing date: 2008-08-12
Publication date: 2009-02-26
Also published as: WO2009026053A3; GB0716322D0

Abstract

The invention provides the crystal structure of the iASPP C-terminal protein molecule. The structure is set out in Table 1. The structure may be used in to model the interaction of compounds such as pharmaceuticals with this protein, and to determine the structure of related iASPP C-terminal molecules.

Description

Inhibitors of iASPP interaction with an iASPP Binding Partner and Methods of Use

Cross -Reference to Related Application

[0001] This application claims the benefit of priority of United Kingdom Patent Application No. 0716322.3, filed August 21, 2007, the disclosure of which is incorporated herein by reference in its entirety.

Field of the Invention

[0002] The present invention relates to the inhibitory apoptosis stimulating protein of p53 (iASPP), crystals of its p53-interacting domain, use of the structure of such crystals and novel peptides that antagonize the function of iASPP.

Background to the Invention

[0003] The tumor suppressor protein p53, the prototypic member of the p53 family, is a transcription factor that responds to oncogenic stress such as DNA damage by activating genes that result in apoptosis and cell cycle arrest; (for a review see Vogelstein et al., 2000). p53 is a primary target for mutation in human cancer; it is estimated that mutations or loss of function of p53 accounts for half of all cancers. The mutation frequency of p53 varies among different tumor types so that it can be much lower than 50%, for example 30% in breast cancer and only 5% in leukemia (Soussi et al., 2005). Most p53 mutations map to the DNA binding domain. These mutants are therefore unable to activate genes upregulated by wild-type p53, a loss of function that ultimately results in unchecked cell division (Cho et al., 1994). Full-length p63 and p73, like p53, have the ability to transactivate p21 and Bax genes causing cell cycle arrest and apoptosis (Di Como et al., 1999; Gaiddon et al., 2001). Unlike p53, p63 and p73 have different isoforms (for review see Yang et al., 2002). Isoforms of p63 and p73 lacking their transactivation domains act as dominant negative inhibitors of p53 (Kaghad et al., 1997; Laan and Paabo, 1998; Yang et al., 1998). p63 and p73 knockout mice show defects in epidermal (Mills et al., 1999; Yang et al., 1999) and neuronal (Pozniak et al., 2002; Yang et al., 2000) development respectively, but do not form spontaneous tumors as is the case for p53 knockout mice (Donehower et al., 1992); the murine knockout phenotypes are mirrored in human disease. Mutations in p63 and p73 are rare in human cancer but a number of mutations in the DNA binding domain and sterile α motif of p63 are associated with genetic epidermal syndromes (van Bokhoven and McKeon, 2002). Overexpression of p73 triggers neuronal cell differentiation in vitro (De Laurenzi et al., 2000). There is also an important functional relationship between the p53 family members: in the absence of p63 and p73, p53-dependent apoptosis in response to DNA damage is severely impaired (Flores et al., 2002).

[0004] The discovery of the apoptosis stimulating protein of p53 (ASPP) family of proteins revealed a new level of p53 regulation. There are three family members in humans ASPPl, ASPP2 and iASPP. ASPPl and ASPP2 selectively stimulate the transactivation function of all three p53 family members p53, p63 and p73, increasing the transactivation of pro-apoptotic genes (Bergamaschi et al., 2004; Samuels-Lev et al., 2001). In contrast, iASPP is an oncoprotein that specifically inhibits p53; its overexpression confers resistance to apoptosis induced by UV radiation and exposure to the chemotherapeutic agent cisplatin in cultured cells (Bergamaschi et al., 2003). There is only one ASPP family member in C.elegans which is functionally interchangeable with human iASPP (Bergamaschi et al., 2003). Thus iASPP is likely to be the most ancient member of the ASPP family. Full length human iASPP consists of 828 amino acids, and is the isoform almost exclusively expressed in cells (Slee et al., 2004). iASPP only shares high sequence homology with ASPPl and ASPP2 in the C-terminus region (-230 amino acids) which is made up of four ankyrin repeats and an SH3 domain, two common structural motifs involved in protein - protein interactions. In all members of the ASPP family this C- terminal region binds to the DNA binding domain of p53, and in iASPP is required for the inhibitory effect (Bergamaschi et al., 2003; Slee et al., 2004). The crystal structure of the C- terminal region of ASPP2 in complex with the DNA binding domain of p53 revealed that the ankyrin repeats and SH3 domain of ASPP2 both contribute to the p53 binding site unit (Gorina and Pavletich, 1996).

[0005] Increased expression of iASPP appears to be a mechanism involved in preventing p53 from working effectively and may be a prognostic marker for human cancers (Sullivan and Lu, 2007). It has also been suggested that targeting iASPP to reduce its expression will allow new therapeutic strategies for the treatment of cancer to be developed.

Summary of the Invention

[0006] The present inventors have crystallized the C-terminal region of inhibitory Apoptosis- Stimulating Protein of P53 (iASPP) to examine its structure in order to understand further its interaction with p53. Crystals of the C-terminal region were examined to determine the regions that interact with p53. During the course of this analysis it was also observed that the N- terminal region of the iASPP fragment occupied the p53-binding site of a neighbouring molecule. An eight amino acid peptide (SEQ ID NO:2, referred to herein as peptide Pl), based on this N-terminal region was shown to bind to iASPP.

[0007] Thus the invention relates, in its broadest aspect, to the three-dimensional structure of iASPP and its use. Particular embodiments relate to the regions of the structure that interact with p53 or with the 8 amino acid region referred to above, and to methods for the modeling and discovery of structures that mimic or compete with such binding in order to antagonize the action of iASPP on p53.

[0008] In another aspect, the invention includes an isolated compound having a spatial arrangement of atoms to bind to iASPP in a manner that interferes with iASPP binding to an iASPP binding partner selected from the group consisting of p53, p63 and p73, wherein the compound interacts with an iASPP polypeptide defined by at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60 , at least 65, at least 75 or more structural coordinates identified in Table 1, wherein the relative atomic positions of the structure identified in Table 1 are varied within a root mean square deviation of less than 1.2A. In some embodiments, the isolated compound interacts with the iASPP in a region of iASPP defined by at least ten coordinates of atoms from at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 or at least 12 different amino acids of Table 3, wherein the relative atomic positions are varied within a root mean square deviation of less than 1.2 A.

[0009] The term "iASPP binding partner" as used herein refers to a macromolecules such proteins or protein complexes that bind to iASPP in a cell that expresses iASPP, including p53, p63 and p73. It should be understood that a fragment of a binding partner that retains iASPP binding activity is considered a binding partner for purposes of such assays.

[0010] In some embodiments, the isolated compound interacts with the iASPP in a region of iASPP defined by at least 10 coordinates of atoms from at least 10 amino acids of Table 3, wherein the relative atomic positions are varied within a root mean square deviation of less than

1.2 A. In one exemplary embodiment, the at least ten amino acids of iASPP are selected from the group consisting of T722, L724, W767, E772, D775, E776, E795, D797. W798, Y809, P811, N813 and Y814. [0011] In some embodiments, the compound is selected from the group consisting of a small molecule, an antibody or an antigen-binding fragment thereof, a nucleic acid molecule and a peptide/polypeptide.

[0012] In some embodiments, the isolated compound is a peptide having a structure selected from the group consisting of

[0013] (a) a compound comprising a sequence of eight amino acids X₁-X₂-X₃-X₄- X₅-X₆-X₇- X₈ (SEQ ID NO: 6), wherein X₁ is selected from Serine or Threonine or conservative substitution threof; X₂ is proline or conservative substitution thereof; X₃, X₄, X₆, and X₇ are each independently selected from Arginine and Lysine; X₅ is Alanine, Serine, Valine, Glycine or conservative substitutions thereof; and X₈ is Alanine, Valine, Glycine or conservative substitutions thereof;

[0014] (b) a compound with the structure of (a), with the proviso that one or more of the X₁ X₂, X₃, X₄, X₅, X₆, X₇, X₈ amino acids are replaced with an unnatural amino acid;

[0015] (c) a compound with the structure of (a) or (b), with the proviso that one or more amide bonds is replaced with an ester or alkyl bond.

[0016] In some embodiments, the peptides described herein are from about 8 amino acid residues in length to about 100 amino acid residues in length. Of course it is contemplated that longer or indeed shorter peptides also may prove useful. Thus, peptides of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 and 100 amino acids in length are contemplated.

[0017] In a further aspect, the invention provides a compound comprises an amino acid an amino acid sequence at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at 81%, at least 82%, at least 83%, at least 84%, at least 85%, least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to a peptide/polypeptide described herein, wherein the compound retains the ability to interact (e.g., bind to) with iASPP.

[0018] In some embodiments, the compound is a peptide consisting of an amino acid sequence at least 50%, at least 62.5%, at least 75%, at least 87.5%, or more identical to SEQ ID NO: 2, wherein the peptide retains the ability to interact (e.g., bind to) with iASPP. In some embodiments, the compound is a peptide consisting of the amino acid sequence SPRKARRA (SEQ ID NO: 2). In some embodiments, the compound is a peptide consisting of an amino acid sequence at least 50%, at least 62.5%, at least 75%, at least 87.5%, or more identical to SEQ ID NO: 3, wherein the peptide retains the ability to interact with (e.g., bind to) iASPP. In some embodiments, the compound is a peptide consisting of the amino acid sequence SPRKSRRA (SEQ ID NO: 3). In some embodiments, the compound is a peptide consisting of an amino acid sequence at least 50%, at least 62.5%, at least 75%, at least 87.5%, or more identical to SEQ ID NO: 4, wherein the peptide retains the ability to interact with (bind to) iASPP. In some embodiments, the compound is a peptide consisting of the amino acid sequence SPRRARRA (SEQ ID NO: 4). In some embodiments, the compound is a peptide consisting of an amino acid sequence at least 50%, at least 62.5%, at least 75%, at least 87.5%, or more identical to SEQ ID NO: 5, wherein the peptide retains the ability to interact with (e.g., bind to) iASPP. In some embodiments, the compound is a peptide consisting of the amino acid sequence SPRRSRRA (SEQ ID NO: 5).

[0019] Cyclized versions of any of the peptides described herein are contemplated.

[0020] Nucleic acid molecules encoding a peptide/polypeptide described herein are also contemplated as another aspect of the inveniton. In some embodiments, the invention provides a nucleic acid molecule comprising a nucleotide sequence that encodes a peptide consisting of an amino acid sequence at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at 81%, at least 82%, at least 83%, at least 84%, at least 85%, least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to a peptide/polypeptide described herein.

[0021] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes a peptide consisting of an amino acid sequence at least 50%, at least 62.5%, at least 75%, at least 87.5% ,or more identical to SEQ ID NO: 2, wherein the peptide retains the ability to interact with (e.g., bind to) iASPP. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes a peptide consisting of the amino acid sequence SPRKARRA (SEQ ID NO: 2). In some embodiments, the a nucleic acid molecule comprises a nucleotide sequence that encodes a peptide consisting of an amino acid sequence at least 50%, at least 62.5%, at least 75%, at least 87.5%, or more identical to SEQ ID NO: 3, wherein the peptide retains the ability to interact with (e.g., bind to) iASPP. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes a peptide consisting of the amino acid sequence SPRKSRRA (SEQ ID NO: 3). In some embodiments, the the nucleic acid molecule comprises a nucleotide sequence encoding a peptide consisting of an amino acid sequence at least 50%, at least 62.5%, at least 75%, at least 87.5%, or more identical to to SEQ ID NO: 4, wherein the peptide retains the ability to interact with (e.g., bind to) iASPP. In some embodiments, the compound comprises a nucleic acid molecule comprises a nucleotide sequence encoding a peptide consisting of the amino acid sequence SPRRARRA (SEQ ID NO: 4). In some embodiments, the compound comprises a nucleic acid molecule comprises a nucleotide sequence that encodes an amino acid sequence at least 50%, at least 62.5%, at least 75%, at least 87.5%, or more identical to SEQ ID NO: 5, wherein the peptide retains the ability to interact with (e.g., bind to) iASPP. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes a peptide consisting of the amino acid sequence SPRRSRRA (SEQ ID NO: 5).

[0022] Any synthesis method can be used for making polypeptides and peptides of the invention. Numerous chemical synthesis methods are known and are especially suitable for shorter peptides. The isolated peptides described herein are optionally made by recombinant techniques in vitro and/or expressed in vivo. Polynucleotides that comprise nucleotide sequences that encode all (or a portion of) a peptide are an additional aspect of the invention. Vectors including expression vectors for in vitro production and gene therapy vectors for in vivo production/expression of the peptide(s), are also considered an aspect of the invention.

[0023] For example, the invention includes polynucleotides comprising a nucleotide sequence that encodes any peptide or polypeptide described herein, including an isolated peptide(s) as discussed above and described in further detail in the description below. In specific embodiments, the polynucleotide further comprises a nucleotide sequence that encodes a signal peptide fused in-frame with the polypeptides described above. The signal peptide facilitates extracellular secretion of the encoded peptide/polypeptide when expressed in a suitable host cell.

[0024] The invention also includes an expression vector comprising a nucleotide sequence that encodes any peptide (or polypeptide) described herein, including an isolated peptide(s) (or isolated polypeptide(s)) operably linked to an expression control sequence or promoter sequence. The expression vector may be any vector used for the expression of a nucleic acid and may for example, be selected from the group consisting of replication deficient adenoviral vectors, adeno-associated viral vectors, and lentivirus vectors. The polynucleotides and vectors described herein may be formulated as compositions in which the polynucleotides or the vector is presented in a pharmaceutically acceptable carrier, excipient or diluent.

[0025] Other aspects of the invention include host cells (including isolated host cells) that have been transformed or transfected with a polynucleotide or vector of the invention. In some variations, the cells are any prokaryotic or eukaryotic cell that can be manipulated (e.g., through transformation or transfection) to express peptides or polypeptides described herein. In some variations, the cells are suitable for ex vivo transfection/transformation and reinplantation into a host organism.

[0026] Another aspect of the invention includes a method of making a peptide or polypeptide described herein comprising growing a host cell described herein in a growth medium under conditions where the cell expresses the peptide or polypeptide encoded by the polynucleotide. In one aspect, the peptide or polypeptide is isolated or purified from the cell or the medium. Isolation of the polypeptide from the cells or from the medium in which the cells are grown is accomplished by purification methods known in the art, e.g., conventional chromatographic methods including immunoaffinity chromatography, receptor affinity chromatography, hydrophobic interaction chromatography, lectin affinity chromatography, size exclusion filtration, cation or anion exchange chromatography, high pressure liquid chromatography (HPLC), reverse phase HPLC, and the like. Still other methods of purification include those wherein the desired protein is expressed and purified as a fusion protein having a specific tag, label, or chelating moiety that is recognized by a specific binding partner or agent. The purified protein can be cleaved to yield the desired protein, or be left as an intact fusion protein. Cleavage of the fusion component may produce a form of the desired protein having additional amino acid residues as a result of the cleavage process.

[0027] Antibodies are contemplated as another aspect of the invention. In one aspect, the antibody is an antibody or antigen-binding fragment thereof that specifically binds to an iASPP epitope defined by at least 10 coordinates of atoms from at least 10 different amino acid residues identified in Table 3, wherein the relative atomic positions are varied within a root mean square deviation of less than 1.2A. In some embodiments, the antibody or antigen binding fragment thereof specifically binds to an epitope that includes one or more iASPP amino acids selected from the group consisting of T722, L724, W767, E772, D775, E776, E795, D797, W798, Y809, P811, N813 and Y814. The antibody or antigen binding fragment can be a monoclonal antibody. Alco contemplated are human, humanized and chimeric antibodies. A hybridoma cell line that produces the monoclonal antibody is also contemplated.

[0028] Compositions comprising a peptide, polypeptide, polynucleotide, or vector described herein and a pharmaceutically acceptable carrier are also contemplated. In certain aspects, the composition further comprises the further therapeutic or therapy. In other aspects, the further therapeutic or therapy is provided in a composition separate from the peptide, polypeptide, polynucleotide or vector.

[0029] In another aspect, the invention provides a method of treating a neoplastic disorder comprising administering to a subject in need of treatment for a neoplastic disorder a composition described herein, in an amount effective to treat the neoplastic disorder. In some embodiments, a subject is identified as having a neoplastic disorder prior to the administration of the composition. In another aspect, the invention provides a method of selecting a therapeutic regimen for a subject comprising identifying a subject as having neoplastic disorder characterized by increased expression of an iASPP polypeptide; and administering to the subject a composition as described herein.

[0030] In yet another aspect, the invention provides a method of treating a condition associated with tumor cell growth in a subject comprising administering to said subject a therapeutically effective amount of a combination therapy comprising agents selected from the group consisting of a composition as described herein and a standard of care anti-neoplastic therapy. In preferred embodiments, the agents are administered in an amount effective to inhibit tumor cell growth.

[0031] In some embodiments, the identifying step may comprise identifying a subject with a neoplastic disorder characterized by elevated expression of an iASPP polypeptide in the neoplastic cells.

[0032] Identifying steps of the methods described herein may comprise screening a biological sample suspected of containing neoplastic cells from the subject for elevated expression of the iASPP polypeptide. Exemplary biological samples suspected of containing neoplastic cells include, but are not limited to, blood, urine, spinal fluid, bone marrow, biopsy tissue, a tumor, and a sample comprising tumor cells. In some embodiments, the screening step comprises comparing expression of iASPP in cells from the sample with expression of iASPP from healthy cells of the same type as the cells in the sample, wherein increased expression of the iASPP in cells from the sample compared to the expression of the iASPP in cells from the healthy cells indicates elevated expression of iASPP.

[0033] In some embodiments, the neoplastic disease is a cancer selected from the group consisting of breast cancer, pancreatic cancer, ovarian cancer, colorectal cancer, esophageal cancer, lung cancer, head cancer, neck cancer, gastric cancer and epithelial cancer.

[0034] Also provided is a method of stimulating p53-mediated apoptosis in a cell comprising contacting the cell with a composition described herein in amount effective to stimulate p53- mediated apoptosis. In some embodiment, the cell expresses wild-type p53.

[0035] In some aspects, and as a variation of all therapies described herein, the methods described herein comprise further administering to the subject or contacting a cell with a standard of care anti-neoplastic therapeutic. In the context of methods of the invention, "standard of care" refers to a treatment that is generally accepted by clinicians for a certain type of patient diagnosed with a type of illness. Exemplary standard of care anti-neoplastic agents include, but are not limited to, a standard of care chemo therapeutic, a standard of care radiotherapeutic, or a standard of care radiation regimen for the neoplastic disorder. For all varieties of cancers and neoplastic disorders, for example, an aspect of the invention is to improve standard of care therapy with co-therapy with agents described herein that modulate (e.g., increase or stimulate) p53-mediated apoptosis.

[0036] The composition and the further therapeutic or therapy can be administered concurrently or separately. In embodiments where the further therapeutic or therapy and composition described herein are administered separately, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the further therapeutic or therapy and the composition described herein would still be able to exert an advantageously combined effect. In such instances, it is contemplated that one would administer both modalities within about 12-24 hours of each other and, more preferably, within about 6-12 hours of each other, with a delay time of only about 12 hours being most preferred. In some situations, it may be desirable to extend the time period for treatment significantly, however, where several days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the respective administrations. Repeated treatments with one or both agents are specifically contemplated. Exemplary routes of administration of the peptides or compositions described herein include, but are not limited to, intradermal, intramuscular, intraperitoneal, intraocular, intravenous, subcutaneous, topical, oral and intranasal administration.

[0037] In another aspect, the invention includes a method of screening for a modulator of binding of iASPP and an iASPP binding partner. An exemplary method comprises, (a) contacting an iASPP polypeptide comprising a structure defines by the structural coordinates provided in Table 1 with an iASPP binding with an iASPP binding partner, in the presence and absence of a test compound, under conditions in which the binding partner binds with the iASPP carboxy- terminal domain; wherein the binding partner comprises a member selected from the group consisting of p53, p63 and p73; and (b) comparing binding between said carboxy- terminal iASPP polypeptide and the binding partner in the presence and absence of the test compound, wherein increased binding (in the presence of the test compound, compared to the absence of the test compound) identifies the test compound as an agonist of binding, and decreased binding identifies the test compound as an antagonist inhibitor of binding.

[0038] Such assays can be performed in vitro in cell-free formats or cell based formats, which often lend themselves to high throughput implementation. In some cell-free variations, the iASPP or the binding partner is attached to a solid support (e.g., a bead, membrane, plate, or chip) and the other, unattached moiety is incubated with the solid support to permit binding. Optionally, the unbound moiety (the iASPP or the binding partner) is labeled (radiolabel, colorimetric label, etc.) or tagged (e.g., a peptide or epitope tag, or GFP fusion or enzymatic fusion) to facilitate measurement of binding.

[0039] In yet another aspect, the invention provides a computer-based method for the analysis of the interaction of a molecular structure with an iASPP structure. Such method comprises fitting a molecular structure to an iASPP structure defined by the structural coordinates identified in Table 1 or selected coordinates thereof, wherein the iASPP structure is optionally varied by a root square mean deviation of not more that 1.2A, and (b) determining at least one interaction between an atom of said iASPP structure and said molecular structure.

[0040] Also provided is a computer-based method of rational drug design comprising: fitting structure of at least two molecular fragments to an iASPP structure defined by the structural coordinates identified in Table 1 or selected coordinates thereof, wherein the iASPP structure is optionally varied by a root square mean deviation of not more that 1.2A, and assembling the fitted molecular structure fragments into a single molecule to form a single molecular structure. [0041] In some embodiments, the selected coordinates of the iASPP structure include atoms from one or more of the amino acid residues identified in Table 3. In one variation, the selected coordinates comprise at least 5 atoms.

[0042] In some embodiments, the molecular structure comprises at least two atoms that are located in the same relative spatial orientation to each other and are of the same elements as a corresponding number of atoms found in any of the amino acids identified in Table 4 or Table 5. In some embodiments, the atoms of Table 5 include at least two atoms that contact iASPP residues via hydrogen bonds.

[0043] In some embodiments, the molecular structure is in the form of a pharmacophore.

[0044] In some embodiments, the iASPP structure is a model constructed from all or a portion of the coordinates of Table 1, optionally varied by a root mean square deviation of not more than 0.5A. The modelay be selected from the group consisting of a wire-frame model, a chicken- wire model, a ball-and- stick model, a space-filling model, a stick- model, a ribbon model, a snake model, an arrow and cylinder model, an electron density map and a molecular surface model.

[0045] In some embodiments, the computer-based methods described herein further comprise modifying the molecular structure to modulate its interaction with the iASPP structure.

[0046] In another aspect, the invention provides a computer system, intended to generate structures and/or perform optimization of compounds that interact with an iASPP C-terminal region, the system containing computer-readable data comprising the atomic coordinate data identified in Table 1 or selected coordinates thereof, optionally varied by a root mean square deviation of not more than 1.2A. In some embodiments, the atomic coordinate data comprises at least one of the atoms provided by the amino acid residues identified in Tables 3 or 4. In some embodiments, the computer-readable data storage medium comprises data storage material encoded with said computer-readable data; a working memory for storing instructions for processing said computer-readable data; and a central-processing unit coupled to said working memory and to said computer-readable storage medium for processing said computer-readable data. In some embodiments, the computer-readable storage medium further comprises a display coupled to yhecentral-processing unit. [0047] In yet another aspect, the invention provides a method for providing data for generating structures and/or performing optimization of compounds which interact with an iASPP C- terminal region, the method comprises establishing communication with a remote device containing computer-readable data comprising atomic coordinate data identified in Table 1 or selected coordinate thereof, optionally varied by a root mean square deviation of not more than 1.2A and receiving the computer readable data fromthe remote device. In some embodiments, the method comprises processing said computer-readable data to display a model of an iASPP C-terminal region.

[0048] Another aspect of the invention includes a computer-readable storage medium comprising a data storage material encoded with computer-readable data, wherein the data are defined by the structure identified in Table 1, optionally varied by a root mean square deviation of not more than 1.2A.

[0049] Yet another aspect of the invention includes a computer-readable storage medium comprising a data storage material encoded with a first set of computer-readable data comprising a Fourier transform of at least a portion of the structural coordinates for the iASPP C-terminal protein defined by the structure identified in Table 1, or selected coordinates thereof, optionally varied by a root mean square deviation of not more than 1.2A; which data, when combined with a second set of machine readable data comprising an X-ray diffraction pattern of a molecule or molecular complex or unknown structure, using a maching programmed with the instructions for usingthe first set of data and the second set of data, can determine at least a portion of the structure coordinates corresponding to the second set of machine readable data.

