WO2002002622A2 - Crystal structure of survivin - Google Patents

Abstract

Provided is the structure of an inhibitor of apoptosis protein (IAP). Å 2.58 A crystal structure of a human survivin point mutant (L54M) determined by Multiple Wavelength Anomalous Dispersion (MAD) using the endogenously bound Zn+2 ions is provided. Methods of using the crystal structure and atomic coordinates fo the development of IAP binding agents is also provided. In addition, the disclosure provides computer programs on computer readable medium for use in developing iAP binding agents.

Description

CRYSTAL STRUCTURE OF SURVIVIN

RELATED APPLICATIONS

This application claims the benefit of U.S. Application No. 09/608,352, filed June 29, 2000, now pending, which is hereby incorporated by reference herein in its entirety.

ACKNOWLEDGMENT

This invention was made with United States Government support under Grants No. GM-57533 and CA-80100, awarded by the National Institutes of Health. The Government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to crystals of the inhibitors of apoptosis protein (IAP) family and more particularly to the high resolution structure of survivin obtained by X-ray diffraction. In addition, the invention relates to methods of using the structure coordinates of the survivin IAP and mutants thereof to screen and design compounds that bind to or interact with IAP proteins and IAP protein family members.

BACKGROUND

Advances in molecular biology have allowed the development of biological agents useful in modulating protein or nucleic acid activity or expression, respectively. Many of these advances are based on identifying the primary sequence of the molecule to be modulated. For example, determining the nucleic acid sequence of DNA or RNA allows the development of antisense or ribozyme molecules. Similarly, identifying the primary sequence allows for the identification of sequences that may be useful in creating monoclonal antibodies. However, often the primary sequence of a protein is insufficient to develop therapeutic or diagnostic molecules due to the secondary, tertiary or quartenary structure of the protein from which the primary sequence is obtained. The process of designing potent and specific inhibitors or activators has improved with the arrival of techniques for

MISSING AT THE TIME OF PUBLICATION

determining the three-dimensional structure of an enzyme or poiypeptide to be modulated.

Cells die as a result of many factors and processes. One process is apoptosis. The apoptosis process, or programmed cell death, often occurs so rapidly that in some biological systems the apoptotic process is difficult to ascertain. Indeed, it has been only in the past few years that the involvement of apoptosis in a wide spectrum of biological processes has become recognized. Apoptosis is a fundamental physiological pathway of cell death, highly conserved throughout evolution, and plays a major role in development, viral pathogenesis, cancer, autoimmune diseases and neurodegenerative disorders.

Inappropriate changes in apoptosis may cause or contribute to a variety of diseases, including AIDS, neurodegenerative diseases (e.g. Alzheimer's Disease, Parkinson's Disease, Amyotrophic Lateral Sclerosis (ALS)), retinitis pigmentosa and other diseases of the retina, myelodysplastic syndrome (e.g., aplastic anemia), toxin- induced liver disease (e.g., alcoholism), ischemic injury (e.g., myocardial infarction, stroke, and reperfusion injury), and the like. In addition, disruption of normally occurring apoptosis has been implicated in the development of some cancers (e.g., follicular lymphoma, p53 carcinomas, and hormone dependent tumors), autoimmune disorders (e.g., lupus erythematosis and multiple sclerosis), viral infections (e.g., herpes virus, poxvirus, and adenovirus infections), and the like.

Survivin (16.5 kDa) is an inhibitor of apoptosis protein (IAP) family member that temporally and spatially localizes to microtubule organizing centers (MTOC) during mitosis (Li, F. et al. Nature 396:580-583, 1998). Localization of survivin to this spindle apparatus is functionally linked to its ability to circumvent both Bax and Fas induced programmed cell death (Tamm, I. et al. Cancer Res. 58:5315-5320, 1998).

IAPs are characterized by the presence of one or more baculovirus IAP repeat (BIR) domains. These 70-residue zinc-binding modules often function as potent inhibitors of cell death proteases (Listen, P. et al. Nature 379:349-353, 1996; Uren, A. G. et al. Proc. Natl. Acad. Sci USA 93:4974-4978, 1996). In many cases, IAPs also contain a caspase recruiting domain (CARD), and a RING finger domain (Deveraux and Reed, Genes Dev. 13:239-252, 1999). Human survivin, 142 residues in length, contains a single BIR domain located in its N-terminal half and a C-terminal region predicted to form a coiled-coil. Survivin is unique among IAPs in that it is undetectable in normal differentiated tissue but highly expressed in the developing embryo and in rapidly dividing cells (Ambrosini et al, Nature Med.3:917-921, 1997).