[0050] These and other aspects of the invention are set out below and in the accompanying claims. The above aspects of the invention, both singly and in combination, all contribute to features of the invention.

[0051] The foregoing summary is not intended to define every aspect of the invention, and additional aspects are described in other sections, such as the Detailed Description. The entire document is intended to be related as a unified disclosure, and it should be understood that all combinations of features described herein are contemplated, even if the combination of features are not found together in the same sentence, or paragraph, or section of this document.

[0052] In addition to the foregoing, the invention includes, as an additional aspect, all embodiments of the invention narrower in scope in any way than the variations defined by specific paragraphs above. For example, certain aspects of the invention that are described as a genus, and it should be understood that every member of a genus is, individually, an aspect of the invention. Also, aspects described as a genus or selecting a member of a genus, should be understood to embrace combinations of two or more members of the genus. Although the applicant(s) invented the full scope of the invention described herein, the applicants do not intend to claim subject matter described in the prior art work of others. Therefore, in the event that statutory prior art within the scope of a claim is brought to the attention of the applicant(s) by a Patent Office or other entity or individual, the applicant(s) reserve the right to exercise amendment rights under applicable patent laws to redefine the subject matter of such a claim to specifically exclude such statutory prior art or obvious variations of statutory prior art from the scope of such a claim. Variations of the invention defined by such amended claims also are intended as aspects of the invention.

Brief Description of the Tables

[0053] Table 1 sets out the coordinate data of the structure of the C-terminal region of iASPP.

[0054] Table 2 sets out alterations to the Pl peptide region amino acids.

[0055] Table 3 sets out the residues of iASPP that form a peptide-binding region.

[0056] Table 4 sets out the residues of iASPP that interact with the peptide-binding region.

[0057] Table 5 sets out atoms of particular interest within Table 4 that interact with the peptide-binding region.

[0058] Table 6 provides crystallographic statistics.

Brief Description of the Drawings

[0059] Figure 1 shows saturation curves and Scatchard plots for the binding of iASPPΔ608 and ASPP2Δ905 to p53_core, p63_core and p73_core- Each value represents the mean and standard deviation of duplicate measurements.

[0060] Figure 2 shows the overall structure of iASPP Δ608 in (A) cartoon presentation and (B) by sequence alignment. Secondary structural features are indicated in relation to both sequences. In Figure 2(B) the symbols above the sequences represent iASPP residues observed to interact with the N-terminal residues (616-623 of SEQ ID NO: 8) of a neighbouring molecule in the crystal lattice. The symbols below the sequences represent ASPP2 residues involved in binding to the p53 DNA binding domain in the ASPP2-p53 crystal structure (PDB code IYCS). Arrow heads depict hydrogen bonds, squares depict hydrophobic interactions and full arrows depict residues involved in both hydrogen bonds and hydrophobic interactions.

[0061] Figure 3 shows a comparison of (A) the interaction between iASPP molecules in the crystal packing and (B) the interaction between iASPP (upper structure) and p53 (lower structure).

[0062] Figure 4 shows the interaction between the peptide Pl region and the iASPP p53- binding site of a neighboring molecule. Dotted lines represent hydrogen bonds with the lengths in A and "eyelashes" represent hydrophobic interactions.

[0063] Figure 5 shows iASPP binds peptide Pl in vitro. A representative ITC experiment showing raw data (upper) and integrated data (lower) for titrations of iASPPΔ625 with the peptide Pl. The dissociation constant is approximately 45 mM.

Detailed Description of the Invention

A. Protein Crystals.

[0064] The present invention provides a crystal of the iASPP C-terminal domain having an space group P2₁2₁2, and unit cell dimensions of about a = 60.0 A, b = 67.5 A, c = 50.5 A, α = β = γ = 90°, with a unit cell variability of 5% in all dimensions. An example of such variability is a crystal of unit cell dimensions a = 59.969 A, b = 67.545 A, c = 50.490 A.

[0065] Such crystals may be obtained using the methods described in the accompanying examples.

[0066] The iASPP C-terminal domain may comprise the sequence of residues 8-228 of SEQ ID NO: 1 illustrated herein, or a variant which retains the ability to form crystals under the conditions illustrated herein. Such variants include those with a number of amino acid substitutions, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids by an equivalent or fewer number of amino acids. Further examples of variants are discussed further herein below.

[0067] Generally the C-terminal domain comprising residues 8-228 of SEQ ID NO:1 will be flanked, at the N- and/or C-terminal by short sequences associated with the expression vector from which the protein is produced. Such vector sequences may include an N-terminal methionine and short tags (such as a polyhistidine tag), e.g. of from 1 to 10 amino acids in length, to facilitate expression, recovery or purification of the sequence. For example in SEQ ID NO:1 there is an N-terminal methionine followed by six histidine residues. It is believed that the presence of from 1 to 10 amino acids at the N- and/or C-termini of the 8-228 region of SEQ ID NO:1 will not substantially alter the folding or structure of this region of the iASPP protein.

[0068] The methodology used to provide the iASPP crystal illustrated herein may be used generally to provide such crystals resolvable at a resolution of at least 2.1 A.

[0069] The present invention contemplates "variants" wherein a "variant" refers to a polypeptide which is obtained by replacing at least one amino acid residue in iASPP of SEQ ID NO:1 with a different amino acid residue and/or by adding and/or deleting amino acid residues within the native polypeptide or at the N- and/or C-terminus of a polypeptide corresponding to iASPP. Variants may be prepared for example by site-specific mutagenesis of nucleic acid coding for the iASPP followed by expression of the iASPP in a host cell. Variants desirably have substantially the same three-dimensional structure as the iASPP from which they are derived.

[0070] By having substantially the same three-dimensional structure is meant having a set of atomic structure co-ordinates that have a root mean square deviation (rmsd) of less than or equal to about 1.2 A when superimposed with the atomic structure co-ordinates of the iASPP of SEQ ID NO:1 from which the variant is derived.

[0071] Amino acid substitutions, deletions and additions which do not significantly interfere with the three-dimensional structure of the iASPP will depend, in part, on the region of the iASPP where the substitution, addition or deletion occurs. In highly variable regions of the molecule, non-conservative substitutions as well as conservative substitutions may be tolerated without significantly disrupting the three-dimensional structure of the molecule. In highly conserved regions, or regions containing significant secondary structure, conservative amino acid substitutions are preferred.

[0072] Conservative amino acid substitutions are well-known in the art, and include substitutions made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphipathic nature of the amino acid residues involved. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; amino acids with uncharged polar head groups having similar hydrophilicity values include the following: leucine, isoleucine, valine; glycine, alanine; asparagine, glutamine; serine, threonine; phenylalanine, tyrosine. Other conservative amino acid substitutions are well known in the art.

[0073] In some instances, it may be particularly advantageous or convenient to substitute, delete and/or add amino acid residues in order to provide convenient cloning sites in the cDNA encoding the polypeptide, to aid in purification of the polypeptide, etc. Such substitutions, deletions and/or additions which do not substantially alter the three dimensional structure of the iASPP will be apparent to those having skills in the art.

[0074] The residues for alteration could easily be identified by those skilled in the art and these alterations can be introduced by site-directed mutagenesis e.g. using a Stratagene QuikChange™ Site-Directed Mutagenesis Kit or cassette mutagenesis methods (see e.g. Ausubel et al., eds., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, and Sambrook et al., Molecular Cloning: a Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (1989)).

[0075] In one aspect, crystals comprising one or more, e.g. 1 to 3 such as 1 or 2 alterations of the region corresponding to peptide Pl are contemplated. Peptide Pl corresponds to amino acids 616-623 of full-length iASPP set forth in SEQ ID NO: 8. In some embodiments, the residues of which may be altered, and the alterations contemplated, may be selected from the those set out in Table 2:

Table 2:

[0076] Examples of such alterations include the sequence alterations of peptide Pl corresponding to the substitutions of SEQ ID NO:3, 4 or 5. Such crystals may be formed using the conditions set out in the accompanying examples, and, if necessary or desirable, variations of these conditions within the routine skill and knowledge of those of ordinary skill in the art.

B. Crystal Coordinates.

[0077] In a further aspect, the invention also provides a crystal of the iASPP C-terminal region having the three dimensional atomic coordinates of Table 1. As indicated in Table 1, this provides the atomic coordinates of non-hydrogen atoms of the amino acids 616 to 822 of full- length iASPP set forth in SEQ ID NO: 8.

[0078] An advantageous feature of the structures defined by the atomic coordinates of Table 1 are that they have a resolution of about 2.1 A.

[0079] Table 1 provides atomic coordinate data for the iASPP C-terminal region. In this Table, the first column is the identifier "ATOM", the second column a contiguous number to uniquely refer to an atom of a particular row, the third column denotes the atom, the fourth the residue type, the fifth the chain identification, the sixth the residue number (the atom numbering is with respect to the full length wild type protein), the seventh, eighth and ninth columns are the X, Y, Z coordinates respectively of the atom in question, the tenth column the occupancy of the atom, the eleventh the temperature factor of the atom, the twelfth the chain identifier.

[0080] Table 1 is set out in an internally consistent format. For example (apart from the first residue, Ser 616) the coordinates of the atoms of each amino acid residue are listed such that the backbone nitrogen atom is first, followed by the C-alpha backbone carbon atom, designated CA, followed by side chain residues (designated according to one standard convention) and finally the carbon and oxygen of the protein backbone. Alternative file formats (e.g. such as a format consistent with that of the EBI Macromolecular Structure Database (Hinxton, UK)) which may include a different ordering of these atoms, or a different designation of the side-chain residues, may be used or preferred by others of skill in the art. However it will be apparent that the use of a different file format to present or manipulate the coordinates of the Table is within the scope of the present invention.

[0081] The coordinates of Table 1 provide a measure of atomic location in Angstroms, given to 3 decimal places. The coordinates are a relative set of positions that define a shape in three dimensions, but the skilled person would understand that an entirely different set of coordinates having a different origin and/or axes could define a similar or identical shape. Furthermore, the skilled person would understand that varying the relative atomic positions of the atoms of the structure within a root mean square deviation of less than 1.2 A when superimposed on the coordinates provided in Table 1 will generally result in a structure which is substantially the same as the structure of Table 1 in terms of both its structural characteristics and usefulness for structure-based analysis of iASPP-interacting molecular structures.

[0082] Table 1 also sets out the position of the oxygen atom of 154 water molecules, in a similar format to the amino acid residue atoms, though with row numbering restarting from 1. Those of skill in the art will understand that changing the number and/or positions of the water molecules of the Tables will not generally affect the usefulness of the structures for structure- based analysis of iASPP-interacting structures.

[0083] Thus for the purposes described herein as being aspects of the present invention, it is within the scope of the invention if: the coordinates of any of Table 1 are transposed to a different origin and/or axes; the relative atomic positions of the atoms of the structure are varied within a root mean square deviation of less than 1.2 A, preferably less than 1.0 A, more preferably less than 0.5 A, more preferably less than 0.3 A, such as less than 0.25 A, or less than 0.2 A, and most preferably less than 0.1 A, when superimposed on the coordinates provided in any of Table 1; and/or the number and/or positions of water molecules is varied.

[0084] Reference herein to the coordinate data of Table 1 and the like thus includes the coordinate data in which one or more individual values of the Table are varied in this way. By "root mean square deviation" we mean the square root of the arithmetic mean of the squares of the deviations from the mean. Reference to a root mean square deviation of less than 1.2 A is to be interpreted as also including reference to any one of the preferred, narrower values set out above.

[0085] Protein structure similarity is routinely expressed and measured by the root mean square deviation (rmsd), which measures the difference in positioning in space between two sets of atoms, based on the square root of the arithmetic mean of the squares of the deviations from the mean. The rmsd measures distance between equivalent atoms after their optimal superposition. The rmsd can be calculated over all atoms, over residue backbone atoms (i.e. the nitrogen-carbon-carbon backbone atoms of the protein amino acid residues), main chain atoms only (i.e. the nitrogen-carbon-oxygen-carbon backbone atoms of the protein amino acid residues), side chain atoms only or more usually over C-alpha atoms only. For the purposes of this invention, the rmsd can be calculated over any of these, using any of the methods outlined below. In one embodiment, rmsd is measured by reference to the C-alpha atoms.

[0086] Reference herein to the iASPP C-terminal structure of Table 1 and the like thus includes the iASPP C-terminal coordinate data of this Table in which one or more individual values of the Table are varied within the above-mentioned rmsd.

[0087] Methods of comparing protein structures are discussed in Methods of Enzymology, vol 115, pg 397-420. The necessary least-squares algebra to calculate rmsd has been given by Rossman and Argos (J. Biol. Chem. , vol 250, pp7525 (1975)) although faster methods have been described by Kabsch (Acta Crystallogr., Section A, A92, 922 (1976)); Acta Cryst. A34, 827-828 (1978)), Hendrickson (Acta Crystallogr., Section A, A35, 158 (1979)); McLachan (J. MoI. Biol., vol 128, pp49 (1979)) and Kearsley (Acta Crystallogr., Section A, A45, 208 (1989)). Some algorithms use an iterative procedure in which the one molecule is moved relative to the other, such as that described by Ferro and Hermans (Ferro and Hermans, Acta Crystallographic, A33, 345-347 (1977)). Other methods e.g. Kabsch's algorithm locate the best fit directly.

[0088] Programs for determining rmsd include MNYFIT (part of a collection of programs called COMPOSER, Sutcliffe, M.J., Haneef, L, Carney, D. and Blundell, T.L. (1987) Protein Engineering, 1, 377-384), MAPS (Lu, G. An Approach for Multiple Alignment of Protein Structures (1998, in manuscript and on http://bioinfol.mbfys.lu.se/TOP/maps.html)).

[0089] It is usual to consider C-alpha atoms and the rmsd can then be calculated using programs such as LSQKAB (Collaborative Computational Project 4. The CCP4 Suite: Programs for Protein Crystallography, Acta Crystallographies D50, (1994), 760-763), QUANTA (Jones et al., Acta Crystallography A47 (1991), 110-119 and commercially available from Accelerys, San Diego, CA), Insight (commercially available from Accelerys, San Diego, CA), Sybyl® (commercially available from Tripos, Inc., St Louis), O (Jones et al., Acta Crystallographica, A47, (1991), 110-119), and other coordinate fitting programs.

[0090] In, for example the programs LSQKAB and O, the user can define the residues in the two proteins that are to be paired for the purpose of the calculation. Alternatively, the pairing of residues can be determined by generating a sequence alignment of the two proteins e.g. using programs for sequence alignment such as those of the BLAST suite of programs. The atomic coordinates can then be superimposed according to this alignment and an rmsd value calculated. The program Sequoia (CM. Bruns, I. Hubatsch, M. Ridderstrδm, B. Mannervik, and J. A. Tainer (1999) Human Glutathione Transferase A4-4 Crystal Structures and Mutagenesis Reveal the Basis of High Catalytic Efficiency with Toxic Lipid Peroxidation Products, Journal of Molecular Biology 288(3): 427-439) performs the alignment of homologous protein sequences, and the superposition of homologous protein atomic coordinates. Once aligned, the rmsd can be calculated using programs detailed above. For sequence identical, or highly identical, the structural alignment of proteins can be done manually or automatically as outlined above. Another approach would be to generate a superposition of protein atomic coordinates without considering the sequence.

[0091] Those of skill in the art will appreciate that in many applications of the invention, it is not necessary to utilise all the coordinates of Table 1, but merely a portion of them. For example, as described below, in methods of modelling Table 1 structures with molecular structures, selected coordinates of the iASPP C-terminal structure of Table 1 may be used.

[0092] By "selected coordinates" it is meant for example at least 5, preferably at least 10, preferably at least 20, more preferably at least 50 and even more preferably at least 100, for example at least 500 or at least 1000 atoms of the iASPP C-terminal structure. Reference herein to selected coordinates in relation to all applications of the invention described herein is to be understood as including reference to the use of any one of these exemplified values.

[0093] In one aspect, the selected coordinates may include all or some of the atoms of the amino acid residues of Table 3. These residues are those to which the Pl peptide region of iASPP binds and are thus of interest in the development of compounds which interact with iASPP in this region in order to interfere with the binding to p53.

Table 3:

Thr 722 Leu 724 Trp 767 GIu 772

Asp 775 GIu 776 GIu 795 Asp 797

Trp 798 Tyr 809 Pro 811 Asn 813

Tyr 814

[0094] In one aspect, the selected coordinates may comprise at least 10 coordinates (e.g. at least 20 coordinates) of atoms from at least 5 different amino acid residues of Table 3. In another aspect, the selected coordinates may comprise at least 10 coordinates (e.g. at least 20, such as at least 50) of atoms from at least 10 different residues of Table 3. References herein below to any use of selected coordinates of Table 1 include the use of selected coordinates of Table 3, including in the above numbers and combinations.

C. Description of Structure.

[0095] In the structure of iASPP C-terminal set out in Table 1 herein, the first resolvable residue is Ser 616 of SEQ ID NO: 8 and the last residue Lys 822 of SEQ ID NO: 8. This corresponds to amino acids 16 to 222 of SEQ ID NO:1. The cDNA and amino acid sequences for full-length iASPP are set forth in SEQ ID NOs: 7 and 8, respectively.

[0096] As expected from the common domain organization of the ASPP proteins, the iASPP structure comprises four ankyrin repeats and a closely juxtaposed SH3 domain (Figure 2A) which together constitutes one structural unit with a buried surface between the ankyrin repeats and SH3 domain of 1312 A². Ankyrin repeats 1-3 are typical in their topology, each folds into two anti-parallel α-helices followed by a β-hairpin loop which juts out at an angle of approximately 90° relative to the α-helices. The fourth repeat lacks the β-hairpin loop and has an extended second helix. The helices of one repeat pack against the helices of the adjacent repeat and the β-hairpin loops form a continuous β-sheet. The four repeats stack together forming an L- shaped structure (the stem of the L being represented by the pairs of helices and the base of the L by the β-hairpins). The inter-repeat interface mainly consists of hydrophobic interactions, and a network of hydrogen bonds connects the β-hairpin loops. There are two regions in the ankyrin repeats that contain insertions compared to the ankyrin consensus; a one residue insertion in the third ankyrin repeat located in the hairpin loop (residues 715-726 of SEQ ID NO: 8) and a second insertion of two residues between the two helices of the fourth ankyrin repeat (residues 734-742 of SEQ ID NO: 8) forming an extended loop; these features are common to ASPP proteins (Figure 2B). When compared to those in ASPP2, the iASPP ankyrin repeats (residues 627-754 of SEQ ID NO: 8) have a root mean square deviation (RMSD) of 1.2 A for 127 equivalent Ca atoms.

[0097] The ankyrin repeats are connected to the SH3 domain via the extended second helix (residues 741-754 of SEQ ID NO: 8) of the fourth ankyrin repeat which is considerably longer than the other helices of the previous repeats. Superpositions of the iASPP and ASPP2 structures considering only the SH3 domains or the ankyrin repeats, reveals a difference in the relative orientation of their two motifs of some 7 degrees, indicating that there is flexibility between these two domains. ASPP2 appears to be in a more open conformation, this is mirrored by a lower buried surface area between the ankyrin repeats and the SH3 domain (1053 A²). [0098] The SH3 domain (residues 758- 828 of SEQ ID NO: 8) of iASPP displays the classical β-barrel-like fold typical of SH3 domains. It comprises five anti-parallel β strands (residues 762- 765 of SEQ ID NO: 8, 784-789 of SEQ ID NO: 8, 798-803 of SEQ ID NO: 8, 806-811 of SEQ ID NO: 8, 815-816 of SEQ ID NO: 8) and a 3₁₀ helical segment (residues 812-814 of SEQ ID NO: 8). SH3 domain sequences have a number of highly conserved residues; the topology of an SH3 domain brings these conserved residues close together, creating a hydrophobic groove on the surface which forms a ligand binding site. Two loops which make a major contribution to this binding site have been termed the RT- loop and the n-Src loop (Figure T). In iASPP the RT- loop is of standard length and the n-Src loop contains a three-residue insertion (residues 793-795 of SEQ ID NO: 8) of mainly acidic residues. The SH3 domain of iASPP has an RMSD of 1.0 A when compared to that of ASPP2 for 61 equivalent Ca atoms.

[0099] The ankyrin repeats and SH3 domain form inter domain hydrogen bonds between GIn 753 of SEQ ID NO: 8 in the second α-helix of the fourth ankyrin repeat and Arg 790 of SEQ ID NO: 8 positioned in the n-Src loop of the SH3 domain, and between Asn 685 in the β-hairpin loop of the second ankyrin repeat and Arg 820 of SEQ ID NO: 8 in the C-terminus of the SH3 domain. The interface is further stabilized by a network of hydrophobic interactions.

Characteristics of the p53 binding site in iASPP

[00100] The putative p53 binding site in iASPP broadly resembles that of ASPP2. The major interactions between the C-terminal region of ASPP2 and the p53 DNA binding domain, as previously reported ((Gorina and Pavletich, 1996); PDB code IYCS), are made via the SH3 domain, which interacts with the L3 loop of p53. Further interactions are made by the β-hairpin loop of the third ankyrin repeat which interacts with the L2 loop of p53 (Gorina and Pavletich, 1996). The ASPP2 residues involved in binding the DNA binding domain of p53 are mapped onto the sequence alignment presented in Figure 2B. Many of the residues involved are conserved in iASPP but there are differences. Of the eight residues of ASPP2 that form hydrogen bonds with p53, five of these residues are identical in iASPP (Ser 725, Tyr 769, Asp 775, GIu 776 and Trp 798 of SEQ ID NO: 8) but residues Thr 722, Phe 773 and GIu 794 of SEQ ID NO: 8, differ from those at the equivalent positions in ASPP2. Of the ten ASPP2 residues observed to make hydrophobic contacts with p53, five are conserved in iASPP (Ser 725, GIu 795, Trp 798, Tyr 809 and Asn 813 of SEQ ID NO: 8). Of those that are not identical, the majority are replaced by residues with similar chemical properties except for iASPP residues Leu724, GIy 727 and Phe 773 of SEQ ID NO: 8 which respectively replace Tyr, Met and Asn in ASPP2. Thus, although a solid phase binding assay (see Examples below) showed similar affinities for p53_core binding to iASPPΔ625 and ASPP2Δ905 their structures contain significant differences which could be exploited for drug design. In contrast, p63_core and p73_core binding affinities discriminate between iASPPΔ625 and ASPP2Δ905.

[00101] The explanation for this may lie in the SH3 domain. iASPP Tyr 814 is a highly conserved residue in SH3 domains, frequently involved in binding to a hydrophobic Po residue (most often Pro, VaI or Leu) in a linear (peptide) binding motif (Wu et al., 1995; Yu et al., 1994). Comparison with the ASPP2-p53 co-structure (Gorina and Pavletich, 1996) suggests that p63 and p73 both have a VaI residue at a suitable position in their L3 loops to form a favourable Po-like interaction to Tyr 814. In the ASPP2 SH3 domain the Tyr is replaced by Leu, removing the potential for a classical P₀-like interaction, a loss consistent with the observed 3-fold reduction in binding affinity to p63_core and p73_core-

A novel interaction potential revealed by iASPP crystal packing

[00102] One unexpected property of the iASPPΔ608 crystal structure is revealed by the crystal packing. The first eight ordered N-terminal residues of one iASPPΔ608 molecule, residues 616-623 of SEQ ID NO: 8 (SPRKARRA (SEQ ID NO:2)), occupy the putative p53 binding site of the neighbouring molecule in the crystal lattice (Figure 3 A, B). The majority of the interactions are made with the SH3 domain but there are also contributions from the third ankyrin repeat (Fig. 4). iASPP residues 616-623 of SEQ ID NO: 8 are orientated with the N- terminus interacting with the ankyrin repeats and the subsequent residues lying in the SH3 peptide binding groove. These residues are set out by name in Table 4.