The design of new, highly specific agents capable of modulating apoptosis represents an important need in the pharmaceutical industry. Such agents can serve as effective chemotherapeutic agents for the treatment of a variety of disorders characterized by inappropriate cell proliferation, including cancer and infectious diseases. The invention disclosed herein addresses this and related needs, as will become apparent upon review of the specification and appended claims.

SUMMARY OF THE INVENTION

In an effort to elucidate IAPs' critical role in proliferating cells, a 2.58 A crystal structure of a human survivin point mutant (L54M) determined by Multiple Wavelength Anomalous Dispersion (MAD) using the endogenously bound Zn⁺² ions is provided. Methods of using the crystal structure and atomic coordinates for the development of IAP binding agents are also provided. In addition, the disclosure provides computer programs on computer readable medium for use in developing IAP binding agents useful in modulating apoptosis and treating cell proliferative disorders.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 collectively shows the overall architecture of human survivin. Figure la shows a ribbon representation of the survivin dime . The Zn⁺² ion is shown as a sphere. Coordination bonds are shown as dotted spheres. Two monomers are depicted. Figure lb is an orthogonal view of the ribbon representation shown in Figure la. Figure lc, shows a GRASP representation of the survivin solvent accessible surface shaded to reflect the underlying electrostatic surface, where shaded areas are positive or negative, and white is neutral. The orientation is the same as in Figure la. Figure Id is an orthogonal view of that shown in Figure lc. Figure le is a close-up view of the dimer interface comprising the intermolecular β-sheet. Alpha carbons are numbered and side chains are omitted for clarity. Boxed numbers correspond to alpha carbons in one survivin monomer. Hydrogen bonds are shown as dotted spheres. The orientation is identical to that shown in Figures la and lc. Figure If is a close-up view of the dimer interface comprising the hydrophobic contacts. Side chains numbered and boxed correspond to the same monomer as in Figure le. The orientation is identical to that shown in Figures lb and Id.

Figure 2 shows the sequence alignment of six representative BIR domain containing proteins. Secondary structural elements for survivin are shown and the analogous features for XIAP BIR2 are depicted in gray shaded text. Residues in dark gray boxes correspond to hydrophobic amino acids at the dimer interface, inverted white on black text corresponds to residues in the basic patch; hatched boxes correspond to amino acids comprising the zinc coordination sphere; boxes with asterisks correspond to residues in the acidic patch; the dark gray boxes in the 5^th and 6^th rows from the bottom are putative phosphorylation sites; light gray boxes delineate positions along α6 forming a hydrophobic patch; and gray-boxed text depict positions previously shown to participate in caspase inhibition, h refers to human survivin (SEQ ID NO: 3), m refers to mouse survivin (SEQ ID NO: 5), c refers to C. elegans survivin.

Figure 3 collectively is an enlarged view of survivin's sub-domains. Figure 3a, is a perspective and close-up view of the Zn⁺² binding site on one survivin monomer. The depicted orientation corresponds to that pictured in Figure la. Figure 3b is a perspective and close-up view of the sulphate binding site. The depicted orientation corresponds to that pictured in Figure lb. Figure 3c shows an expanded view of one survivin monomer illustrating the location of the α6 hydrophobic surface. The orientation corresponds to that shown in Figure lb.

Figure 4 shows an example of a computer system in block diagram form. DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, a method of predicting a binding agent for an inhibitor of apoptosis protein (IAP) is provided. The method comprises modeling a potential binding agent that interacts with one or more functional domains of a survivin poiypeptide (ie., an LAP), defined by a plurality of atomic coordinates of the survivin poiypeptide, and determining the ability of the potential binding agent to modulate a survivin biological function (e.g., apoptosis), thereby predicting an IAP bmding agent.

In another aspect, the invention provides a computer program on a computer readable medium, the computer program having instructions to cause a computer to model a potential binding agent that can bind an LAP molecule defined by a plurality of atomic coordinates.

An IAP poiypeptide typically has at least one BIR domain and a ring zinc finger domain which is capable of modulating (inhibiting or enhancing) apoptosis in a cell or tissue. An IAP gene or poiypeptide also includes any member of the family of apoptosis inhibitory genes characterized by their ability to modulate apoptosis and having at least 20%, preferably 30%, and more preferably 50% amino acid sequence identity to at least one of the conserved regions of one of the IAP members described herein (e.g., either the BIR or ring zinc finger domains from xiap, hiapl and hiap2, m-xiap, a C-terminal helix structure of survivin, and the like). Representative members of the IAP gene family include, for example, the xiap, hiapl, and hiap2 genes of humans, the m-xiap gene of the mouse, and the like. By 'TAP protein" is meant a poiypeptide encoded by an IAP gene.

A "BIR domain" typically has in the range of 65 up to 68 amino acid residues and has an amino acid consensus sequence of: Xaal Xaal Xaal Arg Leu Xaal Thr Phe Xaal Xaal Trp Pro Xaa2 Xaal Xaal Xaa2 Xaa2 Xaal Xaal Xaal Xaal Leu Ala Xaal Ala Gly Phe Tyr Tyr Xaal Gly Xaal Xaal Asp Xaal Val Xaal Cys Phe Xaal Cys Xaal Xaal Xaal Xaal Xaal Xaal Trp Xaal Xaal Xaal Asp Xaal Xaal Xaal Xaal Xaal His Xaal Xaal Xaal Xaal Pro Xaal Cys Xaal Phe Val, wherein Xaal is any a ino acid and Xaa2 is any amino acid or is absent (SEQ ID NO:l). A "ring zinc finger" or "RZF" typically has in the range of 45 to 46 amino acid residues and has a consensus sequence of: Glu Xaal Xaal Xaal Xaal Xaal Xaal Xaa2 Xaal Xaal Xaal Cys Lys Xaa3 Cys Met Xaal Xaal Xaal Xaal Xaal Xaa3 Xaal Phe Xaal Pro Cys Gly His Xaal Xaal Xaal Cys Xaal Xaal Cys Ala Xaal Xaal Xaal Xaal Xaal Cys Pro Xaal Cys, wherein Xaal is any amino acid, Xaa2 is Glu or Asp, and Xaa3 is Val or He (SEQ ID NO: 2).

By "modulating apoptosis" is meant increasing or decreasing the number of cells which undergo apoptosis in a given cell population. Typically the cell population is selected from T-cells, neuronal cells, fibroblasts, or any other cell line known to undergo apoptosis in a laboratory setting (e.g., the baculovirus infected insect cells). It will be appreciated that the degree of modulation provided by an IAP , or modulating agent (e.g., a binding agent, inhibitor, or activator) will vary and will depend upon the assay conditions. An inhibitor of apoptosis includes any agent that decreases the number of cells which undergo apoptosis relative to an untreated control.

A poiypeptide is a chain of amino acids, regardless of length or post- translational modification (e.g., glycosylation or phosphorylation). A poiypeptide or protein refers to a polymer in which the monomers are amino acid residues which are joined together through amide bonds. When the amino acids are alpha-amino acids, either the L-optical isomer or the D-optical isomer can be used, the L-isomers being typical. An IAP or survivin poiypeptide is intended to encompass an amino acid sequence as set forth in SEQ ID NOs: 3 or 4, mutants, variants and conservative substitutions thereof comprising L- or D- a ino acids and include modified forms thereof, such as glycoproteins. Accordingly, the polypeptides of the invention are intended to cover naturally occurring proteins, as well as those which are recombinantly or synthetically synthesized. Poiypeptide or protein fragments are also encompassed by the invention. Fragments can have the same or substantially the same amino acid sequence as the naturally occurring protein. A poiypeptide or peptide having substantially the same sequence means that an amino acid sequence is largely, but not entirely, the same, but retains a functional activity of the sequence to which it is related. In general polypeptides of the invention include peptides, or full length protein, that contain substitutions, deletions, or insertions into the protein backbone, that would still have an approximately 70%-90% homology to the original protein over the corresponding portion. A yet greater degree of departure from homology is allowed if like-a ino acids, ie. conservative amino acid substitutions, do not count as a change in the sequence.

A poiypeptide which is substantially related to a naturally occuring protein but for a conservative variation is also contemplated to be within the scope of the present invention. A conservative variation denotes the replacement of an amino acid residue by another, biologically similar residue. Examples of conservative variations include the substitution of one hydrophobic residue such as isoleucine, valine, leucine or methiorune, for another hydrophobic residue, or the substitution of one polar residue for another, such as the substitution of arginine for lysine, glutamic for aspartic acids, or glutamine for asparagine, and the like. Other illustrative examples of conservative substitutions include the changes of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine, glutamine, or glutamate; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; valine to isoleucine to leucine. The term "conservative variation" also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid provided that antibodies raised to the substituted poiypeptide also immunoreact with the unsubstituted poiypeptide.