Table 4:

[00103] Pro 617 of SEQ ID NO: 8 interacts with the RT-loop and the helical segment of the SH3 domain, Arg 618 of SEQ ID NO: 8 is sandwiched between the β-hairpin loop of the third ankyrin repeat and the 3^ helical segment of the SH3 domain forming hydrogen bonds to the main chain of Thr 722 of SEQ ID NO: 8. Lys 619 of SEQ ID NO: 8 interacts with the 3₁₀ helical segment and is hydrogen bonded to GIu 772 of SEQ ID NO: 8 of the RT loop of the SH3 domain; Ala 620 of SEQ ID NO: 8 is in van-der-Waals contact with the n-Src loop. Arg 622 of SEQ ID NO: 8 is coordinated by hydrogen bonds to Asp775 of SEQ ID NO: 8 and GIu 776 of SEQ ID NO: 8 of the RT loop and GIu 795 of SEQ ID NO: 8 located on the n-Src loop and finally Ala 623 of SEQ ID NO: 8 interacts with the n-Src loop. Figure 4 details all the interactions made between the two iASPP molecules, and these are further summarized by Table 5 below.

Table 5:

* W = hydrogen bond to water molecule; H = hydrogen bond to iASPP amino acid; Ph = hydrophobic interaction with iASPP amino acid. [00104] Residues 616-623 of SEQ ID NO: 8 bury 1100 A² of solvent accessible surface on iASPP, similar to that observed in SH3 domain-peptide co-structures (Harkiolaki et al., 2003; Wu et al., 1995). The residues bind the SH3 domain in the minus orientation (for a definition see (Feng et al., 1994; Lim et al., 1994)), interacting with a surface that shows a high level of conservation (Harkiolaki et al., 2003; Kaneko et al., 2003; Musacchio et al., 1994; Wu et al., 1995). Many of the SH3 residues involved in the interaction (GIu 776 of SEQ ID NO: 8, Trp 798 of SEQ ID NO: 8, Pro 811 of SEQ ID NO: 8, Asn 813 of SEQ ID NO: 8 and Tyr 814 of SEQ ID NO: 8) are highly conserved and mediate ligand binding in classical SH3 domain co- structures. A BLAST search (www.ncbi.nlm.nih.gov) showed that residues 616-623 of SEQ ID NO: 8 are unique to iASPP, and these residues are conserved between the human and mouse proteins.

D. Structure Solution

[00105] The atomic coordinate data of iASPP C-terminal can also be used to solve the crystal structure of other target iASPP C-terminal proteins including other crystal forms of the iASPP C-terminal region, co-complexes of the iASPP C-terminal region, where X-ray diffraction data or NMR spectroscopic data of these target iASPP C-terminal proteins has been generated and requires interpretation in order to provide a structure.

[00106] In the case of the iASPP C-terminal region, this protein may crystallize in more than one crystal form. The data of Table 1, or portions thereof, as provided by this invention, are particularly useful to solve the structure of those other crystal forms of iASPP C-terminal. It may also be used to solve the structure of iASPP variants, co-complexes, or of the crystalline form of any other protein with significant amino acid sequence homology to any functional domain of iASPP C-terminal.

[00107] In the case of other target iASPP C-terminal region proteins, this may include crystals of iASPP C-terminal region proteins which are fragments of SEQ ID NO:1 or are longer versions of iASPP, e.g. truncated nearer the N-terminal region. The present invention allows the structures of such targets to be obtained more readily where raw X-ray diffraction data is generated.

[00108] Thus, where X-ray crystallographic or NMR spectroscopic data is provided for a target iASPP C-terminal region protein of unknown three-dimensional structure, the atomic coordinate data derived from Table 1, may be used to interpret that data to provide a likely structure for the other iASPP C-terminal region protein by techniques which are well known in the art, e.g. phasing in the case of X-ray crystallography and assisting peak assignments in NMR spectra.

[00109] One method that may be employed for these purposes is molecular replacement. In this method, the unknown crystal structure, whether it is another crystal form of an iASPP C- terminal region protein, a iASPP C-terminal variant or an iASPP C-terminal co-complex, or the crystal of a target iASPP C-terminal protein with amino acid sequence homology to any functional domain of iASPP C-terminal, may be determined using the iASPP C-terminal structure coordinates of all or part of Table 1 of this invention. This method will provide an accurate structural form for the unknown crystal more quickly and efficiently than attempting to determine such information ab initio.

[00110] Examples of computer programs known in the art for performing molecular replacement are CNX (Brunger A. T.; Adams P.D.; Rice L.M., Current Opinion in Structural Biology, Volume 8, Issue 5, October 1998, Pages 606-611 (also commercially available from Accelrys San Diego, CA), MOLREP (A.Vagin, A.Teplyakov, MOLREP: an automated program for molecular replacement, J. Appl. Cryst. (1997) 30, 1022-1025, part of the CCP4 suite) or AMoRe (Navaza, J. (1994). AMoRe: an automated package for molecular replacement. Acta Cryst. A50, 157-163).

[00111] Thus, in a further aspect of the invention provides a method for determining the structure of a protein, which method comprises;

[00112] providing the coordinates (or selected coordinates thereof) of the iASPP C- terminal structure of Table 1,

[00113] positioning the coordinates in the crystal unit cell of said protein so as to provide a structure for said protein.

[00114] The invention may also be used to assign peaks of NMR spectra of such proteins, by manipulation of the data of Table 1.

E. Computer Systems.

[00115] In another aspect, the present invention provides systems, particularly a computer system intended to generate structures and/or perform optimization of compounds which interact with an iASPP C-terminal region, the system containing computer-readable data comprising atomic coordinate data of Table 1 optionally varied by a root mean square deviation of not more than 1.2 A, or selected coordinates thereof.

[00116] For example the computer system may comprise: (i) a computer-readable data storage medium comprising data storage material encoded with the computer-readable data; (ii) a working memory for storing instructions for processing said computer-readable data; and (iii) a central-processing unit coupled to said working memory and to said computer-readable data storage medium for processing said computer-readable data and thereby generating structures and/or performing rational drug design. The computer system may further comprise a display coupled to said central-processing unit for displaying said structures.

[00117] The structure of the iASPP region provided by Table 1, or selected coordinates thereof, may be displayed by the computer system in the form of a model, for example (a) a wire-frame model; (b) a chicken-wire model; (c) a ball-and-stick model; (d) a space-filling model; (e) a stick-model; (f) a ribbon model; (g) a snake model; (h) an arrow and cylinder model; (i) an electron density map; (j) a molecular surface model. Such representations will be useful in the practice of various aspects of the invention described further herein.

[00118] The invention also provides such systems containing atomic coordinate data of target iASPP C-terminal proteins wherein such data has been generated according to the methods of the invention described herein based on the starting data provided the data of Table 1 or selected coordinates thereof.

[00119] Such data is useful for a number of purposes, including performing rational drug design of compounds that interact with the iASPP C-terminal region, such as compounds which are potential modulators of the interaction of iASPP with p53, p63 or p73, e.g. inhibitors of these interactions that promote apoptosis.

[00120] In a further aspect, the present invention provides computer-readable storage medium, comprising a data storage material encoded with computer readable data, wherein the data are defined by the structure of Table 1 optionally varied by a root mean square deviation of not more than 1.2 A.

[00121] As used herein, "computer readable media" refers to any medium or media, which can be read and accessed directly by a computer. Such media include, but are not limited to: magnetic storage media such as floppy discs, hard disc storage medium and magnetic tape; optical storage media such as optical discs or CD-ROM; electrical storage media such as RAM and ROM; and hybrids of these categories such as magnetic/optical storage media.

[00122] By providing such computer readable media, the atomic coordinate data of the invention can be routinely accessed to model iASPP C-terminal regions or selected coordinates thereof. For example, RASMOL (Sayle et al., TIBS, Vol. 20, (1995), 374) is a publicly available computer software package, which allows access and analysis of atomic coordinate data for structure determination and/or rational drug design.

[00123] As used herein, "a computer system" refers to the hardware means, software means and data storage means used to analyse the atomic coordinate data of the invention. The minimum hardware means of the computer-based systems of the present invention comprises a central processing unit (CPU), input means, output means and data storage means. Desirably a monitor is provided to visualize structure data. The data storage means may be RAM or means for accessing computer readable media of the invention. Examples of such systems are microcomputer workstations available from Silicon Graphics Incorporated and Sun Microsystems running Unix based, Windows XP or IBM OS/2 operating systems.

[00124] The invention also provides a computer-readable data storage medium comprising a data storage material encoded with a first set of computer-readable data comprising the iASPP C-terminal coordinates of Table 1 or selected coordinates thereof; which, when combined with a second set of machine readable data comprising an X-ray diffraction pattern of a molecule or molecular complex of unknown structure, using a machine programmed with the instructions for using said first set of data and said second set of data, can determine at least a portion of the electron density or structure coordinates corresponding to the second set of machine readable data.

[00125] A further aspect of the invention provides a method of providing data for generating structures and/or performing optimisation of compounds which interact with an iASPP C- terminal region, the method comprising:

[00126] (i) establishing communication with a remote device containing computer- readable data comprising atomic coordinate data of Table 1 optionally varied by a root mean square deviation of not more than 1.2 A, or selected coordinates thereof; and [00127] (ii) receiving said computer-readable data from said remote device.

[00128] When such data is received it may be utilised in the various methods of the invention described herein, including computer-based methods of the analysis of the interaction of a molecular structure with an iASPP structure. The atomic coordinate data may include coordinates of amino acids set out in Table 3 and/or Table 4.

[00129] Thus the remote device may comprise e.g. a computer system or computer readable media of one of the previous aspects of the invention. The device may be in a different country or jurisdiction from where the computer-readable data is received.

[00130] The communication may be via the internet, intranet, e-mail etc, transmitted through wires or by wireless means such as by terrestrial radio or by satellite. Typically the communication will be electronic in nature, but some or all of the communication pathway may be optical, for example, over optical fibers.

F Uses of the Structure.

Summary

[00131] The crystal structures obtained according to the present invention, as well as the structures of target iASPP C-terminal proteins obtained in accordance with the methods described herein, may be used in several ways for drug design. In one aspect, the discovery of a region of the iASPP that can be bound by a short peptide allows a process of rational drug discovery based on the targeting of interactions that replicate at least some of the Pl peptide interactions.

[00132] In the case of the Pl peptide itself (SEQ ID NO:2), or its variants including SEQ ID NOs:3-5, information on the binding orientation by either co-crystallization, soaking or computationally docking such peptides can be used to guide specific modifications to the chemical structure designed to improve the interaction of the peptide with iASPP, or to improve the size or pharmacological properties of the peptide.

[00133] Thus, the determination of the three-dimensional structure of iASPP C-terminal provides a basis for the design of new compounds, which interact with iASPP C-terminal in novel ways. For example, knowing the three-dimensional structure of iASPP C-terminal, computer modelling programs may be used to design different molecules expected to interact with possible or confirmed active sites, such as binding sites or other structural or functional features of iASPP C-terminal. These and further aspects of the invention are described further below:

(i) Computer based methods of analysis

[00134] Although the invention will facilitate the determination of actual crystal structures comprising an iASPP C-terminal region and a compound which interacts with the iASPP C- terminal, current computational techniques provide a powerful alternative to the need to generate such crystals and generate and analyze diffraction data. Accordingly, a particularly preferred aspect of the invention relates to computer-based (sometimes referred to as "in silico") methods directed to the analysis and development of compounds which interact with iASPP C- terminal structures of the present invention.

[00135] Determination of the three-dimensional structure of iASPP C-terminal provides important information about the binding sites of iASPP C-terminal, particularly when comparisons are made with similar proteins. This information may then be used for rational design and modification of iASPP C-terminal inhibitors, e.g. by computational techniques which identify possible binding ligands for the binding sites, by enabling linked-fragment approaches to drug design, and by enabling the identification and location of bound ligands using X-ray crystallographic analysis. These techniques are discussed in more detail below.

[00136] Thus as a result of the determination of the iASPP C-terminal three-dimensional structure, more purely computational techniques for rational drug design may also be used to design structures whose interaction with iASPP C-terminal is better understood (for an overview of these techniques see e.g. Walters et al (Drug Discovery Today, Vol.3, No.4, (1998), 160-178; Abagyan, R.; Totrov, M. Curr. Opin. Chem. Biol. 2001, 5, 375-382). For example, automated ligand-receptor docking programs (discussed e.g. by Jones et al. in Current Opinion in Biotechnology, Vol.6, (1995), 652-656 and Halperin, L; Ma, B.; Wolfson, H.; Nussinov, R. Proteins 2002, 47, 409-443), which require accurate information on the atomic coordinates of target receptors may be used.

[00137] The aspects of the invention described herein which utilize the iASPP C-terminal structure in silico may be equally applied to both the iASPP C-terminal structure of Table 1 or selected coordinates thereof and the models of target iASPP C-terminal proteins obtained by other aspects of the invention. Thus having determined a conformation of a iASPP C-terminal by the method described above, such a conformation may be used in a computer-based method of rational drug design as described herein. In addition the availability of the structure of the iASPP C-terminal will allow the generation of highly predictive pharmacophore models for virtual library screening or compound design.

[00138] Accordingly, the invention provides a computer-based method for the analysis of the interaction of a molecular structure with an iASPP structure, which comprises:

[00139] providing a structure comprising a three-dimensional representation of the C- terminal region of an iASPP structure, said structure being as set out in Table 1 optionally varied by a root mean square deviation of not more than 1.2 A, or selected coordinates thereof;

[00140] providing a molecular structure to be fitted to said iASPP structure or selected coordinates thereof;

[00141] fitting the molecular structure to said iASPP structure.

[00142] By "fitting", it is meant determining at least one interaction between an atom of said iASPP structure and said molecular structure.

[00143] Interactions may be determined by automatic, semi-automatic or manual means. Computer programs can be employed to estimate interactions including the attraction, repulsion, and steric hindrance of the two binding partners (i.e. the iASPP C-terminal and a molecular structure). Various computer-based methods for fitting are described further herein. Generally the process involves calculating the extent to which such an interaction is present or stable.

[00144] In one embodiment, the iASPP C-terminal structure described herein comprises the coordinate data set forth in Table 1, or selected coordinates thereof. In practice, it will be desirable to model a sufficient number of atoms of the iASPP C-terminal as defined by the coordinates of Table 1 or selected coordinates thereof), which represent a region of interest. In the present invention, we have identified that the region of iASPP corresponding to the Pl peptide appears to bind to the p53 binding region of the protein. The residues to which the Pl peptide bind include those set out in Table 3 above. Thus methods of the invention, including those relating to the analysis of the interaction of a molecular structure with an iASPP structure and to methods of rational drug design may utilize selected coordinates which include some or all of those of the amino acid resides of Table 3. [00145] Although every different compound that may bind to the iASPP C-terminal region may interact with different parts of this protein, the structure of this iASPP C-terminal allows the identification of a number of particular sites which are likely to be involved in many of the interactions of iASPP C-terminal with a drug candidate, in particular the residues set out in Table 3. Thus, in this aspect of the invention, the selected coordinates may comprise coordinatesof at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or all of the residues set forth in Table 3.

[00146] In order to provide a three-dimensional structure of compounds to be fitted to a iASPP C-terminal structure of the invention, the compound structure may be modeled in three dimensions using commercially available software for this purpose or, if its crystal structure is available, the coordinates of the structure may be used to provide a representation of the compound for fitting to a iASPP C-terminal structure of the invention.

[00147] Newly designed structures may be synthesized and their interaction with iASPP C- terminal may be determined or predicted as to how the newly designed structure interacts with said iASPP C-terminal structure. This process may be iterated so as to further alter the interaction between it and the iASPP C-terminal.

[00148] More specifically, the interaction of a compound or compounds with iASPP C- terminal can be examined through the use of computer modeling using a docking program such as GOLD (Jones et al., /. MoI. Biol, 245, 43-53 (1995), Jones et al., /. MoI. Biol, 267, 727-748 (1997)), GRAMM (Vakser, I.A., Proteins , Suppl., 1:226-230 (1997)), DOCK (Kuntz et al, J.Mol.Biol. 1982 , 161, 269-288, Makino et al, J.Comput.Chem. 1997, 18, 1812-1825), AUTODOCK (Goodsell et al, Proteins 1990, 8, 195-202, Morris et al, J.Comput.Chem. 1998, 19, 1639-1662.), FlexX, (Rarey et al, J.Mol.Biol. 1996, 261, 470-489) or ICM (Abagyan et al, J.Comput.Chem. 1994, 15, 488-506). This procedure can include computer fitting of compounds to the iASPP C-terminal to ascertain how well the shape and the chemical structure of the compound will bind to the iASPP C-terminal.

[00149] Also computer-assisted, manual examination of the active site structure of iASPP C- terminal may be performed. The use of programs such as GRID (Goodford, /. Med. Chem., 28, (1985), 849-857) - a program that determines probable interaction sites between molecules with various functional groups and an enzyme surface - may also be used to predict, for example, the types of modifications which will alter the binding of a compound. [00150] Detailed structural information can then be obtained about the binding of the compound to iASPP C-terminal, and in the light of this information adjustments can be made to the structure or functionality of the compound, e.g. to alter its interaction with iASPP C- terminal. The above steps may be repeated and re-repeated as necessary.

[00151] Molecular structures, which may be used in the present invention, will usually be compounds under development for pharmaceutical use or pharmacophore structures representative of a molecular framework that carries the essential features responsible for a drug's biological activity. Generally such compounds will be organic molecules, which are typically from about 100 to 2000 Da, more preferably from about 100 to 1000 Da in molecular weight. Such compounds include peptides and derivatives thereof, anti-cancer agents, and the like. In principle, any compound under development in the field of pharmacy can be used in the present invention in order to facilitate its development or to allow further rational drug design to improve its properties.

(H) Fragment linking and growing.

[00152] The provision of the crystal structures of the invention will also allow the development of compounds which interact with the Pl peptide binding region of iASPP based on a fragment linking or fragment growing approach.

[00153] For example, the binding of one or more molecular fragments can be determined in the protein binding pocket by X-ray crystallography. Molecular fragments are typically compounds with a molecular weight between 100 and 200 Da (Carr et al, 2002). This can then provide a starting point for medicinal chemistry to optimize the interactions using a structure- based approach. The fragments can be combined onto a template or used as the starting point for 'growing out' an inhibitor into other pockets of the protein (Blundell et al, 2002). The fragments can be positioned in the binding pocket of the iASPP C-terminal and then 'grown' to fill the space available, exploring the electrostatic, van der Waals or hydrogen-bonding interactions that are involved in molecular recognition. The potency of the original weakly binding fragment thus can be rapidly improved using iterative structure-based chemical synthesis.

[00154] At one or more stages in the fragment growing approach, the compound may be synthesized and tested in a biological system for its activity. This can be used to guide the further growing out of the fragment. [00155] Where two fragment-binding regions are identified, a linked fragment approach may be based upon attempting to link the two fragments directly, or growing one or both fragments in the manner described above in order to obtain a larger, linked structure, which may have the desired properties.

[00156] Where the binding site of two or more ligands are determined they may be connected to form a potential lead compound that can be further refined using e.g. the iterative technique of Greer et al. For a virtual linked- fragment approach see Verlinde et al., /. of Computer-Aided Molecular Design, 6, (1992), 131-147, and for NMR and X-ray approaches see Shuker et al., Science, 21 A, (1996), 1531-1534 and Stout et al., Structure, 6, (1998), 839-848. The use of these approaches to design iASPP C-terminal inhibitors is made possible by the determination of the iASPP C-terminal structure.

[00157] Accordingly, the present invention also provides a computer-based method of rational drug design comprising:

[00158] providing a structure comprising a three-dimensional representation of the C- terminal region of an iASPP structure, said structure being as set out in Table 1 optionally varied by a root mean square deviation of not more than 1.2 A, or selected coordinates thereof;

[00159] providing the structures of a plurality of molecular fragments;

[00160] fitting the structure of at least two of the molecular fragments to the selected coordinates, wherein said fitting includes determining at least one interaction between an atom of said iASPP structure and a molecular fragment; and

[00161] assembling the fitted molecular fragments into a single molecule to form a single molecular structure.

(Ui) Molecular structures and fragments.

[00162] In one aspect of the invention, molecular structures and fragments that are used in soaking, co-crystallization or computer-based methods of design may be based upon the detailed analysis provided herein on the Pl peptide and its interactions with the C-terminal region of iASPP.

[00163] Thus the methods of the invention may utilize said molecular structures or fragments that comprise at least two atoms that are located in the same relative spatial orientation to each other and are of the same elements as a corresponding number of atoms found in any of the amino acids selected from Table 4.

[00164] In particular, the atoms may be selected from the atoms of Table 5. As noted above, Table 5 identifies atoms which interact with iASPP via hydrogen bonds or hydrophobic interactions, as well as with water molecules associated with iASPP. The former two types of interactions are particularly preferred, in particular atoms that contact iASPP residues via hydrogen bonds are of interest.

[00165] For example, molecular structures or fragments may be based upon peptide variants of the Pl peptide, including the peptides of SEQ ID NOs:3-5, or fragments thereof. Mimetics comprising a modified amide bond but retaining two or more side-chain residues which contain atoms of the same element and in the same relative orientation in space as atoms of Table 4 residues, and preferably of Table 5 atoms are also contemplated.

[00166] Thus, one aspect the invention includes a process comprising selecting two atoms of residues of Table 4, preferably atoms of Table 5; calculating the distance between said atoms; screening a library of compound structures to identify compounds comprising two atoms of the same elements as the selected atoms that are an equivalent distance apart; and fitting said compounds to the C-terminal region of an iASPP structure, said structure being as set out in

Table 1 optionally varied by a root mean square deviation of not more than 1.2 A, or selected coordinates thereof.

[00167] The above process may be refined by screening compound structures for three or more atoms in the same spatial orientation as the same three elements of the Pl peptide. Such a process may comprise selecting two atoms of residues of Table 4, preferably atoms of Table 5; calculating the distance between said atoms; screening a library of compound structures to identify compounds comprising two atoms of the same elements as the selected atoms that are an equivalent distance apart; further selecting those compounds which comprise a third atom of an element which is located in an equivalent relative spatial orientation to the first two atoms as an atom of the same element in a Table 4 residue; and fitting said compound to the C-terminal region of an iASPP structure, said structure being as set out in Table 1 optionally varied by a root mean square deviation of not more than 1.2 A, or selected coordinates thereof. (iv) Analysis and modification of compounds bound to iASPP

[00168] In any event, where a molecular structure or fragment is fitted to the C-terminal region of iASPP, e.g. by a method of the invention described herein, it will also be possible to modify the structure to modify, e.g. decrease or increase, its interaction with iASPP.

[00169] The present invention also provides a method which comprises:

[00170] fitting a starting compound to a structure comprising a three-dimensional representation of the C-terminal region of an iASPP structure, said structure being as set out in

Table 1 optionally varied by a root mean square deviation of not more than 1.2 A, or selected coordinates thereof;

[00171] determining at least one interaction between an atom of said iASPP structure and said compound; and

[00172] modifying the structure of the compound so as to alter the interaction between it and the iASPP structure.

[00173] It would be understood by those of skill in the art that modification of the structure will usually occur in silico, allowing predictions to be made as to how the modified structure interacts with the iASPP C-terminal.

[00174] Greer et al. ( /. of Medicinal Chemistry, Vol. 37, (1994), 1035-1054) describes an iterative approach to ligand design based on repeated sequences of computer modeling, protein- ligand complex formation and X-ray crystallographic or NMR spectroscopic analysis. Thus novel thymidylate synthase inhibitor series were designed de novo by Greer et al., and iASPP C- terminal ligands may also be designed or modified in the this way. More specifically, using e.g. GRID on the solved structure of iASPP C-terminal, a ligand for iASPP C-terminal may be designed that complements the functionalities of the iASPP C-terminal binding sites. Alternatively a ligand for iASPP C-terminal may be modified such that it complements the functionalities of the iASPP C-terminal binding sites better or less well. The ligand can then be synthesised, formed into a complex with iASPP C-terminal, and the complex then analyzed by X-ray crystallography to identify the actual position of the bound ligand. The structure and/or functional groups of the ligand can then be adjusted, if necessary, in view of the results of the X- ray analysis, and the synthesis and analysis sequence repeated until an optimized ligand is obtained. Related approaches to structure -based drug design are also discussed in Bohacek et al, Medicinal Research Reviews, Vol.16, (1996), 3-50. Design of a compound with alternative iASPP C-terminal properties using structure based drug design may also take into account the requirements for high affinity to a second, target protein. Gschwend et al, (Bioorganic & Medicinal Chemistry Letters, VoI 9, (1999), 307-312) and Bayley et al, (Proteins: Structure, Function and Genetics, VoI 29, (1997) 29-67) describe approaches where structure based drug design is used to reduce affinity to one protein whilst maintaining affinity for a target protein.