The term "positively charged amino acid" includes any naturally occurring or unnatural amino acid having a positively charged side chain under normal physiological conditions. Examples of positively charged naturally occurring amino acids are arginine, lysine and histidine.

The term "negatively charged amino acid" includes any naturally occurring or unnatural amino acid having a negatively charged side chain under normal physiological conditions. Examples of negatively charged naturally occurring amino acids are aspartic acid and glutamic acid. The term "hydrophobic amino acid" means any amino acid having an uncharged, nonpolar side chain that is relatively insoluble in water. Examples of naturally occurring hydrophobic amino acids are alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine.

The term "hydrophiUc a ino acid" means any amino acid having an uncharged, polar side chain that is relatively soluble in water. Examples of naturally occurring hydrophiUc amino acids are serine, threonine, tyrosine, asparagine, glutamine, and cysteine.

Modifications and substitutions are not limited to replacement of amino acids. For a variety of purposes, such as increased stability, solubiUty, or configuration concerns, one skilled in the art will recognize the potential value of introducing, (by deletion, replacement, or addition) other modifications. Examples of such other modifications include incorporation of rare amino acids, D-amino acids, glycosylation sites, cytosine for specific disulfide bridge formation, and the like. The modified peptides can be chemically synthesized, or the isolated gene can be site-directed mutagenized, or a synthetic gene can be synthesized and expressed in bacteria, yeast, baculovirus, tissue culture, and the like. An example of a modification providing increased solubility includes the L54M mutation (SEQ ID NO: 4) described below. The mutation increases the hydrophilic nature of the survivin poiypeptide compared to the wild type poiypeptide. Accordingly, other modifications which alter the hydrophiUc nature or hydrophobic nature of the survivin poiypeptide are encompassed by the present invention.

IAP or survivin polypeptides of the invention include survivin polypeptides from invertebrates, mammals and humans and include sequences as set forth in SEQ ID NOs: 3, 4, and 5, as well as sequences that have at least 70% homology to the sequences of SEQ ID NOs: 3, 4, and 5, fragments, variants, or conservative substitutions of any of the foregoing sequences. Other survivin related poiypeptide sequences are appUcable to the methods of the present invention (see, for example, Conway et al. Blood, 95(4):1435-1442 (2000), which is incorporated by reference herein). The term "variant" refers to polypeptides which are modified at one or more amino acid residues yet still retain the biological activity of an IAP or survivin poiypeptide. Variants can be produced by any number of means known in the art, including, for example, such methods as error-prone PCR, shuffling, oUgonucleotide- directed mutagenesis, assembly PCR, sexual PCR mutagenesis, and the like, as well as any combination of two or more thereof.

By "substantiaUy identical" is meant a poiypeptide or nucleic acid exhibiting at least 50%, preferably 85%, more preferably 90%, and most preferably 95% homology to a reference amino acid or nucleic acid sequence.

Homology or identity is often measured using sequence analysis software (e.g.,

Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705). Such software matches similar sequences by assigning degrees of homology to various deletions, substitutions and other modifications. The terms "homology" and "identity" in the context of two or more nucleic acids or poiypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same when compared and aligned for maximum correspondence over a comparison window or designated region as measured using any number of sequence comparison algorithms or by manual alignment and visual inspection.

For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

A "comparison window", as used herein, includes reference to a segment of any one of the number of contiguous positions falling in the range of about 20 to about 600, usually from about 50 to about 200, more usually from about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aUgned. Methods of aJignment of sequences for comparison are well-known in the art. Optimal aUgnment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482, 1981, by the homology aUgnment algorithm of Needleman & Wunsch, J. Mol. Biol 48:443, 1970, by the search for similarity method of person & Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WT), by manual aUgnment and visual inspection, and the like. Other algorithms for determining homology or identity include, for example, in addition to a BLAST program (Basic Local AUgnment Search Tool at the National Center for Biological Information), ALIGN, AMAS (Analysis of Multiply AUgned Sequences), AMPS (Protein Multiple Sequence AUgnment), ASSET (Aligned Segment Statistical Evaluation Tool), BANDS, BESTSCOR, BIOSCAN (Biological Sequence Comparative Analysis Node), BLIMPS (BLocks IMProved Searcher), FASTA, Intervals & Points, BMB, CLUSTAL V, CLUSTAL W, CONSENSUS, LCONSENSUS, WCONSENSUS, Smith- Waterman algorithm, DARWIN, Las Vegas algorithm, FNAT (Forced Nucleotide Alignment Tool), FrameaUgn, Framesearch, DYNAMIC, FILTER, FSAP (Fristensky Sequence Analysis Package), GAP (Global AUgnment Program), GENAL, GIBBS, GenQuest, ISSC (Sensitive Sequence Comparison), LALIGN (Local Sequence AUgnment), LCP (Local Content Program), MACAW (Multiple Alignment Construction & Analysis Workbench), MAP (Multiple Alignment Program), MBLKP, MBLKN, PIMA (Pattern-Induced Multi-sequence AUgnment), SAGA (Sequence AUgnment by Genetic Algorithm) and WHAT-IF. Such aUgnment programs can also be used to screen genome databases to identify polynucleotide sequences having substantially identical sequences. A number of genome databases are available, for example, a substantial portion of the human genome is available as part of the Human Genome Sequencing Project (J. Roach, http://weber.u. Washington.edu/~roach/ human_ genome_progress 2.html) (Gibbs, 1995). At least twenty-one other genomes have already been sequenced, including, for example, M. genitalium (Fraser et al, 1995), M. jannaschu (Bult et al, 1996), H. influenz e (Fleischmann et al, 1995), E. coli (Blattner et al, 1997), and yeast (S. ceremsiae) (Mewes et al, 1997), and D. melanogaster (Adams et al, 2000). Significant progress has also been made in sequencing the genomes of model organisms, such as mouse, C. elegans, and Arabadopsis sp. Several databases containing genomic information annotated with some functional information are maintained by different organizations, and are accessible via the internet, for example, http://wwwtigr.org/tdb; http://www.genetics.wisc.edu; http://genome- www.stanford.edu/~baU; http://hiv-web.lanl.gov; http://www.ncbi.nlm.nih.gov; http://www.ebi.ac.uk; http://Pasteur.fr/other/biology; and http:// www.genome.wi.mit.edu.

Examples of useful algorithms are BLAST and BLAST 2.0 algorithms, which are described in Altschul et al, Nuc. Acids Res.25:3389-3402, 1977, and Altschul et al, J. Mol. Biol. 215:403-410, 1990, respectively. Software for performing BLAST analyses is pubUcly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih. gov). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aUgned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative aUgnment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of rmtching residues; always >0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative aUgnment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the aUgnment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=-4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectations (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915, 1989) aUgnments (B) of 50, expectation (E) of 10, M=5, N= -4, and a comparison of both strands.

The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Natl. Acad. Sci. USA 90:5873, 1993). One measure of similarity provided by the BLAST algorithm is the smallest sum probabiUty (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smaUest sum probabiUty in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

In one embodiment, protein and nucleic acid sequence homologies are evaluated using the Basic Local Alignment Search Tool ("BLAST"). In particular, five specific BLAST programs are used to perform the foUowing task:

(1) BLASTP and BLAST3 compare an amino acid query sequence against a protein sequence database;

(2) BLASTN compares a nucleotide query sequence against a nucleotide sequence database;

(3) BLASTX compares the six-frame conceptual translation products of a query nucleotide sequence (both strands) against a protein sequence database;

(4) TBLASTN compares a query protein sequence against a nucleotide sequence database translated in aU six reading frames (both strands); and

(5) TBLASTX compares the six-frame translations of a nucleotide query sequence against the six-frame translations of a nucleotide sequence database. The BLAST programs identify homologous sequences by identifying similar segments, which are referred to herein as "high-scoring segment pairs," between a query amino or nucleic acid sequence and a test sequence which is preferably obtained from a protein or nucleic acid sequence database. High-scoring segment pairs are preferably identified (i.e., aligned) by means of a scoring matrix, many of which are known in the art. Preferably, the scoring matrix used is the BLOSUM62 matrix (Gonnet et al, Science 256:1443-1445, 1992; Henikoff and Henikoff, Proteins 17:49-61, 1993). Less preferably, the PAM or PAM250 matrices may also be used (see, e.g., Schwartz and Dayhoff, eds., 1978, Matrices for Detecting Distance Relationships: Atlas of Protein Sequence and Structure, Washington: National Biomedical Research Foundation). BLAST programs are accessible through the U.S. National Library of Medicine, e.g., atwww.ncbi.nlm.nih.gov.