[00175] Modification will be those conventional in the art known to the skilled medicinal chemist, and will include, for example, substitutions or removal of groups containing residues which interact with the amino acid side chain groups of an iASPP C-terminal structure described herein. For example, the replacements may include the addition or removal of groups in order to decrease or increase the charge of a group in a test compound, the replacement of a group to increase or decrease the size of the group in a test compound, the replacement of a charge group with a group of the opposite charge, or the replacement of a hydrophobic group with a hydrophilic group or vice versa. It will be understood that these are only examples of the type of substitutions considered by medicinal chemists in the development of new pharmaceutical compounds and other modifications may be made, depending upon the nature of the starting compound and its activity.

[00176] Where a potential modified compound has been developed by fitting a starting compound to the iASPP C-terminal structure disclosed herein and predicting from this a modified compound with an altered property, the invention further includes the step of synthesizing the modified compound and testing it in a in vivo or in vitro biological system in order to determine its activity.

[00177] The above-described processes of the invention may be iterated in that the modified compound may itself be the basis for further compound design.

[00178] The various computer-based methods of analysis may be performed using computer systems such as those described in the preceding section. Generally, the computer systems used will be configured to display or transmit a model of the structure of Table 1, or selected coordinates thereof and a molecular structure so as to indicate one or more interactions between the two. A variety of formats of display are known in the art and may be selected by a person of ordinary skill in the art dependent upon a variety of factors including, for example, the nature of the interactions being determined. [00179] Once a molecular structure has been obtained via the computer-based methods of analysis of the present invention, a compound may be obtained or synthesized for further testing or experimental verification.

[00180] Thus in one aspect the invention provides, following the computer-based methods of the invention described above, the steps of:

[00181] obtaining or synthesizing a compound which has said molecular structure; and

[00182] contacting said compound with an iASPP protein to determine the ability of said compound to interact with the iASPP.

[00183] In another aspect the invention provides, following the computer-based methods disclosed herein, the steps of:

[00184] obtaining or synthesizing a compound which has said molecular structure;

[00185] forming a complex of an iASPP C-terminal protein and said compound; and

[00186] analyzing said complex by X-ray crystallography to determine the ability of said compound to interact with the iASPP C-terminal region.

[00187] In a further aspect the invention provides, following the computer-based methods disclosed herein, the steps of:

[00188] obtaining or synthesizing a compound which has said molecular structure; and

[00189] determining or predicting how said compound is bound to said iASPP C-terminal structure; and

[00190] modifying the compound structure so as to alter the interaction between it and the iASPP C-terminal region.

[00191] For the avoidance of doubt, the term "modifying" is used as defined above.

[00192] The ability of a compound to interact with an iASPP protein or C-terminal region thereof may be determined experimentally. For example, the accompanying example demonstrates that iASPP binds to peptide Pl in vitro. A compound obtained or selected according to the present invention may be used in the method described in place of the Pl peptide or in competition with the peptide to determine if the compound can bind to the Pl peptide-binding region with a greater or similar affinity to Pl.

[00193] The compound may also be tested in a culture of mammalian, e.g., human, cells to determine whether or not it enhances p53-mediated apoptosis. Such a method will generally comprise the steps of:

[00194] bringing the compound into contact with a mammalian cell culture; and

[00195] comparing the rate of apoptosis in the presence of the compound compared to its absence.

[00196] The cells may be a tumor cell line. In one embodiment the tumor cell line is be a wild-type p53 tumor cell line.

(v) Obtaining and analysing crystal complexes.

[00197] In one approach, the structure of a compound bound to an iASPP C-terminal region may be determined by experiment. This will provide a starting point in the analysis of the compound bound to iASPP, thus providing those of skill in the art with a detailed insight as to how that particular compound interacts with iASPP C-terminal region.

[00198] Many of the techniques and approaches to structure-based drug design described above rely at some stage on X-ray analysis to identify the binding position of a ligand in a ligand-protein complex. A common way of doing this is to perform X-ray crystallography on the complex, produce a difference Fourier electron density map, and associate a particular pattern of electron density with the ligand. However, in order to produce the map (as explained e.g. by Blundell et al., in Protein Crystallography, Academic Press, New York, London and San Francisco, (1976)), it is necessary to know beforehand the protein 3D structure (or at least the protein structure factors). Therefore, determination of the iASPP C-terminal structure also allows difference Fourier electron density maps of iASPP C-terminal-compound complexes to be produced, determination of the binding position of the drug and hence may greatly assist the process of rational drug design.

[00199] Accordingly, the invention provides a method for determining the structure of a compound bound to an iASPP C-terminal region, said method comprising:

[00200] providing a crystal of an iASPP C-terminal region; [00201] soaking the crystal with the compound to form a complex; and

[00202] determining the structure of the complex by employing the data of Table 1 optionally varied by a root mean square deviation of not more than 1.2 A, or selected coordinates thereof.

[00203] Alternatively, the iASPP C-terminal and compound may be co-crystallized. Thus the invention provides a method for determining the structure of a compound bound to an iASPP C- terminal region, said method comprising:

[00204] mixing iASPP C-terminal protein with the compound;

[00205] crystallizing a iASPP C-terminal protein-compound complex; and

[00206] determining the structure of the complex by employing the data of Table 1 optionally varied by a root mean square deviation of not more than 1.2 A, or selected coordinates thereof.

[00207] In both approaches, the iASPP C-terminal protein may be that of SEQ ID NO: 1 (which corresponds to residues 608-828 of full-length iASPP, Genbank Accession No. NP_006654) or may be an iASPP C-terminal region protein which is a fragment of SEQ ID NO:1, or is a longer version of iASPP, e.g. truncated nearer the N-terminal region.

[00208] In some aspects, a mixture of compounds may be used for soaking or co- crystallization. For example, a diverse mixture of compounds representing pharmacophore structures may be employed to determine which member or members of the mixture bind to iASPP. Where two or more components of the mixture are found to bind, these may be used as the basis for fragment linking analysis such as that described below.

[00209] The analysis of the co-crystal structures obtained by soaking or co-crystallization may employ (i) X-ray crystallographic diffraction data from the complex and (ii) a three-dimensional structure of iASPP C-terminal, or at least selected coordinates thereof, to generate a difference Fourier electron density map of the complex, the three-dimensional structure being defined by atomic coordinate data of Table 1 or selected coordinates thereof. The difference Fourier electron density map may then be analyzed. [00210] Therefore, such complexes can be crystallized and analyzed using X-ray diffraction methods, e.g. according to the approach described by Greer et al., /. of Medicinal Chemistry, Vol. 37, (1994), 1035-1054, and difference Fourier electron density maps can be calculated based on X-ray diffraction patterns of soaked or co-crystallized protein and the solved structure of uncomplexed protein. These maps can then be analyzed e.g. to determine whether and where a particular compound binds to the iASPP C-terminal region and/or changes the conformation of this region.

[00211] Electron density maps can be calculated using programs such as those from the CCP4 computing package (Collaborative Computational Project 4. The CCP4 Suite: Programs for Protein Crystallography, Acta Crystallographica, D50, (1994), 760-763.). For map visualization and model building programs such as "O" (Jones et al., Acta Crystallographica, A47, (1991), 110-119) can be used.

[00212] In addition, iASPP C-terminal variants may be crystallized in co-complex with known iASPP C-terminal substrates or inhibitors or novel compounds, such as peptide Pl. The crystal structures of a series of such complexes may then be solved by molecular replacement and compared with that of the iASPP C-terminal structure of Table 1 or selected coordinates thereof. This information provides an additional tool for determining the most efficient binding interactions, for example, increased hydrophobic interactions, between the iASPP C-terminal region and a chemical entity or compound.

[00213] All of the complexes referred to above may be studied using well-known X-ray diffraction techniques and may be refined against 1.5 to 3.5 A resolution X-ray data to an R value of about 0.30 or less using computer software, such as CNX (Brunger et al., Current Opinion in Structural Biology, Vol. 8, Issue 5, October 1998, 606-611, and commercially available from Accelrys, San Diego, CA), and as described by Blundell et al, (1976) and Methods in Enzymology, vol. 114 & 115, H. W. Wyckoff et al., eds., Academic Press (1985).

[00214] This information may thus be used to optimize molecular structures that bind to the iASPP C-terminal region, and more importantly, to design and synthesize novel classes of such structures.

[00215] Information obtained on complexes of iASPP and a compound may be used to initiate or optimize the computer-based methods described herein. (vi) Compounds of the invention.

[00216] A therapy based on the inhibition of iASPP binding p53 - or either of p63 and p73 - could be a target for cancer therapy (Bergamaschi et al., 2003; Trigiante and Lu, 2006). SH3 domains have proved to be difficult targets for drug design as most SH3 ligand interactions show high cross-reactivity with several SH3 domains (Feng et al., 1994; Feng et al., 1995; Knudsen et al., 1995; Lim et al., 1994; Wu et al., 1995). The uniqueness of the p53 binding site, made up of contributions from both the SH3 domain and the third ankyrin may provide a draggable target. Protein-protein interactions observed in the iASPP crystal structure provide a starting point for structure based drag design. For example, residues Arg 618 of SEQ ID NO: 8 and Lys 619 of SEQ ID NO: 8, from a neighbouring molecule in the crystal lattice, interact with residues unique to iASPP (not present in ASPPl or ASPP2) such as Thr 722 of SEQ ID NO: 8 and Leu 724 of SEQ ID NO: 8. A small molecule that mimicked this binding mode could specifically block the p53 binding site of iASPP and as such provide a novel strategy for inhibition of this oncoprotein.

[00217] Thus where a compound has been developed by fitting a starting compound to the iASPP C-terminal structure of the invention and obtaining from this a modified compound the invention further includes the step of synthesizing the modified compound and testing it in an in vivo or in vitro biological system in order to determine its activity, e.g. in binding to iASPP or modifying apoptosis in a cell.

[00218] In another aspect, the invention includes a compound, which is identified by the methods of the invention described above.

[00219] In another aspect, the invention includes an isolated compound having a spatial arrangement of atoms to bind to iASPP in a manner that interferes with iASPP binding to an iASPP binding partner selected from the group consisting of p53, p63 and p73, wherein the compound interacts with an iASPP polypeptide defined by at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60 , at least 65, at least 75 or more structural coordinates identified in Table 1, wherein the relative atomic positions of the structure identified in Table 1 are varied within a root mean square deviation of less than 1.2A. In some embodiments, the isolated compound interacts with the iASPP in a region of iASPP defined by at least ten coordinates of atoms from at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 or at least 12 different amino acids of Table 3, wherein the relative atomic positions are varied within a root mean square deviation of less than 1.2 A.

[00220] In some embodiments, the isolated compound interacts with the iASPP in a region of iASPP defined by at least 10 coordinates of atoms from at least 10 amino acids of Table 3, wherein the relative atomic positions are varied within a root mean square deviation of less than 1.2 A. In one exemplary embodiment, the at least ten amino acids of iASPP are selected from the group consisting of T722, L724, W767, E772, D775, E776, E795, D797. W798, Y809, P811, N813 and Y814. 00221] In some embodiments, the compound is selected from the group consisting of a small molecule, an antibody or an antigen-binding fragment thereof, a nucleic acid molecule and a polypeptide.

[00222] In some embodiments, the isolated compound has a structure selected from the group consisting of

[00223] (a) a compound comprising a sequence of eight amino acids X₁-X₂-X₃-X₄-X₅-X₆-X₇- X₈ (SEQ ID NO: 6), wherein X₁ is selected from Serine or Threonine or conservative substitution threof; X₂ is proline or conservative substitution thereof; X₃, X₄, X₆, and X₇ are each independently selected from Arginine and Lysine; X₅ is Alanine, Serine, Valine, Glycine or conservative substitutions thereof; and X₈ is Alanine, Valine, Glycine or conservative substitutions thereof;

[00224] (b) a compound with the structure of (a), with the proviso that one or more of the X₁ X₂, X₃, X₄, X₅, X₆, X₇, X8 amino acids are replaced with an unnatural amino acid;

[00225] (c) a compound with the structure of (a) or (b), with the proviso that one or more amide bonds is replaced with an ester or alkyl bond.

[00226] In some embodiments, the amino acid sequence set forth in SEQ ID NO: 6 includes the amino acids set forth below in Table 6 below.

[00227] Any amino acid identified as X₁ in Table 6 above can be paired with any amino acid identified as X₂, X₃, X₄, X₅, X₆, X₇ or X₈ in Table 6, for a total of 23,382 individual peptides. All individual peptides are contemplated as aspects of the invention.

[00228] In another embodiment, the compound is a peptide comprising a sequence of eight amino acids X₁-X₂-X₃-X₄-X₅- X₆-X₇-X₈ (SEQ ID NO: 6), wherein

[00229] X₁ is selected from Serine or Threonine or a conservative substitution thereof;

[00230] X₂ is Proline or a conservative substitution thereof;

[00231] X₃, X₄, X₆, and X₇ are each independently selected from Arginine and Lysine or a conservative substitution thereof;

[00232] X₅ is Alanine, Serine, Valine or Glycine or a conservative substitution thereof; and

[00233] X₈ is Alanine, Valine or Glycine or a conservative substitution thereof,

[00234] wherein the compound inhibits binding between iASPP and an iASPP binding partner selected from the group consisting of p53, p63 and p73.

[00235] In some embodiments, the peptides described herein are from about 8 amino acid residues in length to about 100 amino acid residues in length. Of course it is contemplated that longer or indeed shorter peptides also may prove useful. Thus, peptides of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 and 100 amino acids in length are contemplated. In some embodiments, a peptide described herein comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or more amino acids from iASPP added to its N-terminus. For example, the amino acid at position 615 of SEQ ID NO: 8 could be added to a peptide described herein (e.g., SPRKARRA (SEQ ID NO: 2 or residues 616-623 of SEQ ID NO: 8), SPRKSRRA (SEQ ID NO: 3) or SPRRARRA (SEQ ID NO: 4)) if the addition of 1 amino acid to the N-terminus of a peptide sequence described herein is desired. Similarly, the amino acids at positions 611-615, 606-615, 601-615, or 596-615 of SEQ ID NO: 8 could be added to a peptide described herein (e.g., SPRKARRA (SEQ ID NO: 2 or residues 616-623 of SEQ ID NO: 8), SPRKSRRA (SEQ ID NO: 3) or SPRRARRA (SEQ ID NO: 4)) if the addition of 5, 10, 15, or 20 amino acids, respectively, to the N-terminus of the peptide is desired.

[00236] In some embodiments, a peptide described herein comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or more amino acids added to its C-terminus. For example, the amino acid at position 624 of SEQ ID NO: 8 could be added to a peptide described herein (e.g., SPRKARRA (SEQ ID NO: 2 or residues 616-623 of SEQ ID NO: 8), SPRKSRRA (SEQ ID NO: 3) or SPRRARRA (SEQ ID NO: 4)) if the addition of 1 amino acid to the C-terminus of a peptide sequence described herein is desired. Similarly, the amino acids at positions 624-628, 624-633, 624-638 or 624-643 of SEQ ID NO: 8 could be added to a peptide described herein (e.g., SPRKARRA (SEQ ID NO: 2 or residues 616-623 of SEQ ID NO: 8), SPRKSRRA (SEQ ID NO: 3) or SPRRARRA (SEQ ID NO: 4)) if the addition of 5, 10, 15, or 20 amino acids, respectively, to the C-terminus of the peptide is desired.

[00237] In some embodiments, the addition of amino acids to both the N- and C-termini of a peptide described herein is contemplated. For example, the amino acids at positions 615 and 624 of SEQ ID NO: 8 can be added to a peptide described herein (e.g., SPRKARRA (SEQ ID NO: 2 or residues 616-623 of SEQ ID NO: 8), SPRKSRRA (SEQ ID NO: 3) or SPRRARRA (SEQ ID NO: 4)) if the addition of 1 amino acid to both the N- and C-termini of the peptide is desired. Similarly, the amino acids at positions 611-615 and 624-628; 606-615 and 624-633; 601-615 and 624-638; or 595-615and 624-643 of SEQ ID NO: 8 can be added to a peptide described herein (e.g., SPRKARRA (SEQ ID NO: 2 or residues 616-623 of SEQ ID NO: 8), SPRKSRRA (SEQ ID NO: 3) or SPRRARRA (SEQ ID NO: 4)) if the addition of 5, 10, 15, or 20 amino acids, respectively, to the N- and C-termini of the peptide is desired.

[00238] In some embodiments, the peptide consists of an amino acid sequence at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at 81%, at least 82%, at least 83%, at least 84%, at least 85%, least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to a peptide/polypeptide described herein.

[00239] In some embodiments, the compound is a peptide consisting of an amino acid sequence at least 50%, at least 62.5%, at least 75%, at least 87.5% ,or more identical to SEQ ID NO: 2, wherein the peptide retains the ability to interact with (e.g., bind to) iASPP. In some embodiments, the compound is a peptide consisting of the amino acid sequence SPRKARRA (SEQ ID NO: T). In some embodiments, the compound is a peptide consisting of an amino acid sequence at least 50%, at least 62.5%, at least 75%, at least 87.5% , or more identical to SEQ ID NO: 3, wherein the peptide retains the ability to interact with (e.g., bind to) iASPP. In some embodiments, the compound is a peptide consisting of the amino acid sequence SPRKSRRA (SEQ ID NO: 3). In some embodiments, the compound is a peptide consisting of an amino acid sequence at least 50%, at least 62.5%, at least 75%, at least 87.5% ,or more identical to SEQ ID NO: 4, wherein the peptide retains the ability to interact with (bind to) iASPP. In some embodiments, the compound is a peptide consisting of the amino acid sequence SPRRARRA (SEQ ID NO: 4). In some embodiments, the compound is a peptide consisting of an amino acid sequence at least 50%, at least 62.5%, at least 75%, at least 87.5% ,or more identical to SEQ ID NO: 5, wherein the peptide retains the ability to interact with (e.g., bind to) iASPP. In some embodiments, the compound is a peptide consisting of the amino acid sequence SPRRSRRA (SEQ ID NO: 5).

[00240] Compounds of the invention include the peptide of SEQ ID NO: 2 and variants thereof. In some embodiments, variants of this peptide include those with one or more, e.g. 1, 2, 3, 4, 5 or more amino acid alterations. In some embodiments, the residues of which may be altered, and the alterations contemplated, may be selected from, for example, those set out in Table 2 above. For example, the alteration of position 5 of peptide Pl (iASPP residue 620 of SEQ ID NO: 8) to Ser (serine) provides the peptide of SEQ ID NO:3. The alteration of position 4 of the peptide Pl (iASPP residue 619 of SEQ ID NO: 8) to Arg (arginine) provides the peptide of SEQ ID NO:4. Alteration of both residues in the same peptide provides the peptide of SEQ ID NO:5. Other alterations, both individually and combinations thereof, may be made based on the alterations set out in Table 2 above.

[00241] In some embodiments, the variant peptides may differ in amino acid sequence by one or more substitutions, additions, deletions, truncations which may be present in any combination. In one aspect, the modification is a conservative substitution. The term "conservative substitution" as used herein denotes the replacement of an amino acid residue by another, biologically similar residue with respect to hydrophobicity, hydrophilicity, cationic charge, anionic charge, shape, polarity and the like. Examples of conservative substitutions include the substitution of one hydrophobic residue such as isoleucine, valine, leucine, alanine, cysteine, glycine, phenylalanine, proline, tryptophan, tyrosine, norleucine or methionine for another, or the substitution of one polar residue for another, such as the substitution of arginine for lysine, glutamic acid for aspartic acid, or glutamine for asparagine, and the like. Neutral hydrophilic amino acids which can be substituted for one another include asparagine, glutamine, serine and threonine. The term "conservative substitution" also includes the use of a substituted or modified amino acid in place of an unsubstituted parent amino acid provided that antibodies raised to the substituted polypeptide also immunoreact with the unsubstituted polypeptide. By "substituted" or "modified" the present invention includes those amino acids that have been altered or modified from naturally occurring amino acids.

[00242] As such, it should be understood that in the context of the present invention, a conservative substitution is recognized in the art as a substitution of one amino acid for another amino acid that has similar properties. Exemplary conservative substitutions are set out in the Table A below:

[00243] Alternatively, conservative amino acids can be grouped as described in Lehninger, [Biochemistry, Second Edition; Worth Publishers, Inc. NY:NY (1975), pp.71-77] as set out in Table B, immediately below.

[00244] As still an another alternative, exemplary conservative substitutions are set out in Table 9, immediately below.

[00245] Any conservative substitution variant of SPRKARRA (SEQ ID NO: 2) is contemplated to be a useful peptide of the present invention as long as such a variant retains its property of being an inhibitor of binding of iASPP to an iASPP binding partner. Additionally it is contemplated that non-conservative substitution variants of these peptides also may be designed that may prove to be more efficient inhibitors of p53/p63/p73 binding to iASPP than the original SPRKARRA (SEQ ID NO: 2) described herein.

[00246] In some embodiments, the substituted amino acids are unnatural amino acids. For example, this includes D-amino acids of the amino acids of peptide Pl (SEQ ID NO: 2) or the alterations discussed above. Other unnatural amino acids include ornithine, diaminobutyric acid ornithine, norleucine ornithine, pyriylalanine, thienylalanine, naphthylalanine, phenylglycine, alpha and alpha-disubstituted amino acids, N-alkyl amino acids, lactic acid, halide derivatives of natural amino acids, such as trifluorotyrosine, p-Cl-phenylalanine, p-Br-phenylalanine, p-I- phenylalanine, L-allyl-glycine, b-alanine, L-a-amino butyric acid, L-g-amino butyric acid, L-a- amino isobutyric acid, L-e-amino caproic acid, 7-amino heptanoic acid, L methionine sulfone, L-norleucine, L-norvaline, p-nitro-L-phenylalanine, L-hydroxyproline, L-thioproline, methyl derivatives of phenylalanine - such as 1-methyl-Phe, pentamethyl-Phe, L-Phe(4-amino), L- Tyr(methyl), L-Phe(4-isopropyl), L-Tic(l,2,3,4-tetrahydroisoquinoline-3-carboxyl acid), L- diaminopropionic acid and L-Phe(4-benzyl).

[00247] Also contemplated are derivatives of SEQ ID NO: 2. Derivatives of such peptides form a further aspect of the invention. By "derivative" it is meant one of more of (a) addition of one or more polyalkyleneglycol (e.g. polyethylene glycol (PEG)) moities to the peptide, e.g. to the N- or C-terminal of the peptide, (b) modification of an amino acid side-chain residue by, for example, esterification of an acid group (e.g. with a Cl-6 alkyl moiety) or the modification of an amine group to a mono- or di-Cl-6alkyl amine, (c) replacement of one or more amide bonds by ester or alkyl backbone bonds.

[00248] Peptides can be modified by a variety of chemical techniques to produce derivatives having essentially the same activity as the unmodified peptides, and optionally having other desirable properties. For example, carboxylic acid groups of the protein, whether carboxyl- terminal or side chain, may be provided in the form of a salt of a pharmaceutically-acceptable cation or esterified to form a C₁-C₁₆ ester, or converted to an amide of formula NR₁R₂ wherein R₁ and R₂ are each independently H or C₁-C₁₆ alkyl, or combined to form a heterocyclic ring, such as a 5- or 6-membered ring. Amino groups of the peptide, whether amino-terminal or side chain, may be in the form of a pharmaceutically-acceptable acid addition salt, such as the HCl, HBr, acetic, benzoic, toluene sulfonic, maleic, tartaric and other organic salts, or may be modified to C₁-C₁₆ alkyl or dialkyl amino or further converted to an amide. Peptides can also be modified by methylation. A peptide is considered methylated when methyl group is added at one or more nucleophilic side chains. Methylation in the proteins negates the negative charge on it and increase the hydrophobicity of the protein.

[00249] Hydroxyl groups of the peptide side chains may be converted to C₁-C₁₆ alkoxy or to a C₁-C₁₆ ester using well-recognized techniques. Phenyl and phenolic rings of the peptide side chains may be substituted with one or more halogen atoms, such as fluorine, chlorine, bromine or iodine, or with C₁-C₁₆ alkyl, C₁-C₁₆ alkoxy, carboxylic acids and esters thereof, or amides of such carboxylic acids. Methylene groups of the peptide side chains can be extended to homologous C₂-C₄ alkylenes. Thiols can be protected with any one of a number of well- recognized protecting groups, such as acetamide groups. Those skilled in the art will also recognize methods for introducing cyclic structures into the peptides of this invention to select and provide conformational constraints to the structure that result in enhanced stability. For example, a carboxyl-terminal or amino-terminal cysteine residue can be added to the peptide, so that when oxidized the peptide will contain a disulfide bond, generating a cyclic peptide. Other peptide cyclizing methods include the formation of thioethers and carboxyl- and amino-terminal amides and esters.