The parameters used with the above algorithms may be adapted depending on the sequence length and degree of homology studied. Ln some embodiments, the parameters may be the default parameters used by the algorithms in the absence of instructions from the user.

By a "substantially pure poiypeptide" is meant an IAP poiypeptide which has been separated from components which naturaUy accompany it. TypicaUy, the poiypeptide is substantiaUy pure when it is at least 60%, by weight, free from the proteins and naturally-occurring molecules with which it is naturaUy associated. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, IAP poiypeptide. A substantiaUy pure IAP poiypeptide may be obtained, for example, by extraction from a natural source; by expression of a recombinant nucleic acid encoding an IAP poiypeptide; or by chemicaUy synthesizing the protein. Purity can be measured by any appropriate method (e.g., column chromatography, polyacrylamide gel electrophoresis, by HPLC analysis, and the Uke).

One aspect of the invention resides in obtaining crystals of the IAP poiypeptide survivin of sufficient quaUty to determine the three dimensional (tertiary) structure of the protein by X-ray diffraction methods. The knowledge obtained concerning the three-dimensional structure of survivin can be used in the determination of the three dimensional structure of other IAP proteins. The binding agent can also be predicted by various computer models. Based on the structural coordinates of the survivin poiypeptide (i.e., the three dimensional protein structure), as described herein, small molecules which mimic or are capable of interacting with a functional domain of an IAP molecule can be designed and synthesized to modulate IAP biological functions (e.g., modulate apoptosis). Accordingly, in one embodiment, the invention provides a method of "rational" drug design. Another approach to "rational" drug design is based on a lead compound that is discovered using high throughput screens; the lead compound is further modified based on a crystal structure of the binding regions of the molecule in question. Accordingly, another aspect of the invention is to provide material which is a starting material in the rational design of drugs which mimic or prevent the action of an IAP (e.g., a survivin molecule).

In one embodiment, a survivin monomer has an amino acid sequence as set forth in SEQ ID NO: 3. The term "amino acids" means the L-isomers of the naturaUy occurring amino acids or unnatural amino acids. The naturaUy occurring amino acids are glycine, alanine, valine, leucine, isoleucine, serine, rnethionine, threonine, phenylalanine, tyrosine, tryptophan, cysteine, proline, histidine, aspartic acid, asparagine, glutamic acid, glutamine, γ-carboxyglutamic acid, arginine, ornithine and lysine. Unless specificaUy indicated, all amino acids referred to in this application are in the L-f orm.

The term "unnatural amino acids" means amino acids that are not naturaUy found in proteins. Examples of unnatural amino acids used herein, include racemic mixtures of selenocysteine and selenomethionine. In addition, unnatural amino acids include the D forms of amino acids, D or L forms of nor-leucine, para- nitiophenylalanine, homophenylalanine, para-fluorophenylalanine, 3-amino-2- benzylpropionic acid, homoarginine, and D-phenylalanine.

The term "crystal structure coordinates" refers to mathematical coordinates derived from mathematical equations related to the patterns obtained on diffraction of a monochromatic beam of X-rays by the atoms (scattering centers) of an IAP poiypeptide (e.g., a survivin protein molecule) in crystal form. The diffraction data are used to calculate an electron density map of the repeating unit of the crystal. The electron density maps are used to estabUsh the positions of the individual atoms within the unit cell of the crystal. The crystal structure coordinates of an IAP can be obtained from a Survivin protein crystal having space group C2 (a = 114.040 A, b = 71.45 A, c = 86.63, β = 133.370°). The coordinates of the survivin poiypeptide can also be obtained by means of computational analysis.

The term "selenon ethionine substitution" refers to the method of producing a chemicaUy modified form of a crystal of survivin. The survivin protein is expressed by bacteria in media that is depleted in methionine and supplemented with selenoniethionine. Selenium is thereby incorporated into the crystal in place of the sulfur of methionine. The location(s) of selenium are determined by X-ray diffraction analysis of the crystal. This information is used to generate the phase information used to construct a three-dimensional structure of the protein.

The term "heavy atom derivatization" refers to the method of producing a chemicaUy modified form of a crystal of survivin. A crystal is soaked in a solution containing heavy metal atom salts or organometaUic compounds, which can diffuse through the crystal and bind to the surface of the protein. The location(s) of the bound heavy metal atom(s) are determined by X-ray diffraction analysis of the soaked crystal. This information is used to generate the phase information used to construct a three-dimensional structure of the protein.