[00250] Peptidomimetic and organomimetic embodiments are also contemplated, whereby the three-dimensional arrangement of the chemical constituents of such peptido- and organomimetics mimic the three-dimensional arrangement of the peptide backbone and component amino acid side chains, resulting in such peptido- and organomimetics of the proteins of this disclosure having measurable or enhanced ability to bind an antibody. For computer modeling applications, a pharmacophore is an idealized, three-dimensional definition of the structural requirements for biological activity. Peptido- and organomimetics can be designed to fit each pharmacophore with current computer modeling software (using computer assisted drug design or CADD). See Walters, "Computer-Assisted Modeling of Drugs", in Klegerman & Groves, eds., 1993, Pharmaceutical Biotechnology, Interpharm Press: Buffalo Grove, IL, pp. 165-174 and Principles of Pharmacology Munson (ed.) 1995, Ch. 102, for descriptions of techniques used in CADD. Also included within the scope of the disclosure are mimetics prepared using such techniques.

[00251] Peptides of the invention defined above may further be cyclisized. Methods are well known in the art for introducing cyclic structures into the peptides of the present invention to select and provide conformational constraints to the structure that result in enhanced stability. For example, a C- or N-terminal cysteine can be added to the peptide, so that when oxidized the peptide will contain a disulfide I bond, generating a cyclic peptide. Other peptide cyclizing methods include the formation of thioethers and carboxyl- and amino- terminal amides and esters. A number of synthetic techniques have been developed to generate synthetic circular peptides (Tarn and Lu, Protein ScL, 7(7) 1583-1592 (1998); Romanovskis and Spatola, J. Pept. Res., 52(5) 356-374 (1998); Camarero and Muir, J. Amer. Chem. Soc, 121 5597-5598 (1999); Valero, et al., J. Pept. Res., 53(1) 56-67 (1999)). The role of cyclising peptides is two-fold: a) to reduce hydrolysis in vivo and b) to thermodynamically destabilize the unfolded state and promote secondary structure formation.

[00252] Preferably, the peptides described herein are non-hydrolyzable. To provide such peptides, one may select peptides from a library non-hydrolyzable peptides, such as peptides containing one or more D-amino acids or peptides containing one or more non-hydrolyzable peptide bonds linking amino acids. Alternatively, one can select peptides which are optimal for inhibiting iASPP binding to an iASPP binding partner and then modify such peptides as necessary to reduce the potential for hydrolysis by proteases. For example, to determine the susceptibility to proteolytic cleavage, peptides may be labeled and incubated with cell extracts or purified proteases and then isolated to determine which peptide bonds are susceptible to proteolysis, e.g., by sequencing peptides and proteolytic fragments. Alternatively, potentially susceptible peptide bonds can be identified by comparing the amino acid sequence of the inhibitory peptides of the present invention with the known cleavage site specificity of a panel of proteases. Based on the results of such assays, individual peptide bonds which are susceptible to proteolysis can be replaced with non-hydrolyzable peptide bonds by in vitro synthesis of the peptide.

[00253] Many non-hydrolyzable peptide bonds are known in the art, along with procedures for synthesis of peptides containing such bonds. Non-hydrolyzable bonds include -[CH₂NH]- reduced amide peptide bonds, -[COCH₂ ]— ketomethylene peptide bonds, -[CH(CN)NH]- (cyanomethylene)amino peptide bonds, -[CH₂ CH(OH)]- hydroxyethylene peptide bonds, — [CH₂O]- peptide bonds, and -[CH₂ S]- thiomethylene peptide bonds (see e.g., U.S. Patent 6,172,043).

[00254] Furthermore, nonpeptide analogs of peptides which provide a stabilized structure or lessened biodegradation, are also contemplated. Peptide mimetic analogs can be prepared based on a selected inhibitory peptide by replacement of one or more residues by nonpeptide moieties. Preferably, the nonpeptide moieties permit the peptide to retain its natural confirmation, or stabilize a preferred, e.g., bioactive, confirmation. One example of methods for preparation of nonpeptide mimetic analogs from peptides is described in Nachman et al, Regul. Pept. 57:359- 370 (1995). Peptide as used herein embraces all of the foregoing.

[00255] In particular, it is anticipated that the peptides described herein can be conjugated to a reporter group, including, but not limited to a radiolabel, a fluorescent label, an enzyme (e.g., that catalyzes a colorimetric or fluorometric reaction), a substrate, a solid matrix, or a carrier (e.g., biotin or avidin). The invention accordingly provides a molecule comprising a peptide inhibitor of iASPP binding to an iASPP binding partner, wherein the molecule preferably further comprises a reporter group selected from the group consisting of a radiolabel, a fluorescent label, an enzyme, a substrate, a solid matrix, and a carrier. Such labels are well known to those of skill in the art, e.g., biotin labels are particularly contemplated. The use of such labels is well known to those of skill in the art and is described in, e.g., U.S. No. Patent 3,817,837; U.S. Patent No. 3,850,752; U.S. Patent No. 3,996,345 and U.S. Patent No. 4,277,437. Other labels that will be useful include but are not limited to radioactive labels, fluorescent labels and chemiluminescent labels. U.S. Patents concerning use of such labels include for example U.S. Patent No. 3,817,837; U.S. Patent No. 3,850,752; U.S. Patent No. 3,939,350 and U.S. Patent No. 3,996,345. Any of the peptides of the present invention may comprise one, two, or more of any of these labels.

[00256] The peptides described herein can be used as therapeutic compositions either alone or in combination with other therapeutic agents. For such therapeutic uses small molecules are generally preferred because the reduced size renders such peptides more accessible for uptake by the target.

[00257] Peptides of the invention may be prepared using methods known in the art. For example, peptides may be produced by chemical synthesis, eg. solid phase techniques and automated peptide synthesisers, or by recombinant means (using nucleic acids such as those described herein). For example, peptides may be synthesized using solid phase strategies on an automated multiple peptide synthesizer (Abimed AMS 422) using 9- fluorenylmethyloxycarbonyl (Fmoc) chemistry. The peptides can then be purified by reversed phase-HPLC and lyophilized. The peptide may be prepared by cleavage of a longer peptide. Thus, the peptide may be a fragment of the iASPP C-terminal sequence described herein.

[00258] Peptides may also be prepared by recombinant expression of the polynucleotides described herein. Peptides are expressed in suitable host cells and isolated using methods known in the art.

[00259] Fusion proteins comprising a peptide described herein, and a heterologous polypeptide, are a specifically contemplated. Nonlimiting examples of heterologous polypeptides which can be fused to polypeptides of interest include proteins with long circulating half-life, such as, but not limited to, immunoglobulin constant regions (e.g., Fc region); transthyretin (WO 2003/086444, the disclosure of which is incorporated herein by reference in its entirety), marker sequences that permit identification of the polypeptide of interest; sequences that facilitate purification of the polypeptide of interest; and sequences that promote formation of multimeric proteins. In some embodiments, a receptor fragment is fused to alkaline phosphatase (AP). Methods for making Fc or AP fusion constructs are found in WO 02/060950, the disclosure of which is incorporated herein in its entirety. [00260] Methods of making antibody fusion proteins are well known in the art. See, e.g., U.S. Patent No. 6,306,393, the disclosure of which is incorporated herein by reference in its entirety. In certain embodiments of the invention, fusion proteins are produced which may include a flexible linker, which connects the chimeric scFv antibody to the heterologous protein moiety. Appropriate linker sequences are those that do not affect the ability of the resulting fusion protein to be recognized and bind the epitope specifically bound by the V domain of the protein (see, e.g., WO 98/25965, the disclosure of which is incorporated herein by reference in its entirety).

[00261] For further discussions of fusion proteins see, for example, WO 95/04076, published 9 February 1995; US. Patent 5,629, 172 issued 13 May 1997; WO 94/23040, published 13 October 1994; Flaschel et al., Biotech Adv., 11:31-78 (1993); European patent application 207,044, published 30 December 1986; US Patent 5,322,930, issued 21 June 1994; European Patent 293,249, published 30 November 1988; US Patent 5,654, 176, issued 5 August 1997; WO 95/16044, published 15 June 1995; WO 94/02502, published 3 February 1994; and WO 92/13955, published 20 August 1992, the disclosures of which are incorporated herein by reference in their entireties.

(vii) Nucleic acids, vectors and host cells

[00262] In a further aspect, the invention provides a nucleic acid molecule comprising a nucleotide sequence that encodes a peptide consisting of an amino acid sequence at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at 81%, at least 82%, at least 83%, at least 84%, at least 85%, least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to a peptide/polypeptide described herein.

[00263] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes a peptide consisting of an amino acid sequence at least 50%, at least 62.5%, at least 75%, at least 87.5% ,or more identical to SEQ ID NO: 2, wherein the peptide retains the ability to interact with (e.g., bind to) iASPP. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes a peptide consisting of the amino acid sequence SPRKARRA (SEQ ID NO: T). In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes a peptide consisting of an amino acid sequence at least 50%, at least 62.5%, at least 75%, at least 87.5% ,or more identical to SEQ ID NO: 3, wherein the peptide retains the ability to interact with (e.g., bind to) iASPP. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes a peptide consisting of the amino acid sequence SPRKSRRA (SEQ ID NO: 3). In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding a peptide consisting of an amino acid sequence at least 50%, at least 62.5%, at least 75%, at least 87.5% ,or more identical to to SEQ ID NO: 4, wherein the peptide retains the ability to interact with (e.g., bind to) iASPP. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding a peptide consisting of the amino acid sequence SPRRARRA (SEQ ID NO: 4). In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes a peptide consisting of an amino acid sequence at least 50%, at least 62.5%, at least 75%, at least 87.5% ,or more identical to SEQ ID NO: 5, wherein the peptide retains the ability to interact with (e.g., bind to) iASPP. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes a peptide consisting of the amino acid sequence SPRRSRRA (SEQ ID NO: 5).

[00264] In one embodiment, the nucleic acid is operably linked to a promoter. In a further embodiment, the nucleic acid, optionally operably linked to a promoter, has a stop codon immediately adjacent to the final codon of the sequence encoding SEQ ID NO: 2, 3, 4 or 5.

[00265] Complementary molecules are useful as templates for synthesizing coding molecules, and for making stable double-stranded polynucleotides. Due to the well-known degeneracy of the universal genetic code, one can synthesize numerous polynucleotide sequences that encode each polypeptide of the present invention. All such polynucleotides are contemplated as part of the invention. Such polynucleotides are useful for recombinant expression of polypeptides of the invention in vivo or in vitro (e.g., for gene therapy).

[00266] This genus of polynucleotides embraces polynucleotides comprises nucleotide sequences that encode peptides/polypeptides with one or a few amino acid differences (additions, insertions, or deletions) relative to amino acid sequences specifically depicted herein. Such changes are easily introduced by performing site directed mutagenesis, for example.

[00267] Polynucleotides of the invention (and peptides/polypeptides encoded thereby) can be defined by molecules that hybridize under specified conditions to a polynucleotide sequence complementary to a sequence that encodes a construct of the invention.

[00268] Exemplary highly stringent hybridization conditions are as follows: hybridization at 65°C for at least 12 hours in a hybridization solution comprising 5X SSPE, 5X Denhardt's, 0.5% SDS, and 2 mg sonicated non homologous DNA per 100 ml of hybridization solution; washing twice for 10 minutes at room temperature in a wash solution comprising 2X SSPE and 0.1% SDS; followed by washing once for 15 minutes at 65°C with 2X SSPE and 0.1% SDS; followed by a final wash for 10 minutes at 65°C with 0.1X SSPE and 0.1% SDS. Moderate stringency washes can be achieved by washing with 0.5X SSPE instead of 0.1X SSPE in the final 10 minute wash at 65°C. Low stringency washes can be achieved by using IX SSPE for the 15 minute wash at 65°C, and omitting the final 10 minute wash. It is understood in the art that conditions of equivalent stringency can be achieved through variation of temperature and buffer, or salt concentration as described Ausubel, et al. (Eds.), Protocols in Molecular Biology, John Wiley & Sons (1994), pp. 6.0.3 to 6.4.10. Modifications in hybridization conditions can be empirically determined or precisely calculated based on the length and the percentage of guanosine/cytosine (GC) base pairing of the probe. The hybridization conditions can be calculated as described in Sambrook et al., (Eds.), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York (1989), pp. 9.47 to 9.51.

[00269] Preferred variants exhibit the activities of native molecules described herein, such as the ability to inhibit the binding between iASPP and an iASPP binding partner (i.e., p53, p63 or p73).

[00270] In a related embodiment, the invention provides vectors comprising a polynucleotide of the invention. Such vectors are useful, e.g., for amplifying the polynucleotides in host cells to create useful quantities thereof, and for expressing polypeptides of the invention using recombinant techniques. There is a significant amount of published literature with respect to expression vector construction and recombinant DNA techniques in general. Please see, Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory, Cold Spring Harbour, NY and references therein; Marston, F (1987) DNA Cloning Techniques: A Practical Approach VoI EU IRL Press, Oxford UK; DNA Cloning: F M Ausubel et al, Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994). In preferred embodiments, the vector is an expression vector wherein the polynucleotide of the invention is operatively linked to a polynucleotide comprising an expression control sequence. Autonomously replicating recombinant expression constructs such as plasmid and viral DNA vectors incorporating polynucleotides of the invention are specifically contemplated. Expression control DNA sequences include promoters, enhancers, and operators, and are generally selected based on the expression systems in which the expression construct is to be utilized. Preferred promoter and enhancer sequences are generally selected for the ability to increase gene expression, while operator sequences are generally selected for the ability to regulate gene expression. Expression vectors are useful for recombinant production of polypeptides of the invention. Expression constructs of the invention may also include sequences encoding one or more selectable markers that permit identification of host cells bearing the construct. Expression constructs may also include sequences that facilitate, and preferably promote, homologous recombination in a host cell. Preferred constructs of the invention also include sequences necessary for replication in a host cell.

[00271] In some embodiments, polynucleotides of the invention further comprise additional sequences to facilitate the gene therapy. In one embodiment, a "naked" transgene encoding a polypeptide of the invention (i.e., a transgene without a viral, liposomal, or other vector to facilitate transfection) is employed for gene therapy. In this embodiment, the polynucleotide of the invention preferably comprises a suitable promoter and/or enhancer sequence (e.g., cytomegalovirus promoter/enhancer [Lehner et al., J. Clin. Microbiol., 29:2494 2502 (1991); Boshart et al., Cell, 41:521 530 (1985)]; Rous sarcoma virus promoter [Davis et al., Hum. Gene Ther., 4:151 (1993)]; Tie promoter [Korhonen et al., Blood, 86(5): 1828 1835 (1995)]; or simian virus 40 promoter) for expression in the target mammalian cells, the promoter being operatively linked upstream (i.e., 5') of the polypeptide coding sequence. The polynucleotides of the invention also preferably further includes a suitable polyadenylation sequence (e.g., the SV40 or human growth hormone gene polyadenylation sequence) operably linked downstream (i.e., 3') of the polypeptide coding sequence. The polynucleotides of the invention also preferably comprise a nucleotide sequence encoding a secretory signal peptide fused in frame with the polypeptide sequence. The secretory signal peptide directs secretion of the polypeptide of the invention by the cells that express the polynucleotide, and is cleaved by the cell from the secreted polypeptide. The signal peptide sequence can be that of another secreted protein, or can be a completely synthetic signal sequence effective to direct secretion in cells of the mammalian subject.

[00272] The polynucleotide may further optionally comprise sequences whose only intended function is to facilitate large scale production of the vector, e.g., in bacteria, such as a bacterial origin of replication and a sequence encoding a selectable marker. However, in a preferred embodiment, such extraneous sequences are at least partially cleaved off prior to administration to humans according to methods of the invention. One can manufacture and administer such polynucleotides for gene therapy using procedures that have been described in the literature for other transgenes. See, e.g., Isner et al., Circulation, 91: 2687-2692 (1995); and Isner et al., Human Gene Therapy, 7: 989-1011 (1996); incorporated herein by reference in their entirety. [00273] Vectors also are useful for "gene therapy" treatment regimens, wherein a polynucleotide that encodes a polypeptide of the invention is introduced into a subject in need of treatment involving the inhibition of the binding of iASPP to an iASPP binding partner (i.e., p53, p63 or p73) in a form that causes cells in the subject to express the polypeptide of the invention in vivo. Gene therapy aspects that are described in WO 2007/006573, the disclosure of which is incorporated herein by reference, are also applicable herein.

[00274] Any suitable vector may be used to introduce a polynucleotide that encodes a polypeptide of the invention encoding one of the polypeptides of the invention, into the host. Exemplary vectors that have been described in the literature include replication deficient retroviral vectors, including but not limited to lentivirus vectors [Kim et al., J. Virol., 72(1): 811-816 (1998); Kingsman & Johnson, Scrip Magazine, October, 1998, pp. 43 46.]; adeno- associated viral (AAV) vectors [ U.S. Patent No. 5,474,935; U.S. Patent No. 5, 139,941; U.S. Patent No. 5,622,856; U.S. Patent No. 5,658,776; U.S. Patent No. 5,773,289; U.S. Patent No. 5,789,390; U.S. Patent No. 5,834,441; U.S. Patent No. 5,863,541; U.S. Patent No. 5,851,521; U.S. Patent No. 5,252,479; Gnatenko et al., J. Invest. Med., 45: 87 98 (1997)]; adenoviral (AV) vectors [See, e.g., U.S. Patent No. 5,792,453; U.S. Patent No. 5,824,544; U.S. Patent No. 5,707,618; U.S. Patent No. 5,693,509; U.S. Patent No. 5,670,488; U.S. Patent No. 5,585,362; Quantin et al., Proc. Natl. Acad. Sci. USA, 89: 2581 2584 (1992); Stratford Perricadet et al., J. Clin. Invest., 90: 626 630 (1992); and Rosenfeld et al., Cell, 68: 143 155 (1992)]; an adenoviral adenoassociated viral chimeric (see for example, U.S. Patent No. 5,856,152) or a vaccinia viral or a herpesviral (see for example, U.S. Patent No. 5,879,934; U.S. Patent No. 5,849,571; U.S. Patent No. 5,830,727; U.S. Patent No. 5,661,033; U.S. Patent No. 5,328,688; Lipofectin mediated gene transfer (BRL); liposomal vectors [See, e.g., U.S. Patent No. 5,631,237 (Liposomes comprising Sendai virus proteins)] ; and combinations thereof. All of the foregoing documents are incorporated herein by reference in their entirety. Replication deficient adenoviral vectors constitute a preferred embodiment.

[00275] In another related embodiment, the invention provides host cells, including prokaryotic and eukaryotic cells, that are transformed or transfected (stably or transiently) with polynucleotides of the invention or vectors of the invention. Polynucleotides of the invention may be introduced into the host cell as part of a circular plasmid, or as linear DNA comprising an isolated protein coding region or a viral vector. Methods for introducing DNA into the host cell, which are well known and routinely practiced in the art include transformation, transfection, electroporation, nuclear injection, or fusion with carriers such as liposomes, micelles, ghost cells, and protoplasts. As stated above, such host cells are useful for amplifying the polynucleotides and also for expressing the polypeptides of the invention encoded by the polynucleotide. The host cell may be isolated and/or purified. The host cell also may be a cell transformed in vivo to cause transient or permanent expression of the polypeptide in vivo. The host cell may also be an isolated cell transformed ex vivo and introduced post-transformation, e.g., to produce the polypeptide in vivo for therapeutic purposes. The definition of host cell explicitly excludes a transgenic human being.

[00276] Such host cells are useful in assays as described herein. For expression of polypeptides of the invention, any host cell is acceptable, including but not limited to bacterial, yeast, plant, invertebrate (e.g., insect), vertebrate, and mammalian host cells. For developing therapeutic preparations, expression in mammalian cell lines, especially human cell lines, is preferred. Use of mammalian host cells is expected to provide for such post-translational modifications (e.g., glycosylation, truncation, lipidation, and phosphorylation) as may be desirable to confer optimal biological activity on recombinant expression products of the invention. Glycosylated and non-glycosylated forms of polypeptides are embraced by the present invention. Similarly, the invention further embraces polypeptides described above that have been covalently modified to include one or more water soluble polymer attachments such as polyethylene glycol, polyoxyethylene glycol, or polypropylene glycol.

[00277] In a further embodiment, the nucleic acid encodes a cleavable leader sequence in- between the promoter and the sequence encoding SEQ ID NO:2, 3, 4 or 5 such that on expression in a host cell, the leader sequence is cleaved in order to provide a peptide consisting of any one of SEQ ID NO:2, 3, 4, and 5. The leader sequence may be heterologous to the peptide sequence. A number of leader sequences of such types are known in the art and may be selected to be compatible with the host cell in which the nucleic acid is expressed.

[00278] Alternatively, as noted above, foreign proteins can also be secreted from the cell into the growth media by creating chimeric DNA molecules that encode a fusion protein comprised of a leader sequence fragment that provides for secretion of the foreign protein in mammalian cells. Preferably, there are processing sites encoded between the leader fragment and the foreign gene that can be cleaved either in vivo or in vitro. The leader sequence fragment usually encodes a signal peptide comprised of hydrophobic amino acids which direct the secretion of the protein from the cell. The adenovirus triparite leader is an example of a leader sequence that provides for secretion of a foreign protein in mammalian cells. [00279] Usually, transcription termination and polyadenylation sequences recognized by mammalian cells are regulatory regions located 3' to the translation stop codon and thus, together with the promoter elements, flank the coding sequence. The 3' terminus of the mature mRNA is formed by site-specific post-transcriptional cleavage and polyadenylation [Birnstiel et al. (1985) Cell 41:349; Proudfoot and Whitelaw (1988) "Ternmination and 3' end processing of eukaryotic RNA. In Transcription and splicing (ed. B. D. Hames and D. M. Glover); Proudfoot (1989) Trends Biochem. Scip. 14:105]. These sequences direct the transcription of an mRNA which can be translated into the polypeptide encoded by the DNA. Examples of transcription terminater/polyadenylation signals include those derived from SV40 [Sambrook et al (1989) "Expression of cloned genes in cultured mammalian cells." In Molecular Cloning: A Laboratory Manual] .

[00280] Gene therapy vehicles for delivery of constructs including a nucleic acid of the invention, to be delivered to the mammal for expression in the mammal, can be administered either locally or systemically. These constructs can utilize viral or non- viral vector approaches in in vivo or ex vivo modality. Expression of such coding sequence can be induced using endogenous mammalian or heterologous promoters. Expression of the coding sequence in vivo can be either constitutive or regulated. The gene delivery vehicle is preferably a viral vector and, more preferably, a retroviral, adenoviral, adeno-associated viral (AAV), herpes viral, or alphavirus vector. The viral vector can also be an astrovirus, coronavirus, orthomyxovirus, papovavirus, paramyxovirus, parvovirus, picomavirus, poxvirus, or togavirus viral vector. See generally, Jolly (1994) Cancer Gene Therapy 1:51-64; Kimura (1994) Human Gene Therapy 5:845-852; Connelly (1995) Human Gene Therapy 6:185-193; and Kaplitt (1994) Nature Genetics 6:148-153.

[00281] Thus, in one embodiment, the invention provides a method of treatment of a subject, which method comprises administration of a gene therapy vector encoding a peptide of any one of SEQ ID NO :2, 3, 4 and 5 or any other peptiude described herein. The gene therapy vector may incorporate a nucleic acid of the invention in any of the forms described above. The treatment may be the treatment of a cancer, as described above. The treatment may be preceded by a diagnostic step of determining a sample of a tumor present in the patient, whether or not at least a portion of the tumor cells have wild-type p53. Optionally the status (i.e. wild-type or mutant) of p63 and/or p73 may be determined. (viii) Antibodies

[00282] In another embodiment, the isolated compound is an antibody. Thus, the invention contemplates use of antibodies (e.g., monoclonal and polyclonal antibodies, single chain antibodies, chimeric antibodies, humanized antibodies, human antibodies, and complementarity determining region (CDR)-grafted antibodies, including compounds that include CDR sequences specifically recognizing a polypeptide of the invention). Antibodies can be human antibodies which are produced and identified according to methods described in WO 93/11236, which is incorporated herein by reference in its entirety. Antibody fragments, including Fab, Fab', F(ab')₂, and Fv, and single-chain antibodies are also provided by the invention. The terms "specific for" or "specifically binds," when used to describe antibodies of the invention, indicates that the variable regions of the antibodies of the invention recognize and bind the polypeptide of interest with a detectable preference (i.e., able to distinguish the polypeptide of interest from other known polypeptides of the same family, by virtue of measurable differences in binding affinity, despite the possible existence of localized sequence identity, homology, or similarity between family members). It will be understood that specific antibodies may also interact with other proteins (for example, S. aureus protein A or other antibodies in ELISA techniques) through interactions with sequences outside the variable region of the antibodies, and in particular, in the constant region of the molecule. Screening assays to determine binding specificity of an antibody of the invention are well known and routinely practiced in the art. For a comprehensive discussion of such assays, see Harlow et al. (Eds), Antibodies A Laboratory Manual; Cold Spring Harbor Laboratory; Cold Spring Harbor, NY (1988), Chapter 6. Antibodies of the invention can be produced using any method well known in the art.

[00283] Various procedures known in the art may be used for the production of polyclonal antibodies to peptides described herein. For the production of antibodies, various host animals (including but not limited to rabbits, mice, rats, hamsters, and the like) are immunized by injection with a phosphorylated iASPP protein or peptide. Various adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete) adjuvant, mineral gels such as aluminium hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (Bacillus Calmette-Guerin) and Corynebacterium parvum. [00284] A monoclonal antibody to a peptide described herein (e.g., SEQ ID NOs: 2-5) may be prepared by using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include but are not limited to the hybridoma technique originally described by Kδhler et al, Nature, 256: 495-497 (1975), and the more recent human B-cell hybridoma technique [Kosbor et al, Immunology Today, 4: 72 (1983)] and the EBV- hybridoma technique [Cole et al, Monoclonal Antibodies and Cancer Therapy, Alan R Liss, Inc., pp. 77-96 (1985), all specifically incorporated herein by reference]. Antibodies against a peptide described herein also may be produced in bacteria from cloned immunoglobulin cDNAs. With the use of the recombinant phage antibody system it may be possible to quickly produce and select antibodies in bacterial cultures and to genetically manipulate their structure.

[00285] When the hybridoma technique is employed, myeloma cell lines may be used. Such cell lines suited for use in hybridoma-producing fusion procedures preferably are non-antibody- producing, have high fusion efficiency, and exhibit enzyme deficiencies that render them incapable of growing in certain selective media which support the growth of only the desired fused cells (hybridomas). For example, where the immunized animal is a mouse, one may use P3-X63/Ag8, P3-X63-Ag8.653, NS 1/1.Ag 4 1, Sp210-Agl4, FO, NSO/U, MPC-Il, MPCIl- X45-GTG 1.7 and S194/5XX0 BuI; for rats, one may use R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210; and U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6 are all useful in connection with cell fusions. It should be noted that the hybridomas and cell lines produced by such techniques for producing the monoclonal antibodies are contemplated compositions of the present invention.

[00286] In addition to the production of monoclonal antibodies, techniques developed for the production of "chimeric antibodies," the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity can be used [Morrison et al, Proc. Natl. Acad. ScL 81:6851-6855 (1984); Neuberger et al, Nature 312:604-608 (1984); Takeda et al, Nature 314:452-454 (1985)]. Alternatively, techniques described for the production of single-chain antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce phosphorylated-iASPP peptide- specific single chain antibodies.

[00287] Antibody fragments which contain the idiotype of the molecule may be generated by known techniques. For example, such fragments include, but are not limited to, the F(ab')₂ fragment which may be produced by pepsin digestion of the antibody molecule; the Fab' fragments which may be generated by reducing the disulfide bridges of the F(ab')₂ fragment, and the two Fab fragments which may be generated by treating the antibody molecule with papain and a reducing agent.

[00288] Non-human antibodies may be humanized by any methods known in the art. A preferred "humanized antibody" has a human constant region, while the variable region, or at least a CDR, of the antibody is derived from a non-human species. Methods for humanizing non-human antibodies are well known in the art. {see U.S. Patent Nos. 5,585,089, and 5,693,762). Generally, a humanized antibody has one or more amino acid residues introduced into its framework region from a source which is non-human. Humanization can be performed, for example, using methods described in Jones et al, Nature 321: 522-525, (1986), Riechmann et al, Nature, 332: 323-327, (1988) and Verhoeyen et al, Science 239:1534-1536, (1988), by substituting at least a portion of a rodent complementarity-determining region for the corresponding regions of a human antibody. Numerous techniques for preparing engineered antibodies are described, e.g., in Owens et al, J. Immunol. Meth., 168:149-165, (1994). Further changes can then be introduced into the antibody framework to modulate affinity or immunogenicity.

[00289] Rapid, large-scale recombinant methods for generating antibodies may be employed, such as phage display [Hoogenboom et al, J. MoI Biol. 227: 381, (1991); Marks et al, J. MoI Biol. 222: 581, (1991)] or ribosome display methods, optionally followed by affinity maturation [see, e.g., Ouwehand et al, Vox Sang 74(Suppl 2):223-232 (1998); Rader et al., Proc. Natl. Acad. ScL USA 95:8910-8915 (1998); Dall'Acqua et al, Curr. Opin. Struct. Biol. 8:443-450, (1998)]. Phage-display processes mimic immune selection through the display of antibody repertoires on the surface of filamentous bacteriophage, and subsequent selection of phage by their binding to an antigen of choice. One such technique is described in WO 99/10494, which describes the isolation of high affinity and functional agonistic antibodies for MPL and msk receptors using such an approach.

[00290] Recombinant antibody fragments, e.g., scFvs, can also be engineered to assemble into stable multimeric oligomers of high binding avidity and specificity to different target antigens. Such diabodies (dimers), triabodies (trimers) or tetrabodies (tetramers) are well known in the art, see e.g., Kortt et al, Biomol Eng. 2001 18:95-108, (2001) and Todorovska et al, J Immunol Methods. 248:47-66, (2001). Further therapeutically active moieties

[00291] In a further embodiment of the present invention the compound is linked to a therapeutically active moiety, preferably the moiety is cytotoxic.

[00292] The term "therapeutically active moiety" encompasses a moiety having beneficial, prophylactic and/or therapeutic properties. Methods of conjugating polypeptides to therapeutic agents are well known in the art. In one embodiment the therapeutically active moiety is a cytotoxic chemotherapeutic agent.

[00293] Cytotoxic chemotherapeutic agents are well known in the art. Exemplary cytotoxic chemotherapeutic agents, such as anticancer agents, include: alkylating agents including nitrogen mustards such as mechlorethamine (HN2), cyclophosphamide, ifosfamide, melphalan (L- sarcolysin) and chlorambucil; 10 ethylenimines and methylmelamines such as hexamethylmelamine, thiotepa; alkyl sulphonates such as busulfan; nitrosoureas such as carmustine (BCNU), lomustine (CCNLJ), semustine (methyl-CCN-U) and streptozoein (streptozotocin); and triazenes such as decarbazine (DTIC; dimethyltriazenoimidazolecarboxamide); Antimetabolites including folic acid analogues such as methotrexate (amethopterin); pyrimidine analogues such as fluorouracil (5- fluorouracil; 5-FU), floxuridine (fluorodeoxyuridine; FUdR) and cytarabine (cytosine arabinoside); and purine analogues and related inhibitors such as mercaptopurine (6-mercaptopurine; 6-MP), thioguanine (6-thioguanine; TG) and pentostatin (2'-deoxycofonnycin). Natural Products including vinca alkaloids such as vinblastine (VLB) and vincristine; epipodophyllotoxins such as etoposide and teniposide; antibiotics such as dactinomycin (actinomycin D), daunorabicin (daunomycin; rubidomycin), doxorubicin, bleomycin, plicamycin (mithramycin) and mitomycin (mitomycin Q; enzymes such as L-asparaginase; and biological response modifiers such as interferon alphenomes. Miscellaneous agents including platinum coordination complexes such as cisplatin (cis-DDP) and carboplatin; anthracenedione such as mitoxantrone and antbracycline; substituted urea such as hydroxyurea; methyl hydrazine derivative such as procarbazine (N- methylhydrazine, MIH); and adrenocortical suppressant such as mitotane (o, p'-DDD) and aminoglutethimide; taxol and analogues/derivatives; and hormone agonists/antagonists such as flutamide and tamoxifen.

[00294] In a further embodiment of the invention, the cytotoxic moiety is a cytotoxic peptide or polypeptide moiety by which we include any moiety which leads to cell death. [00295] Cytotoxic peptide and polypeptide moieties are well known in the art and include, for example, ricin, abrin, Pseudomonas exotoxin, tissue factor and the like. The use of ricin as a cytotoxic agent is described in Burrows & Thorpe (1993) Proc. Natl. Acad. Sci. USA 90, 8996- 9000, incorporated herein by reference, and the use of tissue factor, which leads to localised blood clotting and infarction of a tumour, has been described by Ran et al (1998) Cancer Res. 58, 4646-4653 and Huang et al (1997) Science 275, 25 547-550. Tsai et al (1995) Dis. Colon Rectum 38, 1067- 1074 describes the abrin A chain conjugated to a monoclonal antibody and is incorporated herein by reference. Other ribosome inactivating proteins are described as cytotoxic agents in WO 96/06641. Pseudomonas exotoxin may also be used as the cytotoxic polypeptide moiety (see, for example, Aiello et al (1995) Proc. Nad. Acad. Sci. USA 92, 10457- 1046 1).

[00296] Certain cytokines, such as TNFa and IL-2, may also be useful as cytotoxic agents.

[00297] Certain radioactive atoms may also be cytotoxic if delivered in sufficient doses. Thus, the cytotoxic moiety may comprise a radioactive atom which, in use, Io delivers a sufficient quantity of radioactivity to the target site so as to be cytotoxic. Suitable radioactive atoms include phosphorus-32, iodine-125, iodine-131, indium-Ill, rhenium-186, rhenium- 188 or yttrium-90, or any other isotope which emits enough energy to destroy neighboring cells, organelles or nucleic acid. Preferably, the isotopes and density of radioactive atoms in the compound of the invention are such that a dose of more than 4000 cGy (preferably at least 6000, 8000 or 10000 cGy) is delivered to the target site and, preferably, to the cells at the target site and their organelles, particularly the nucleus. The radioactive atom may be attached to the binding moiety in known ways. For example EDTA or another chelating agent may be attached to the binding moiety and used to attach ¹¹¹In or ⁹⁰Y. Tyrosine residues may be labelled with ¹²⁵ I or ¹³¹I.

[00298] Alternatively, any of these systems can be incorporated into a prodrug system. Such prodrug systems are well known in the art.

(v) Screening methods for modulators of binding ofiASPP to an iASPP binding partner

[00299] Methods are provided for screening for modulators of binding of an iASPP polypeptide comprising a structure defined by the structural coordinates provided in Table 1 with an iASPP binding partner. Generally, such methods involve introducing one or more test compounds (alone or simultaneously) into a controlled system containing an iASPP polypeptide and one of its binding partners, to determine whether the test compound affects binding between the iASPP and its natural binding partner. The assays can be quantitative in an absolute sense, involving measurements of binding, or quantitative in a relative sense, involving comparative measurements of binding, such as comparative evaluation of the intensity of a radioactive or colorimetric signal. It is often beneficial, but not absolutely necessary, to compare the results involving a test compound with a control involving no test compound to evaluate the effects of the compound. However, even absent such a control, the binding measurement can provide an indication of the effect of the test compound, and the effect of the test compound can alternatively be compared to results involving previous test compounds or known binding inhibitors.

[00300] Such assays can be performed in vitro in cell-free formats or cell based formats, which often lend themselves to high throughput implementation. In some cell-free variations, the iASPP or the binding partner is attached to a solid support (e.g., a bead, membrane, plate, or chip) and the other, unattached moiety is incubated with the solid support to permit binding. Optionally, the unbound moiety (the iASPP or the binding partner) is labeled (radiolabel, colorimetric label, etc.) or tagged (e.g., a peptide or epitope tag, or GFP fusion or enzymatic fusion) to facilitate measurement of binding.

[00301] Cell-based binding assays can involve cells that naturally express one or both of the proteins (iASPP and its binding partner), or cells in which expression is induced, or cells which express one or both proteins due to recombinant modification, e.g., the introduction of an expression vector. Prokaryotic (e.g., bacteria such as E. coli) and Eukaryotic (e.g., animal, plant, yeast/fungal, etc.) cells can be employed. The test compound is introduced into the cell growth media or directly into the cell to determine its effect on binding.

[00302] In one aspect, the method comprises (a) contacting an iASPP polypeptide comprising a structure defined by the structural coordinates provided in Table 1 with an iASPP binding partner, in the presence and absence of a test compound, under conditions in which the binding partner binds with the iASPP polypeptide; and (b) comparing binding of the polypeptide and the binding partner in the presence and absence of the test compound. In another aspect, the method comprises measuring binding between a polypeptide that comprises comprising a structure defined by the structural coordinates provided in Table 1 with an iASPP binding partner with an iASPP binding partner, in the presence and absence of a test compound, under conditions in which the binding partner binds with the iASPP polypeptide. Increased binding of the polypeptide to the binding partner identifies the test compound as an agonist of binding and decreased binding of the polypeptide to the binding partner identifies the test compound of as an antagonist of binding. In one aspect, the binding partner comprises a member selected from the group consisting of p53, p63 and p73.

[00303] In the case of binding partners identified herein, experiments can be performed with binding partners from any organism in which iASPP and the binding partner are expressed. Mammalian iASPP and binding partners are preferred, with primate highly preferred and human very highly preferred. Numerous sequences have already been reported in the scientific literature, public (Genbank, patent), and commercial databases.

[00304] In certain aspects, the binding partner comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98%, at least 99% or more identical an amino acid sequence of a mammalian protein selected from the group consisting of p53 (Genbank Accession Nos. BAC16799 (human), BAA82344 (mouse), BAA788379 (canine)), p63 (Genbank Accession Nos. AAB21139 (human) and Q9JJP6 (rat) and p73 (Genbank Accession Nos.: CAA72221 (human) and AAD33213 (mouse).

[00305] In some variations, a full-length iASPP polypeptide (i.e., amino acids 1-828 of SEQ ID NO: 7) and the complete binding partner is employed in the assay. However, evidence, including that described below, indicates that the iASPP binding partners identified herein interact with a carboxyl-terminal portion of iASPP, and thus it is unnecessary to use the complete iASPP for binding assays of the invention. In the context of assays of the invention, the term "iASPP carboxyl-terminal domain" refers to a portion of iASPP that is sufficient to exhibit the activity (e.g., binding activity) needed for the assay. It is possible to determine a minimum effective iASPP carboxyl-terminal domain by screening deletion fragments of iASPP according to binding assay procedures described herein. The iASPP carboxyl-terminal domain can be fused to other sequences to make fusion proteins, and/or contain additional moieties such as labels and tags.

[00306] In one aspect, the polypeptide comprising an iASPP carboxyl-terminal domain comprises amino acids 1-828 of SEQ ID NO: 7 or comprises an amino acid sequence at least at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to amino acids 1-828 of SEQ ID NO: 7.

[00307] In another aspect, the polypeptide comprising an iASPP carboxyl-terminal domain comprises amino acids 608-828 of SEQ ID NO: 7 or comprises an amino acid sequence at least at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to amino acids 608-828 of SEQ ID NO: 7.

[00308] In other aspects, the polypeptide comprising an iASPP carboxyl-terminal domain comprises amino acids 616-623 of SEQ ID NO: 7 or comprises an amino acid sequence at least at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to amino acids 616-623 of SEQ ID NO: 7.

[00309] Exemplary assays for screening test compounds include, but are not limited to, labeled in vitro protein-protein binding assays, electrophoretic mobility shift assays, immunoassays, and cell-based assays such as two- or three -hybrid screens and those used in high-throughput screening and expression assays. For example, hybrid screens can be used to rapidly examine the effect of transfected nucleic acids on the intracellular binding of iASPP polypeptides or fragments thereof to specific intracellular targets. The transfected nucleic acids can encode, for example, combinatorial peptide libraries or antisense molecules. Convenient reagents for such assays, such as GAL4 fusion proteins, are known in the art.

[00310] The polypeptides comprising an iASPP carboxyl-terminal domain used in the screening methods, when not produced by a transfected nucleic acid molecule in a cell based assay, can be added to an assay mixture as an isolated polypeptide. A polypeptide comprising an iASPP carboxyl-terminal domain can be produced recombinantly or isolated from biological extracts. Full-length or functional fragments of a polypeptide comprising an iASPP carboxyl- terminal domain can be used, as can mimetics and analogs thereof, as long as the portion, mimetic or analog provides binding affinity and avidity measurable in the assay.

[00311] The assay mixture also includes a test compound. In particular examples, a plurality of assay mixtures are run in parallel with different agent concentrations to obtain a different response to the various concentrations. Typically, one of these concentrations serves as a negative control (such as at zero concentration of agent or at a concentration of agent below the limits of assay detection). Exemplary test compounds are discussed below.

[00312] Additional reagents can be included in the mixture. Reagents such as salts, buffers, neutral proteins (such as albumin), and detergents, can be used to facilitate optimal protein- protein and/or protein-nucleic acid binding. Such a reagent can also reduce non-specific or background interactions of the reaction components. Other reagents that improve the efficiency of the assay such as protease, inhibitors, nuclease inhibitors, antimicrobial agents, and the like can also be used. Exemplary conditions include conditions that approximate reaction conditions inside a cell, and thus may include an isotonic level of salts, buffered pH, and avoidance of extreme temperatures.

[00313] The mixture of assay materials is incubated under conditions whereby, but for the presence of the test compound, the polypeptide comprising an iASPP carboxyl-terminal domain specifically binds the cellular binding target. Incubation temperatures typically are between 4°C and 40°C. Incubation times can be minimized to facilitate rapid, high throughput screening and such as about 0.1 to 10 hours.

[00314] After incubation, the presence or absence of specific binding between the polypeptide comprising an iASPP carboxyl-terminal domain and one or more binding partners is detected by any convenient method available to the user. Exemplarary binding targets include, but are not limited to, p53, p63 and p73. For example, in a cell free binding assays, a separation step can be used to separate bound from unbound components. The separation step can be accomplished in a variety of ways. For example, at least one of the components can be immobilized on a solid substrate, from which the unbound components may be easily separated. The solid substrate can be made of a wide variety of materials and in a wide variety of shapes, such as a microtiter plate, a microbead, a dipstick, or a resin particle. Ideally, the substrate provides maximum signal to noise ratios, to minimize background binding.

[00315] In one example, separation is achieved by removing a bead or dipstick from a reservoir, emptying or diluting a reservoir such as a microtiter plate well, rinsing a bead, particle, chromatographic column or filter with a wash solution or solvent. The separation step can include multiple rinses or washes. For example, when the solid substrate is a microtiter plate, the wells can be washed several times with a washing solution, which typically includes those components of the incubation mixture that do not participate in specific bindings such as salts, buffer, detergent, non-specific protein. Where the solid substrate is a magnetic bead, the beads can be washed one or more times with a washing solution and isolated using a magnet.

[00316] Detection of the presence or absence of a polypeptide comprising an iASPP carboxyl- terminal domain complex with a binding partner can be achieved using any method known in the art. For example, the transcript resulting from a reporter gene transcription assay of a polypeptide comprising an iASPP carboxyl-terminal domain interacting with a target molecule (e.g., binding partner) typically encodes a directly or indirectly detectable product (such as β- galactosidase activity, luciferase activity, and the like). For cell-free binding assays, one of the components usually includes, or is coupled to, a detectable label. A wide variety of labels can be used, such as those that provide direct detection (such as radioactivity, luminescence, optical or electron density) or indirect detection (such as epitope tag such as the FLAG epitope, enzyme tag such as horseradish peroxidase). The label can be bound to an iASPP binding partner, or incorporated into the structure of the binding partner.

[00317] A variety of methods can be used to detect the label, depending on the nature of the label and other assay components. For example, the label can be detected while bound to the solid substrate or subsequent to separation from the solid substrate. Labels can be directly detected through optical or electron density, radioactive emissions, nonradiative energy transfers or indirectly detected with antibody conjugates, or strepavidin-biotin conjugates. Methods for detecting the labels are well known in the art.

[00318] A test compound employed in a method of the invention can be any organic or inorganic chemical or biological molecule known in the art, such as small organic or inorganic molecules preferably found in small molecule libraries containing compounds of synthetic or natural origin, or combinatorial libraries as described below. Further, peptides, preferably found in peptide libraries, are contemplated as candidate modulators such as inhibitors. Test compounds suitable for administration as therapeutics will exhibit acceptable toxicity levels as would be known in the art or determinable by one of skill in the art using routine experimentation. Toxicity can be determined in subsequent assays, however, and often "designed out" of molecules by pharmaceutical chemists. Screening of chemical libraries such as those developed and maintained by pharmaceutical companies, consisting of both chemically synthesized and natural compounds, and combinatorial libraries, are specifically contemplated. [00319] Chemical libraries may contain known compounds, proprietary structural analogs of known compounds, or compounds that are identified from natural product screening.

[00320] Natural product libraries are collections of materials isolated from natural sources, typically, microorganisms, animals, plants, or marine organisms. Natural products are isolated from their sources by fermentation of microorganisms followed by isolation and extraction of the fermentation broths or by direct extraction from the microorganism or tissue (plant or animal) themselves. Natural product libraries include polyketides, non-ribosomal peptides, and variants (including non-naturally occurring variants) thereof. See Cane et al., Science, 252:63-68 (1998), incorporated herein by reference.

[00321] Combinatorial libraries are composed of large numbers of related compounds, such as peptides, oligonucleotides, or other organic compounds as a mixture. Such compounds are relatively straightforward to design and prepare by traditional automated synthesis protocols, PCR, cloning or proprietary synthetic methods. Of particular interest are peptide and oligonucleotide combinatorial libraries.

[00322] Still other libraries of interest include peptide, protein, peptidomimetic, multiparallel synthetic collection, recombinatorial, and polypeptide libraries. For a review of combinatorial chemistry and libraries created thereby, see Myers, Curr. Opin. BiotechnoL, 5:701-707 (1997), incorporated herein by reference.

[00323] Modulators of iASPP binding to an iASPP binding partner identified by assessment of the test compounds may be formulated into compositions which include pharmaceutically acceptable {i.e., sterile and non-toxic) liquid, semisolid, or solid diluents that serve as pharmaceutical vehicles, excipients, or media.

[00324] Compositions comprising one or more modulators of iASPP binding to an iASPP binding partner can be packaged in forms convenient for delivery. The compositions can be enclosed within a capsule, caplet, sachet, cachet, gelatin, paper, or other container. The dosage units can be packaged, e.g., in tablets, capsules, suppositories or cachets.

Therapeutic uses of the Compounds

[00325] Following identification of a compound by the present invention, including a peptide of the invention referred to above, it may be manufactured and/or used in the preparation, i.e. manufacture or formulation, of a composition such as a medicament, pharmaceutical composition or drug. Small peptides may be manufactured by recombinant DNA technology, as described further herein, or synthetically. These may be administered to individuals.

[00326] Thus, the present invention extends in various aspects not only to a compound as provided by the invention, but also a pharmaceutical composition, medicament, drug or other composition comprising such a compound for the treatment of a neoplastic disorder. Thus, in one aspect, a method of treating a neoplastic disorder comprising administering to a subject in need of treatment a composition comprising a compound of the invention in an amount effective to treat the neoplastic disorder is contemplated. In some embodiments, prior to the administering step, a subject having a neoplastic disorder is identified. The identifying step may comprise identifying a subject with a neoplactic disorder characterized by elevated expression of an iASPP polypeptide in neoplastic cells. In some embodiments, the identifying step comprises screening a biological sample suspected of containing neoplastic cells from the subject for elevated expression of the iASPP polypeptide. The screening step comprises comparing expression of iASPP in cells from the sample with expression of iASPP from healthy cells of the same type of cells in the sample, wherein increased expression of the iASPP in cells from the sample compared to the expression of the iASPP in cells from the healthy cells indicates elevated expression of iASPP.