Those of skill in the art understand that a set of structure coordinates determined by X-ray crystaUography is not without standard error.

The term "unit ceU" refers to the basic paraUeUpiped shaped block. The entire volume of a crystal may be constructed by regular assembly of such blocks.

The term "space group" refers to the arrangement of symmetry elements of a crystal.

The crystal structure coordinates of the IAP poiypeptide survivin can be used to design compounds that bind to the protein and alter its physical or physiological properties in a variety of ways. The structure coordinates of the protein can also be used to computationally screen smaU molecule data bases for agents that bind to the poiypeptide to develop IAP modulating or binding agents.

Those of skill in the art may identify binding agents or modulatory agents as inhibitors or activators by computer fitting kinetic data using standard equations according to Segel, I. H., Enzyme Kinetics, J. Wiley & Sons, (1975).

Methods of using crystal structure data to design inhibitors or binding agents are known in the art. Thus, the crystal structure data provided herein can be used in the design of new or improved inhibitors. For example, the survivin poiypeptide coordinates can be superimposed onto other available coordinates of similar enzymes which have inhibitors bound to them to give an approximation of the way these and related inhibitors might bind to survivin. Alternatively, computer programs employed in the practice of rational drug design can be used to identify compounds that reproduce interaction characteristics similar to those found between a survivin poiypeptide and a co-crystaUized substrate. Furthermore, detaUed knowledge of the nature of binding site interactions aUows for the modification of compounds to alter or improve solubiUty, pharmacokinetics, etc. without affecting binding activity.

Computer programs are widely available that are capable of carrying out the activities necessary to design agents using the crystal structure information provided herein. Examples include, but are not Umited to, the computer programs listed below:

Catalyst Databases™ - an information retrieval program accessing chemical databases such as BioByte Master File, Derwent WDI and ACD;

Catalyst/ HYPO™ - generates models of compounds and hypotheses to explain variations of activity with the structure of drug candidates;

Ludi™ - fits molecules into the active site of a protein by identifying and matching complementary polar and hydrophobic groups;

Leapfrog™ - "grows" new Ugands using a genetic algorithm with parameters under the control of the user. In addition, various general purpose machines may be used with programs written in accordance with the teachings herein, or it may be more convenient to construct more specialized apparatus to perform the operations. However, preferably the embodiment is implemented in one or more computer programs executing on programmable systems each comprising at least one processor, at least one data storage system (including volatile and non-volatile memory and/ or storage elements), at least one input device, and at least one output device. The program is executed on the processor to perform the functions described herein.

Each such program may be implemented in any desύed computer language (including machine, assembly, high level procedural, or object oriented prograrnrning languages) to communicate with a computer system. In any case, the language may be a compiled or interpreted language. The computer program will typicaUy be stored on a storage media or device (e.g., ROM, CD-ROM, or magnetic or optical media) readable by a general or special purpose programmable computer, for configuring and operating the computer when the storage media or device is read by the computer to perform the procedures described herein. The system may also be considered to be implemented as a computer-readable storage medium, configured with a computer program, where the storage medium so configured causes a computer to operate in a specific and predefined manner to perform the functions described herein.

Embodiments of the invention include systems (e.g., internet based systems), particularly computer systems which store and manipulate the coordinate and sequence information described herein. One example of a computer system 100 is iUustrated in block diagram form in Figure 4. As used herein, "a computer system" refers to the hardware components, software components, and data storage components used to analyze the coordinates and sequences such as those set forth in Table 1. The computer system 100 typicaUy includes a processor for processing, accessing and manipulating the sequence data. The processor 105 can be any weU- known type of central processing unit, such as, for example, the Pentium III from Intel Corporation, or similar processor from other suppUers such as Sun, Motorola, Compaq, AMD or International Business Machines. TypicaUy the computer system 100 is a general purpose system that comprises the processor 105 and one or more internal data storage components 110 for storing data, and one or more data retrieving devices for retrieving the data stored on the data storage components. A skilled artisan can readily appreciate that any one of the currently available computer systems are suitable.

In one particular embodiment, the computer system 100 includes a processor 105 connected to a bus which is connected to a main memory 115 (preferably implemented as RAM) and one or more internal data storage devices 110, such as a hard drive and/ or other computer readable media having data recorded thereon. In some embodiments, the computer system 100 further includes one or more data retrieving device(s) 118 for reading the data stored on the internal data storage devices 110.