[00327] In some embodiments, the neoplastic disorder is a cancer selected form the group consisting of breast cancer, pancreatic cancer, ovarian cancer, colorectal cancer, esophageal cancer, lung cancer, head cancer, neck cancer, gastric cancer and epithelial cancer,

[00328] In another embodiment, the invention provides a method of selecting a therapeutic regimen for a subject comprising identifying a subject as having a neoplastic disorder characterized by increased expression of an iASPP polypeptide and administering to the subject a compound that inhibits binding between iASPP and an iASPP binding partner. In some embodiments, the identifying step comprises screening a biological sample suspected of containing neoplastic cells from the subject for elevated expression of the iASPP polypeptide. The screening step comprises comparing expression of iASPP in cells from the sample with expression of iASPP from healthy cells of the same type of cells in the sample, wherein increased expression of the iASPP in cells from the sample compared to the expression of the iASPP in cells from the healthy cells indicates elevated expression of iASPP. [00329] Also provided is a method of stimulating p53-mediated apoptosis in a cell comprising contacting the cell with a compound that inhibits binding between iASPP and an iASPP binding partner in an amount effective to stimulate p53-mediated apoptosis in the cell. In one embodiment, the cell expresses wild-type p53.

[00330] The compositions may be used for treatment (which may include preventative treatment) of disease such as cancer. Cancers include lung cancer, small cell lung cancer, non- small cell lung cancer, gastrointestinal cancer, stomach cancer, bowel cancer, colon cancer, rectal cancer, colorectal cancer, thyroid cancer, breast cancer, ovarian cancer, endometrial cancer, prostate cancer, testicular cancer, liver cancer, kidney cancer, renal cell carcinoma, bladder cancer, pancreatic cancer, brain cancer, glioma, sarcoma, osteosarcoma, bone cancer, skin cancer, squamous cancer, Kaposi's sarcoma, melanoma, malignant melanoma, lymphoma, and leukemia.

[00331] In one embodiment, the treatment is treatment of:

[00332] a carcinoma, for example a carcinoma of the bladder, breast, colon (e.g., colorectal carcinomas such as colon adenocarcinoma and colon adenoma), kidney, epidermal, liver, lung (e.g., adenocarcinoma, small cell lung cancer and non-small cell lung carcinomas), oesophagus, gall bladder, ovary, pancreas (e.g., exocrine pancreatic carcinoma), stomach, cervix, thyroid, prostate, skin (e.g., squamous cell carcinoma);

[00333] a hematopoietic tumour of lymphoid lineage, for example leukemia, acute lymphocytic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, non- Hodgkin's lymphoma, hairy cell lymphoma, or Burkett's lymphoma;

[00334] a hematopoietic tumor of myeloid lineage, for example acute and chronic myelogenous leukemias, myelodysplastic syndrome, or promyelocytic leukemia;

[00335] a tumour of mesenchymal origin, for example fibrosarcoma or habdomyosarcoma;

[00336] a tumor of the central or peripheral nervous system, for example astrocytoma, neuroblastoma, glioma or schwannoma;

[00337] melanoma; seminoma; teratocarcinoma; osteosarcoma; xenoderoma pigmentoum; keratoctanthoma; thyroid follicular cancer; or Kaposi's sarcoma. [00338] In one embodiment, the treatment is treatment of solid tumour cancer (e.g., cancer characterized by the appearance of solid tumours).

[00339] The term "treatment," as used herein in the context of treating a condition, pertains generally to treatment and therapy, whether of a human or an animal (e.g., in veterinary applications), in which some desired therapeutic effect is achieved, for example, the inhibition of the progress of the condition, and includes a reduction in the rate of progress, a halt in the rate of progress, alleviation of symptoms of the condition, amelioration of the condition, and cure of the condition. Treatment as a prophylactic measure (i.e., prophylaxis) is also included. For example, use with patients who have not yet developed the condition, but who are at risk of developing the condition, is encompassed by the term "treatment."

[00340] For example, treatment of cancer includes the prophylaxis of cancer, reducing the incidence of cancer, alleviating the symptoms of cancer, etc.

[00341] Such a treatment may comprise administration of such a composition to a patient, e.g. for treatment of disease; the use of such an inhibitor in the manufacture of a composition for administration, e.g. for treatment of disease; and a method of making a pharmaceutical composition comprising admixing such an compound with a pharmaceutically acceptable excipient, vehicle or carrier, and optionally other ingredients. The administration of the agent will be of a therapeutically-effective amount, i.e. that amount of an active compound, or a material, composition or dosage form comprising an active compound, which is effective for producing some desired therapeutic effect, commensurate with a reasonable benefit/risk ratio, when administered in accordance with a desired treatment regimen.

Combination Therapy

[00342] In some embodiments, the methods of the invention further comprise the administration of a standard of care anti-neoplastic therapy selected from the grou consisting of a standard of care therapeutic, a standard of care radio therapeutic, or a standard of care radiation regimen for the neoplastic disorder. Thus, in one aspect, methods of treatment of a condition associated with tumor cell growth in a subject are provided comprising administering to the subject a compound that inhibits binding between iASPP and an iASPP binding partner and a standard of care anti-neoplastic therapy. [00343] Administration of a combination of a compound that inhibits binding between iASPP and an iASPP binding partner with one or more additional therapeutics/second agents in methods of the invention may reduce the amount of either agent needed as a therapeutically effective dosage, and thereby reduce any negative side effects the agents may induce in vivo. Thus, a method of treating a neoplastic disorder comprising administering to a subject in need of treatment a composition comprising a compound of the invention in an amount effective to treat the neoplastic diseaorder and an additional therapeutic agent is specifically contemplated. Additional therapeutics or second agents contemplated for use in combination with an iASPP antagonist or an iASPP inhibitor peptide include a tyrosine kinase inhibitor, a cytokine, a chemotherapeutic agent, a radiotherapeutic agent, or radiation therapy.

[00344] Any tyrosine kinase inhibitor may be suitable for use in combination with an iASPP antagonist or an iASPP inhibitor peptide in a compostition or method of the invention. Inhibitors of non-receptor tyrosine kinases are specifically contemplated. Examples of suitable tyrosine kinase inhibitors include, but are not limited to, SKI-606 (4-anilino-3- quinolinecarbonitrile), PD173955 (pyrido[2,3-d]pyrimidine), AZD0530, AZM475271, PP2 (4- amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4,d]pyrimidine), PPl (4-Amino-5-(4- methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine), CGP76030 (substituted 5,7-diphenyl- pyrrolo[2,3d]pyrimidine), dasatinib, TG100435 ([7-(2,6-dichloro-phenyl)-5-methyl- benzo[l,2,4]triazin-3-yl]-[4-(2-pyrrolidin-l-yl-ethoxy)-phenyl]-amine), TG100855 [7-(2,6- dichloro-phenyl)-5 -methyl-benzo [ 1 ,2,4] triazin-3-yl] - { 4- [2-( 1 -oxy-pyrrolidin- 1 -yl)-ethoxy] - phenyl} -amine), AZM 475271, M475271, SU6656 (2,3-Dihydro-N,N-dimethyl-2-oxo-3- [(4,5,6,7-tetrahydro-lH-indol-2-yl)methylene]-lH-indole-5-sulfonamide), staurosporine from Streptomyces staurosporeus, adaphostin (4-Amino-N-(2,5-dihydroxybenzyl)adamantyl Benzoate, 4-(2,5-Dihydroxy-benzylamino)benzoic Acid Adamantan-1-yl Ester), Tyrphostin AG 957 (4-Amino-N-(2,5-dihydroxybenzyl)methyl benzoate), Imatinib mesylate (Gleevec®, Glivec®), aminogenistein (4'-Amino-6-Hydroxyflavone), Indirubin-3'-oxime (3-[l,3-Dihydro-3- (hydroxyimino)-2H-indol-2-ylidene]-l,3-dihydro-2H-indol-2-one), Kenpaullone (9-Bromo- 7, 12-dihydroindolo[3,2-d][l]benzazepin-6(5H)-one), LFM-A13 (2-Cyano-N-(2,5- dibromophenyl)-3-hydroxy-2-butenamide), Bisindolylmaleimide IX, (Methanesulfonate Salt), edelfosine (rac-2-methyl-l-octadecyl-glycero-(3)-phosphocholine), Rapamycin, Quercetin (3,3',4',5,7-Pentahydroxyflavone . 2H2O and 2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-l- benzopyrano-4-one), TBB (4,5,6,7-Tetrabromotriazole), and Triciribine (6-Amino-4-methyl-8- (β-D-ribofuranosyl)4H,8H-pyrrolo[4,3,2-de]pyrimido[4,5-c] pyridazine). [00345] Any chemotherapeutic or radiotherapeutic agent may be suitable for use in combination with an iASPP antagonist or an iASPP inhibitor peptide in a composition or method of the invention, and may be identified by means well known in the art. Examples of suitable chemotherapeutic and radiotherapeutic agents include, but are not limited to: an antimetabolite; a DNA-damaging agent; a cytokine useful as a chemotherapeutic agent; a covalent DNA-binding drug; a topoisomerase inhibitor; an anti-mitotic agent; an anti-tumor antibiotic; a differentiation agent; an alkylating agent; a methylating agent; a hormone or hormone antagonist; a nitrogen mustard; a radiosensitizer; a photosensitizer; a radiation source, optionally together with a radiosensitizer or photosensitizer; or other commonly used therapeutic agents.

[00346] Specific examples of chemotherapeutic agents useful in methods of the present invention are listed in Table 10 below.

[00347] Cytokines that are effective in inhibiting tumor metastasis are also contemplated for use in the combination therapy. Such cytokines, lymphokines, or other hematopoietic factors include, but are not limited to, M-CSF, GM-CSF, TNF, IL-I, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-Il, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IFN, TNFα, TNFl, TNF2, G-CSF, Meg-CSF, GM-CSF, thrombopoietin, stem cell factor, and erythropoietin.

[00348] Alternatively, the therapeutic treatment employing a compound that inhibits binding between iASPP and an iASPP binding partner described herein or other therapeutic agent described herein may precede or follow the second agent treatment by intervals ranging from minutes to weeks. In embodiments where the second agent and the compound that inhibits binding between iASPP and an iASPP binding partner are administered separately, one would generally ensure that a significant period of time did not expire between the times of each delivery, such that the agent and the compound that inhibits binding between iASPP and an iASPP binding partner would still be able to exert an advantageously combined effect. In such instances, it is contemplated that one would administer both modalities within about 12-24 hours of each other and, more preferably, within about 6-12 hours of each other, with a delay time of only about 12 hours being most preferred. In some situations, it may be desirable to extend the time period for treatment significantly, however, where several days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the respective administrations. Repeated treatments with one or both agents are specifically contemplated.

[00349] The term "treatment" includes combination treatments and therapies, in which two or more treatments or therapies are combined, for example, sequentially or simultaneously. For example, the compounds described herein may also be used in combination therapies, e.g., in conjunction with other agents, for example, cytotoxic agents, anticancer agents, etc. Examples of treatments and therapies include, but are not limited to, chemotherapy (the administration of active agents, including, e.g., drugs, antibodies (e.g., as in immunotherapy), prodrugs (e.g., as in photodynamic therapy, GDEPT, ADEPT, etc.); surgery; radiation therapy; photodynamic therapy; gene therapy; and controlled diets.

[00350] The particular combination would be at the discretion of the physician who would select dosages using his common general knowledge and dosing regimens known to a skilled practitioner. [00351] The agents (e.g., a compound of the invention; plus one or more other agents) may be administered simultaneously or sequentially, and may be administered in individually varying dose schedules and via different routes. For example, when administered sequentially, the agents can be administered at closely spaced intervals (e.g., over a period of 5-10 minutes) or at longer intervals (e.g., 1, 2, 3, 4 or more hours apart, or even longer periods apart where required), the precise dosage regimen being commensurate with the properties of the therapeutic agent(s).

[00352] The agents may be formulated together in a single dosage form, or alternatively, the individual agents may be formulated separately and presented together in the form of a kit, optionally with instructions for their use, as described below.

[00353] In one embodiment, the patient may be suffering from a cancer condition in which iASPP is over-expressed. In another embodiment, the patient may be suffering from a cancer condition in which wild-type p53 function is present in at least a portion of the tumour cells.

[00354] In one aspect, the patient may be suffering from a breast cancer in which iASPP is over-expressed. This may be against a wild-type or non- wild-type p53 background.

[00355] In another aspect, the patient may be suffering from leukaemia in which iASPP is over-expressed. This may be against a wild-type or non-wild-type p53 background.

[00356] In a further embodiment, the treatment of the patient may be preceded by the step of determining on a sample of a tumour present in the patient, whether or not at least a portion of the tumour cells have wild-type p53. Optionally the status (i.e. wild-type or mutant) of p63 and/or p73 may be determined.

[00357] Thus a further aspect of the present invention provides a method for preparing a medicament, pharmaceutical composition or drug, the method comprising:

[00358] (a) identifying or modifying a compound by a method of any one of the other aspects of the invention disclosed herein; (b) optimising the structure of the molecule; and (c) preparing a medicament, pharmaceutical composition or drug containing the optimised compound.

[00359] The above-described processes of the invention may be iterated in that the modified compound may itself be the basis for further compound design. [00360] By "optimising the structure" we mean e.g. adding molecular scaffolding, adding or varying functional groups, or connecting the molecule with other molecules (e.g. using a fragment linking approach) such that the chemical structure of the modulator molecule is changed while its original modulating functionality is maintained or enhanced. Such optimisation is regularly undertaken during drug development programmes to e.g. enhance potency, promote pharmacological acceptability, increase chemical stability etc. of lead compounds.

[00361] Modification will be those conventional in the art known to the skilled medicinal chemist, and will include, for example, substitutions or removal of groups containing residues which interact with the amino acid side chain groups of a iASPP C-terminal structure of the invention. For example, the replacements may include the addition or removal of groups in order to decrease or increase the charge of a group in a test compound, the replacement of a charge group with a group of the opposite charge, or the replacement of a hydrophobic group with a hydrophilic group or vice versa. It will be understood that these are only examples of the type of substitutions considered by medicinal chemists in the development of new pharmaceutical compounds and other modifications may be made, depending upon the nature of the starting compound and its activity.

Formulations and Routes of Administration

[00362] In order to prepare peptide-containing compositions for clinical use, it will be necessary to prepare the therapeutic peptides of the present invention as pharmaceutical compositions, i.e., in a form appropriate for in vivo applications. Generally, this will entail preparing compositions that are essentially free of pyrogens, as well as other impurities that could be harmful to humans or animals.

[00363] One will generally desire to employ appropriate salts and buffers to render delivery vectors stable and allow for uptake by target cells. Buffers also will be employed when recombinant cells are introduced into a patient. Aqueous compositions of the present invention comprise an effective amount of the peptide or an expression vector to cells, dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. Such compositions also are referred to as inocula. The phrase "pharmaceutically acceptable" or "pharmacologically acceptable" refer to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the vectors or cells of the present invention, its use in therapeutic compositions is contemplated. Supplementary active ingredients also can be incorporated into the compositions.

[00364] The compositions of the present invention include classic pharmaceutical preparations. Administration of these compositions according to the present invention will be via any common route so long as the target tissue is available via that route. The pharmaceutical compositions may be introduced into the subject by any conventional method, e.g., by intravenous, intradermal, intramusclar, intramammary, intraperitoneal, intrathecal, retrobulbar, intrapulmonary (e.g., term release); by oral, sublingual, nasal, anal, vaginal, or transdermal delivery, or by surgical implantation at a particular site. The treatment may consist of a single dose or a plurality of doses over a period of time.

[00365] The compositions may be prepared for administration as solutions of free base or pharmacologically acceptable salts in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions also can be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[00366] The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[00367] Sterile injectable solutions are prepared by incorporating the composition in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle that contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[00368] For oral administration the peptides of the present invention may be incorporated with excipients and used in the form of non-ingestible mouthwashes and dentifrices. A mouthwash may be prepared incorporating the active ingredient in the required amount in an appropriate solvent, such as a sodium borate solution (Dobell's Solution). Alternatively, the active ingredient may be incorporated into an antiseptic wash containing sodium borate, glycerin and potassium bicarbonate. The active ingredient may also be dispersed in dentifrices, including: gels, pastes, powders and slurries. The active ingredient may be added in a therapeutically effective amount to a paste dentifrice that may include water, binders, abrasives, flavoring agents, foaming agents, and humectants.

[00369] The compositions described herein may be formulated in a neutral or salt form. Pharmaceutically-acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups also can be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.

[00370] Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms such as injectable solutions, drug release capsules and the like. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.

[00371] "Unit dose" is defined as a discrete amount of a therapeutic composition dispersed in a suitable carrier. For example, where polypeptides are being administered parenterally, the polypeptide compositions are generally injected in doses ranging from lμg/kg to 100mg/kg body weight/day, preferably at doses ranging from 0.1mg/kg to about 50 mg/kg body weight/day. Parenteral administration may be carried out with an initial bolus followed by continuous infusion to maintain therapeutic circulating levels of drug product. Those of ordinary skill in the art will readily optimize effective dosages and administration regimens as determined by good medical practice and the clinical condition of the individual patient.

[00372] The frequency of dosing will depend on the pharmacokinetic parameters of the agents and the routes of administration. The optimal pharmaceutical formulation will be determined by one of skill in the art depending on the route of administration and the desired dosage. See for example Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publ. Co, Easton PA 18042) pp 1435-1712, incorporated herein by reference. Such formulations may influence the physical state, stability, rate of in vivo release and rate of in vivo clearance of the administered agents. Depending on the route of administration, a suitable dose may be calculated according to body weight, body surface areas or organ size. Further refinement of the calculations necessary to determine the appropriate treatment dose is routinely made by those of ordinary skill in the art without undue experimentation, especially in light of the dosage information and assays disclosed herein as well as the pharmacokinetic data observed in animals or human clinical trials.

[00373] Appropriate dosages may be ascertained through the use of established assays for determining blood clotting levels in conjunction with relevant dose-response data. The final dosage regimen will be determined by the attending physician, considering factors that modify the action of drugs, e.g., the drug's specific activity, severity of the damage and the responsiveness of the patient, the age, condition, body weight, sex and diet of the patient, the severity of any infection, time of administration and other clinical factors. As studies are conducted, further information will emerge regarding appropriate dosage levels and duration of treatment for specific diseases and conditions. [00374] In gene therapy embodiments employing viral delivery, the unit dose may be calculated in terms of the dose of viral particles being administered. Viral doses include a particular number of virus particles or plaque forming units (pfu). For embodiments involving adenovirus, particular unit doses include 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³ or 10¹⁴ pfu. Particle doses may be somewhat higher (10 to 100-fold) due to the presence of infection defective particles.

[00375] It will be appreciated that the pharmaceutical compositions and treatment methods described herein may be useful in fields of human medicine and veterinary medicine. Thus the subject to be treated may be a mammal, (e.g., human) or other animal. For veterinary purposes, subjects include for example, farm animals including cows, sheep, pigs, horses and goats, companion animals such as dogs and cats, exotic and/or zoo animals, laboratory animals including mice rats, rabbits, guinea pigs and hamsters; and poultry such as chickens, turkey ducks and geese.

[00376] The invention is illustrated by the following examples:

Examples

Summary of Examples

[00377] The expression levels of ASPP proteins are altered in tumours. Whilst ASPPl and ASPP2 are down regulated in a large percentage of tumours (Agirre et al., 2006; Bergamaschi et al., 2003; Liu et al., 2004; Lossos et al., 2002; Samuels-Lev et al., 2001), iASPP is up regulated in human breast carcinomas expressing wild-type p53 (Bergamaschi et al., 2003). iASPP is also overexpressed in acute leukemias regardless of p53 mutation status (Zhang et al., 2005), suggesting that iASPP may promote carcinogenesis by other mechanisms additional to p53 inhibition. As iASPP is functionally distinct from the other family members, and an inhibitor of p53 dependent apoptosis up regulated in cancer, a strategy based on antagonising the ability of iASPP to bind p53 could be an important target for treating cancer. With the exception of the well characterised ASPP2-p53 interaction (Gorina and Pavletich, 1996; Tidow et al., 2006) there is a paucity of information at the molecular level on the interactions of these protein families.

[00378] A solid phase binding assay (Example 1) revealed that the C-terminal region of iASPP (residues 625-828, named iASPPΔ625), like its homologues, is able to interact with the DNA binding domains of p63 and p73. However, there were significant differences in the equilibrium binding constants for iASPPΔ625 and the homologous region in ASPP2. To further characterise the p53 binding site of iASPP we determined a high resolution (2.1 A) crystal structure of the iASPP C-terminal domain (residues 608-828, named 1ASPPΔ6O8) - see Example 2. A fortuitous lattice packing in the iASPPΔ608 crystals revealed that the N-terminal residues of one molecule can interact with the putative p53 binding site of a second molecule.

[00379] Further studies set out in Example 3 using isothermal titration calorimety (ITC) confirmed that an 8-mer iASPP peptide corresponding to residues 616-623 can indeed bind the C-terminal domain of iASPP with a binding affinity comparable to previously measured SH3- peptide interactions (Dalgarno et al., 1997)..

Example 1. Differential binding of iASPP andASPP2 to the p53 family

[00380] We investigated whether the C-terminal domains of iASPP and ASPP2; iASPP residues 625-828 (iASPPΔ625) and ASPP2 residues 905-1128 (ASPP2Δ905) bind the core

DNA binding domains of p53, p63 and p73; p53 residues 94-292 (p53_core), p63 residues 123-323

(p63_core), and p73 residues 112-312 (p73_core), with varying affinities using a solid phase binding assay.

[00381] For solid phase binding experiments two ASPP constructs; iASPPΔ625 and ASPP2Δ905 encoding iASPP residues 625-628 and ASPP2 residues 905-1128, respectively were made using the bacterial expression vector pET22b (Novagen). Nucleotides encoding the required amino acids were amplified by PCR introducing a hexa-histidine sequence in 5' and a stop codon in 3', cloned into the expression vector using Ndel and EcoRI restriction sites and sequenced. Constructs encoding the DNA binding domain of p53 residues 94-292 (p53_core), p63 residues 123-323 (p63_core) and p73 residues 112-312 (p73_core), used in solid phase binding assays, were also made using pET22b essentially as for ASPP constructs but without the incorporation of a hexa-histidine coding sequence.

[00382] ASPP proteins were expressed in the E.coli strain Rosetta pLysS (Invitrogen), bacteria were grown in Luria broth (LB) with 100 μg/ml ampicillin and protein expression induced by addition of 0.25 mM ispropyl-β-D-thiogalactopyranoside (IPTG). Bacterial pellets were suspended in ice cold high salt PBS (500 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄/NaH₂PO₄ pH 8.0), lysed by sonication and clarified by centrifugation. Recombinant proteins were purified from the soluble fraction by Ni²⁺ affinity chromatography then further purified by size exclusion chromatography into the appropriate buffer. The recombinant proteins p53_core, p63_core and p73_core were purified as previously described for the DNA binding domain of p53 (Derbyshire et al., 2002).

[00383] Purified iASPPΔ625, ASPP2Δ905, p53_core, p63_core and p73_core were buffer exchanged by size exclusion chromatography into PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄/NaH₂PO₄ pH 7.4). The DNA binding domains of p53, p63 and p73 were immobilised on 96-well microtiter plates (Nunc, PolySorp), 10 pM per well, over-night at 4 °C. Remaining binding sites were saturated by one hour incubation at 4 °C in PBS containing 1% bovine serum albumin (BSA). Microtiter plates were incubated for one hour at 4 °C with a dilution series of either iASPPΔ625 or ASPP2Δ905 then incubated with 1/1000 dilution of monoclonal antibody solution (anti-iASPP LX142.1 [raised against iASPPΔ625] or anti-ASPP2 LX141.2 [raised against ASPP2Δ905]), plates were then probed for one hour at 4 °C with an anti-mouse IgG alkaline phosphatase conjugate antibody (Sigma- Aldrich). The bound alkaline phosphatase conjugate antibody was detected by the addition of the substrate p-Nitrophenyl Phosphate. Progress curves were recorded for forty minutes at room temperature, at 405 nm, using a Thermo Labsystems Multiskan Ascent microplate reader and the initial rates were determined using the SigmaPlot 10 software (Systat Software Inc.). Each data point represents the average of duplicate wells, + the standard deviation. Non specific binding was determined from iASPPΔ625 or ASPP2Δ905 bound to BSA and was subtracted from each data point.

[00384] Data points were plotted, rate term against ligand concentration, resulting in saturation curves. Scatchard analysis was also undertaken. In order to determine K_d values, saturation curves were fitted by non-linear regression analysis using the SigmaPlot 10 software.

[00385] Non linear regression fitting of saturation curves revealed that iASPPΔ625 and ASPP2Δ905 bind the DNA binding domains all three p53 family members (Figure 1). Both iASPPΔ625 and ASPP2Δ905 bind p53_core with a similar high affinity, equilibrium dissociation constants ( K_d values) measured were 26.4 + 1.7 nM and 23.3 + 1.6 nM respectively. Values measured for iASPPΔ625 and ASPP2Δ905 binding p63_core and p73_core showed significant differences; iASPPΔ625 binds p63_core and p73_core with an affinity approximately three fold higher than that of ASPP2Δ905 and both iASPPΔ625 and ASPP2Δ905 have a three fold higher affinity for p63_core over p73_core. K_d values for iASPPΔ625 and ASPP2Δ905 binding p63_core were 128.1 + 11 nM and 343.1 + 39.8 nM respectively and for p73_core, 331.3 + 66 nM and 984.4 + 196.1 nM respectively. Example 2. Crystal Structure of the C-terminal region ofiASPP [00386] The crystal structure of the C-terminal domain of iASPP residues 608-828 (iASPPΔ608), was determined by molecular replacement and refined to 2.1 A resolution. For crystallization experiments the construct iASPPΔ608 encoding human iASPP residues 608-828 of SEQ ID NO: 8 was made using the same strategy as for the other ASPP constructs as described in Example 1.

[00387] Prior to crystallization iASPPΔ608 was concentrated by ultrafiltration in size exclusion buffer (150 mM NaCl, 25 mM Tris pH 8.0, 1 mM DTT) to 8 mg/ml. Sitting drop vapour diffusion crystallization trials (drop size: 100 nl protein plus 100 nl reservoir solution) were set up with a Cartesian robot as previously reported (Brown et al., 2003; Walter et al., 2003) . iASPPΔ608 initially crystallized at 20 °C out of a mother liquor containing 20% (w/v) PEG 3350, 0.2 M potassium chloride (Hampton Peg/Ion screen, reagent 8). Optimised crystals grow at 20 °C out of a mother liquor containing 22 % (w/v) PEG 3350, 0.3 M potassium chloride and 17.5 % MPD. The crystals cryo-protected in mother liquor diffracted to 2.1 A after flash-freezing at 100 K on beamline ID14EH2 at the ESRF. X-ray data were processed and scaled with the HKL suite (Otwinowski and Minor, 1997). The program XPREP (www.bruker- nonius.com) was used to calculate quality indicators and to merge data. Crystallographic statistics are shown in Table 11.

[00388] Numbers in parentheses refer to the appropriate outer shell, r.m.s.d.: root mean square deviation from ideal geometry.

[00389] ^aR_merge = ∑hkl ∑_ilI(hkl;ϊ) - <I(hkl)>\/Σ_hkl ∑_il(hkl;i), where I(hkl;i) is the intensity of an individual measurement and <I(hkl)> is the average intensity from multiple observations.

[00390] ^bR_factor =Σ_hklllF_obsl - kIF_calcll/Σ_hkl IF_obsl

[00391] ^cR_free equals the R-factor against 5% of the data removed prior to refinement.

[00392] The iASPPΔ608 crystal structure was determined using the molecular replacement method. Real space cross -rotation function, PC refinement of the rotation peaks, and fast F2F2 translation searches within a data range of 15-3 A with the program CNS (Briinger et al., 1998) using the structure coordinates of ASPP2 (PDB ID code IYCS, sequence identity: 53%) as the search model resulted in the correct orientation and position of the iASPP monomer in the crystallographic asymmetric unit. Refinement was carried out using program CNS by iterative cycles of simulated annealing, conjugate gradient minimization, individual B-factor refinement and manual rebuilding using program O (Jones et al., 1991). Final refinement resulted in an R- factor (R_free) of 20.7 % (25.2 %) (see also crystallographic statistics in Table 11). The stereochemical properties of the structure were assessed by PROCHECK (Laskowski et al., 1993) and WHATCHECK (Hooft et al., 1996) and showed no residue in disallowed regions of the Ramachandran plot. Structural superpositions were made using the program SHP (Stuart et al., 1979) and all figures were produced using Pymol (www.pymol.org).

[00393] The iASPPΔ608 crystal structure shows that the C-terminal domain of iASPP is made up of four ankyrin repeats and a closely juxtaposed SH3 domain that form a single structural unit with a significant buried surface area between the two protein motifs. The close proximity of the ankyrin repeats and the SH3 domain is a characteristic feature of ASPP proteins, indeed, so far only ASPP proteins have been shown to adopt a structural unit consisting of ankyrin repeats closely juxtaposed to an SH3 domain. Without structural information it is difficult to determine whether this domain arrangement is present in other proteins, as terminal ankyrin repeats usually deviate from the established consensus sequence and are often not recognised (Mosavi et al., 2004). However, the proteins CASKINl and CASKIN2 (Tabuchi et al., 2002) may contain a similar structural arrangement to that observed in the crystal structures of the C- terminal domains of iASPP and ASPP2. They are predicted to have six ankyrin repeats, then a 34 residue linker (the approximate average length of an ankyrin repeat) whose sequence is predicted to contain two helices, followed by an SH3 domain. The amino acid sequences which make up the C-terminal ankyrin repeat of iASPP and ASPP2 constitute two α-helices and lack a β-hairpin loop.

[00394] The data show that iASPPΔ625 and ASPP2Δ905 have a similar high affinity for p53_core, the Kd value determined for the ASPP2Δ905- p53_core interaction in our solid state binding assay (23 nM) is very similar to results obtained by (Gorina and Pavletich, 1996) using surface plasmon resonance (30 nM). iASPPΔ625 is able to interact with p63_core and p73_core with a three fold higher affinity than ASPP2Δ905, raising the possibility that iASPP, like ASPP2, plays a role in the regulation of p63 and p73 dependent apoptosis. ASPPl and ASPP2 have been shown to bind all p53 family members and stimulate the transactivation of function of p53, p63 and p73 on the promoters of apoptotic genes (Bergamaschi et al., 2004; Samuels-Lev et al., 2001). Our data now provide comparable biochemical affinity measurements for iASPP and ASPP2 binding p53, p63 and p73. Relative to functional data, analysis of our crystal structure suggests some reasons why compared to ASPP2Δ905, iASPPΔ625 has an approximate three fold greater affinity for p63_core and p73_core- In particular the SH3 domain of iASPP, unlike that of ASPP2, has a tyrosine (Tyr 814) that is likely to form classical interactions with the hydrophobic Po residue in the linear (peptide) motif of the L3 loop of p63 and p73. The 1ASPPΔ6O8 crystal packing suggests that a peptide based on the sequence of iASPP residues 616-623 would have the appropriate properties to bind to the C-terminal part of iASPP. This peptide binding site overlaps the putative binding site on iASPP for the DNA binding domain of p53 and thus such peptides (or polypeptide sequences) would be predicted to compete with p53 for iASPP binding.

Example 3: Binding of the Pl peptide

[00395] To further characterize the binding properties of the iASPPΔ608 N-terminus by isothermal titration calorimetry (ITC), the construct iASPPΔ625 lacking the interacting N- terminal residues was used and a peptide synthesized comprising iASPP residues 616-623 (peptide Pl). [00396] The Pl peptide (SPRKARRA; SEQ ID NO:2) was synthesized using standard technology and purified by high-pressure liquid chromatography (ALTA Bioscience, Birmingham). Experiments were performed using a VP-ITC microcalorimeter (MicroCal, Northampton, MA). Purified iASPPΔ625 was dialysed into a buffer consisting of 50 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄/NaH₂PO₄ pH 8.0. The dialysis buffer was also used to dissolve the peptide. At a concentration of 1.5 mM Pl was titrated from a syringe (300 μl total volume) into a sample cell containing 1.4 ml iASPPΔ625 (0.1 mM). Prior to experiments both peptide and protein solutions were clarified for 10 min at 16100 g and degassed (Thermo Vac; MicroCal). At the equilibrium temperature of 15°C, peptide Pl was titrated into iASPPΔ625 by 44 injections of 5 μl each. Resulting peaks of measured deviations from the equilibrium temperature were integrated to yield the quantity of heat generated. The control experiment; injecting the peptide at the above concentration into buffer alone, using the same experimental conditions, to find the heat generated by peptide dilution, was performed and the integrated data were subtracted from peptide into protein data. Heat generated by protein dilution was determined by injecting buffer alone into a sample chamber filled with iASPPΔ625 and was found to be negligible. Data were fitted using χ2 minimization on a model assuming a single set of binding sites to calculate the dissociation constant, K_d. All steps of the data analysis were performed using ORIGIN (W.0) software provided by MicoCal.

[00397] As illustrated in Figure 5, the results indicate that each iASPP molecule binds one peptide molecule with an estimated K_d of 45 μM. This is the average of three separate experiments.

Sequences

[00398] SEQ ID NO: 1 - Fragment of iASPP. Residues 8 to 228 of SEQ ID NO: 1 correspond to amino acids 608-828 of iASPP (NP_006654, SEQ ID NO: 8). Residues 2-7 are a hexahistidine tag.

MHHHHHHRSV LRKAGSPRKA RRARLNPLVL LLDAALTGEL EVVQQAVKEM NDPSQPNEEG 60

ITALHNAICG ANYSIVDFLI TAGANVNSPD SHGWTPLHCA ASCNDTVICM ALVQHGAAIF 120

ATTLSDGATA FEKCDPYREG YADCATYLAD VEQSMGLMNS GAVYALWDYS AEFGDELSFR 180

EGESVTVLRR DGPEETDWWW AALHGQEGYV PRNYFGLFPR VKPQRSKV 228

[00399] SEQ ID NO:2 - Peptide Pl - SPRKARRA

[00400] SEQ ID NO:3 - Peptide Pl Ala5Ser - SPRKSRRA [00401] SEQ ID NO:4 - Peptide Pl Lys4Arg - SPRRARRA

[00402] SEQ ID NO:5 - Peptide Pl Lys4Arg+Ala5Ser - SPRRSRRA

[00403] SEQ ID NO: 6- X₁X₂X₃X₄X₅X₆X₇X₈, wherein X₁ is selected from Serine or Threonine or conservative substitution threof; X₂ is proline or conservative substitution thereof; X₃, X₄, X₆, and X₇ are each independently selected from Arginine and Lysine; X₅ is Alanine, Serine, Valine, Glycine or conservative substitutions thereof; and X₈ is Alanine, Valine, Glycine or conservative substitutions thereof;

[00404] SEQ ID NO 7 - full-length iASPP polynucleotide sequence

[00405] SEQ ID NO: 8 - full-length iASPP amino acid sequence

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[00458] All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described invention will be apparent to those of skill in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments.

Claims

What is claimed is:

1. An isolated compound having a spatial arrangement of atoms to bind to iASPP in a manner that interferes with iASPP binding to an iASPP binding partner selected from the group consisting of p53, p63 and p73,

wherein the compound interacts with an iASPP polypeptide defined by at least 10 structural coordinates identified in Table 1, wherein the relative atomic positions of the structure identified in Table 1 are varied within a root mean square deviation of less than 1.2A.

2. The isolated compound of claim 1, wherein the compound interacts with the iASPP in a region of iASPP defined by at least 10 coordinates of atoms from at least five different amino acids of Table 3, wherein the relative atomic positions are varied within a root mean square deviation of less than 1.2A.

3. The isolated compound of claim 1, wherein the compound interacts with the iASPP in a region of iASPP defined by at least 10 coordinates of atoms from at least 10 different amino acid residues identified in Table 3, wherein the relative atomic positions are varied within a root mean square deviation of less than 1.2A.

4. The isolated compound of claim 3, wherein the at least 10 amino acids of iASPP are selected from the group consisting of T722, L724, W767, E772, D775, E776, E795, D797, W798, Y809, P811, N813 and Y814.

5. The isolated compound of claim 1, wherein the compound interacts with the iASPP in a region of iASPP defined by at least 20 coordinates of atoms from at least ten different amino acids of Table 3, wherein the relative atomic positions are varied within a root mean square deviation of less than 1.2A.

6. The isolated compound of claim 1, wherein the compound interacts with the iASPP in a region of iASPP defined by at least 50 coordinates of atoms from at least ten different amino acids of Table 3, wherein the relative atomic positions are varied within a root mean square deviation of less than 1.2A.

7. An isolated compound of any one of claims 1-6 having a structure selected from the group consisting of: (a) a compound that comprises a sequence of eight amino acids X1-X2-X3-X4- X6-X7-X8, wherein Xl is selected from Ser or Thr; X2 is Pro; X3, X4, X6, and X7 are each independently selected from Arg and Lys; X5 is Ala, Ser; VaI, or GIy; and X8 is Ala, VaI, or GIy;

(b) a compound with the structure of (a), with the proviso that one or more of X1-X2-X3-X4-X6-X7-X8 amino acids are replaced with an unnatural amino acid; and

(c) a compound with the structure of (a) or (b), with the proviso that one or more amide bonds is replaced with an ester or alkyl bond.

8. An isolated compound of claim 7 that comprises a sequence of eight amino acids X1-X2-X3-X4-X6-X7-X8, wherein Xl is selected from Ser or Thr; X2 is Pro; X3, X4, X6, and X7 are each independently selected from Arg and Lys; X5 is Ala, Ser; VaI, or GIy; and X8 is Ala, VaI, or GIy.

9. The isolated compound of any one of claims 1-6, wherein the compound is selected from the group consisting of a small molecule, an antibody or an antigen-binding fragment thereof, a nucleic acid molecule, and a polypeptide.

10. The isolated compound of claim 9, wherein the compound is a polypeptide.

11. The isolated compound of claim 10, wherein the polypeptide comprises an amino acid sequence at least 80% identical to SEQ ID NO: 2.

12. The isolated compound of claim 9, wherein the compound is a nucleic acid.

13. The isolated compound of claim 9, wherein the compound is an antibody or an antigen binding fragment thereof.

14. The isolated compound of claim 13, wherein the antibody or antigen binding fragment thereof specifically binds to an iASPP epitope defined by at least 10 coordinates of atoms from at least 10 different amino acid residues identified in Table 3, wherein the relative atomic positions are varied within a root mean square deviation of less than 1.2A.

15. The isolated compound of claim 13, wherein the antibody or antigen binding fragment thereof specifically binds to an epitope that includes one or more iASPP amino acids selected from the group consisting of T722, L724, W767, E772, Oil 5, E116, E795, D797, W798, Y809, P811, N813 and Y814.

16. The isolated compound of any one of claims 13-15, wherein the antibody is a monoclonal antibody.

17. The isolated compound of claim 16, wherein the antibody is a human antibody or a humanized antibody.

18. A hybridoma cell that produces the antibody of claim 16 or 17.

19. An isolated compound that comprises a sequence of eight amino acids X1-X2-X3-X4-X6-X7-X8, wherein

Xl is selected from Ser or Thr;

X2 is Pro;

X3, X4, X6, and X7 are each independently selected from Arg and Lys;

X5 is Ala, Ser; VaI, or GIy; and

X8 is Ala, VaI, or GIy;

wherein the compound inhibits binding between iASPP and an iASPP binding partner selected from the group consisting of p53, p63, and p73.

20. The compound of claim 19 that comprises an amino acid sequence at least 80% identical to iASPP amino acids 616-623 (SEQ ID NO: 2).

21. The compound of claim 19 that comprises iASPP amino acids 616-623 (SEQ ID NO: 2).

22. The isolated compound of claim 20, wherein the polypeptide comprises the amino acid sequence SPRKSRRA (SEQ ID NO: 3).

23. The isolated compound of claim 20, wherein the polypeptide comprises the amino acid sequence SPRRARRA (SEQ ID NO: 4).

24. The isolated compound of claim 20, wherein the polypeptide comprises the amino acid sequence SPRRSRRA (SEQ ID NO: 5).

25. The isolated compound of any one of claims 1-24, wherein the compound further comprises at least one attached polyalkyleneglycol moiety.

26. The isolated compound of any one of claims 1-25, wherein the compound further includes at least one esterified acid side chain.

27. The isolated compound of any one of claims 1-26, wherein the compound is cyclized.

28. The isolated compound of any one of claims 1-27, wherein the compound further comprises an attached cytotoxic moiety.

29. The isolated compound of any one of claims 1-28, wherein the compound comprises a peptide.

30. A composition comprising the compound of any one of claims 1-29, and a pharmaceutically acceptable carrier, diluent or excipient.

31. A nucleic acid molecule comprising a nucleotide sequence that encodes the isolated peptide of claim 29.

32. The nucleic acid of claim 31, further comprising a promoter that promotes expression of the nucleotide sequence in cells of the mammalian subject.

33. A vector comprising the nucleic acid of claim 31 or 32.

34. The vector of claim 33, selected from the group consisting of a replication deficient retrovirus, adenovirus, adeno-associated virus, or lentivirus vector.

35. A host cell transformed or transfected with a nucleic acid or vector according to any one of claims 31-34

36. A method of treating a neoplastic disorder comprising:

administering to a subject in need of treatment for a neoplastic disorder the composition of claim 30, in an amount effective to treat the neoplastic disorder.

37. The method of claim 36 further comprising a step, prior to the administering step, of identifying a subject with a neoplastic disorder.

38. The method of claim 37, wherein the identifying step comprises identifying a subject with a neoplastic disorder characterized by elevated expression of an iASPP polypeptide in the neoplastic cells.

39. The method of claim 38, wherein the identifying step comprises screening a biological sample suspected of containing neoplastic cells from the subject for elevated expression of the iASPP polypeptide.

40. The method of claim 39, wherein the screening comprises comparing expression of iASPP in cells from the sample with expression of iASPP from healthy cells of the same type as the cells in the sample, wherein increased expression of the iASPP in cells from the sample compared to the expression of the iASPP in cells from the healthy cells indicates elevated expression of iASPP.

41. The method of any one of claims 36-40, wherein the neoplastic disorder is a cancer selected from the group consisting of breast cancer, pancreatic cancer, ovarian cancer, colorectal cancer, esophageal cancer, lung cancer, head cancer, neck cancer, gastric cancer and epithelial cancer.

42. The method of any one of claims 36-40, further comprising administering to the mammalian subject a standard of care antineoplastic therapy.

43. The method of claim 42, wherein the antineoplastic therapy is selected from the group consisting of a standard of care chemo therapeutic, a standard of care radiotherapeutic, or a standard of care radiation regimen for the neoplastic disorder.

44. A method of stimulating p53-mediated apoptosis in a cell, comprising contacting the cell with the composition according to claim 30 in an amount effective to stimulate p53-mediated apoptosis in the cell.

45. The method of claim 44, wherein the cell expresses wild type p53.

46. A method of selecting a therapeutic regimen for a subject comprising (a) identifying a subject as having neoplastic disorder characterized by increased expression of an iASPP polypeptide; and

(b) administering to the subject the composition according to claim 30.

47. The method of claim 46, wherein the identifying step comprises screening a biological sample from the subject for increased expression of the iASPP polypeptide.

48. The method of claim 47, wherein the screening comprises comparing expression of iASPP in cells from the sample with expression of iASPP from healthy cells of the same type as the cells in the sample, wherein increased expression of the iASPP in cells from the sample compared to the expression of the iASPP in cells from the healthy cells indicates that the cell is characterized by increased expression of iASPP.

49. A method of treating a condition associated with tumor cell growth in a subject comprising administering to said subject a therapeutically effective amount of a combination therapy comprising agents selected from the group consisting of:

(a) a composition according to claim 30; and

(b) a standard of care anti-neoplastic therapy.

50. The method of claim 49, wherein the antineoplastic therapy is selected from the group consisting of a standard of care chemo therapeutic, a standard of care radiotherapeutic, or a standard of care radiation regimen for the neoplastic disorder.

51. The method of claim 49, wherein the agent(s) are administered in amounts effective to inhibit neoplastic cell growth in the subject.

52. A method of screening for a modulator of binding of iASPP with an iASPP binding partner, comprising:

(a) contacting an iASPP polypeptide comprising a structure defined by the structural coordinates provided in Table 1 with an iASPP binding partner, in the presence and absence of a test compound, under conditions in which the binding partner binds with the iASPP polypeptide; wherein the binding partner comprises a member selected from the group consisting of p53, p63 and p73;

(b) comparing binding between said iASPP polypeptide and the binding partner in the presence and absence of the test compound, wherein increased binding identifies the test compound as an agonist of binding, and decreased binding identifies the test compound as an antagonist of binding.

53. A computer-based method for the analysis of the interaction of a molecular structure with an iASPP structure, the method comprising

(a) fitting a molecular structure to an iASPP structure defined by the structural coordinates identified in Table 1 or selected coordinates thereof, wherein the iASPP structure is optionally varied by a root square mean deviation of not more that 1.2A, and

(b) determining at least one interaction between an atom of said iASPP structure and said molecular structure.

54. A computer-based method of rational drug design comprising:

(a) fitting structure of at least two molecular fragments to an iASPP structure defined by the structural coordinates identified in Table 1 or selected coordinates thereof, wherein the iASPP structure is optionally varied by a root square mean deviation of not more that 1.2A, and

(b) assembling the fitted molecular structure fragments into a single molecule to form a single molecular structure.

55. The method of claim 53 or 54, wherein said selected coordinates of the iASPP structure include atoms from one or more of the amino acid residues identified in Table

3.

56. The method of any one of claims 53-55, wherein the selected coordinates comprise at least 5 atoms.

57. The method of any one of claims 53-56, wherein said molecular structure comprises at least two atoms that are located in the same relative spatial orientation to each other and are of the same elements as a corresponding number of atoms found in any of the amino acids identified in Table 4.

58. The method of claim 57, wherein the atoms are selected from the atoms identified in Table 5.

59. The method of claim 58, wherein said atoms of Table 5 include at least twp atoms that contact iASPP residues via hydrogen bonds.

60. The method of any one of claims 53-59, wherein the molecular structure is in the form of a pharmacophore.

61. The method of any one of claims 53-60, wherein the iASPP structure is a model constructed from all or a portion of the coordinates of Table 1, optionally varied by a root mean square deviation of not more than 0.5A.

62. The method of claim 61, wherein the model is selected from the group consisting of a wire-frame model, a chicken- wire model, a ball-and-stick model, a space-filling model, a stick-model, a ribbon model, a snake model, an arrow and cylinder model, an electron density map and a molecular surface model.

63. The method of any one of claims 53-62, further comprising modifying the molecular structure to modulate its interaction with the iASPP structure.

64. A computer system, intended to generate structures and/or perform optimization of compounds that interact with an iASPP C-terminal region, the system containing computer-readable data comprising the atomic coordinate data identified in Table 1 or selected coordinates thereof, optionally varied by a root mean square deviation of not more than 1.2A.

65. The computer system of claim 64, wherein said atomic coordinate data comprises at least one of the atoms provided by the amino acid residues identified in Tables 3 or

4.

66. The computer system of claim 64 or 65 comprising:

(a) a computer-readable data storage medium comprising data storage material encoded with said computer-readable data;

(b) a working memory for storing instructions for processing said computer- readable data; and

(c) a central-processing unit coupled to said working memory and to said computer-readable storage medium for processing said computer-readable data.

67. The computer system of claim 66, further comprising a display coupled to said central-processing unit.

68. A method for providing data for generating structures and/or performing optimization of compounds which interact with an iASPP C-terminal region, the method comprising

(a) establishing communication with a remote device containing computer- readable data comprising atomic coordinate data identified in Table 1 or selected coordinate thereof, optionally varied by a root mean square deviation of not more than 1.2A. and

(b) receiving said computer readable data from said remote device.

69. The method of claim 68, further comprising processing said computer- readable data to display a model of an iASPP C-terminal region.

70. A computer-readable storage medium comprising a data storage material encoded with computer-readable data, wherein the data are defined by the structure identified in

Table 1, optionally varied by a root mean square deviation of not more than 1.2A.

71. A computer-readable storage medium comprising a data storage material encoded with a first set of computer-readable data comprising a Fourier transform of at least a portion of the structural coordinates for the iASPP C-terminal protein defined by the structure identified in Table 1, or selected coordinates thereof, optionally varied by a root mean square deviation of not more than 1.2A; which data, when combined with a second set of machine readable data comprising an X-ray diffraction pattern of a molecule or molecular complex or unknown structure, using a maching programmed with the instructions for using said first set of data and said second set of data, can determine at least a portion of the structure coordinates corresponding to the second set of machine readable data.