The data retrieving device 118 may represent, for example, a floppy disk drive, a compact disk drive, a magnetic tape drive, a modem capable of connection to a remote data storage system (e.g., via the internet), and the Uke. Ln some embodiments, the internal data storage device 110 is a removable computer readable medium such as a floppy disk, a compact disk, a magnetic tape, and the Uke, containing control logic and/ or data recorded thereon. The computer system 100 may advantageously include or be programmed by appropriate software for reading the control logic and/ or the data from the data storage component once inserted in the data retrieving device.

The computer system 100 includes a display 120 which is used to display output to a computer user. It should also be noted that the computer system 100 can be linked to other computer systems 125a-c in a network or wide area network to provide centralized access to the computer system 100.

Software for accessing and processing the coordinate and sequences of Table 1,

(such as search tools, compare tools, and modeling tools etc.) may reside in main memory 115 during execution.

For the first time, the present invention permits the use of molecular design techniques to design, select and synthesize chemical entities and compounds, including inhibitory compounds, capable of binding to an IAP poiypeptide (e.g., a survivin poiypeptide), in whole or in part.

One approach enabled by this invention, is to use the structure coordinates as set forth in Table 1 to design compounds that bind to the poiypeptide and alter the physical properties of the compounds in different ways, e.g., solubiUty. For example, the present invention enables the design of compounds that act as inhibitors of IAP biological function by binding to aU, or a portion of, an LAP molecule.

Ln another approach a survivin poiypeptide crystal is probed with a variety of different chemical entities to determine optimal sites for interaction between candidate binding molecules (e.g., inhibitors) and the survivin (ie., IAP poiypeptide).

In another embodiment, an approach made possible and enabled by the present invention, is to screen computationaUy small molecule data bases for chemical entities or compounds that can bind in whole, or in part, to an LAP poiypeptide or fragment thereof. Ln this screening, the quaUty of fit of such entities or compounds to the binding site may be judged in a variety of ways, e.g., by shape complementarity or by estimated interaction energy (Meng, E. C. et al, J. Comp. Chem., 13:505-524, 1992).

Survivin is one member of a family of LAP polypeptides, many of which have similar functional activities. Various IAP polypeptides may crystallize in more than one crystal form. Accordingly, the structure coordinates of survivin, or portions thereof, as provided by this invention are particularly useful to solve the structure of other crystal forms of LAP molecules. They may also be used to solve the structure of an IAP or a survivin mutant.

One method that may be employed for this purpose is molecular replacement. The term "molecular replacement" refers to a method that involves generating a preliminary model of a crystal whose structure coordinates are not known, by orienting and positioning a molecule whose structure coordinates are known. Phases are then calculated from this model and combined with observed ampUtudes to give an approximate Fourier synthesis of the structure whose coordinates are known. Using this method, the unknown crystal structure, whether it is another LAP crystal form, an LAP or survivin mutant, or an IAP complexed with a substrate or other molecule, or the crystal of some other protein with significant amino acid sequence homology to any IAP poiypeptide, may be determined using the structure coordinates as provided in Table 1. This method wiU provide an accurate structural form for the unknown crystal more quickly and efficiently than attempting to determine such information ab initio.

TABLE 1. Atomic Coordinates

In addition, in accordance with this invention, an IAP or survivin poiypeptide mutant may be crystallized in association or complex with known IAP binding agents, substrates, or inhibitors. The crystal structures of a series of such complexes may then be solved by molecular replacement and compared with that of a wild-type IAP molecule. Potential sites for modification within the LAP molecule may thus be identified. This information provides an additional tool for deterrnining the most efficient binding interactions, for example, increased hydrophobic interactions, between an IAP and a chemical entity or compound.

AU of the complexes referred to above may be studied using weU-known X-ray diffraction techniques and may be refined versus 2-3 A resolution X-ray data to an R value of about 0.20 or less using computer software, such as X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc.). See, e.g., Blundel & Johnson, supra; Methods in Enzymology, vol. 114 and 115, H. W. Wyckoff et al, eds., Academic Press (1985). This information may thus be used to optimize known classes of LAP binding agents or substrates (e.g., inhibitors), and to design and synthesize novel classes of IAP binding agents (e.g., inhibitors).

The design of compounds or binding agents that bind to or inhibit an LAP poiypeptide according to the invention generaUy involves consideration of two factors. First, the compound or binding agent must be capable of physicaUy and structuraUy associating with an IAP molecule. Non-covalent molecular interactions important in the association of an LAP with a substrate include hydrogen bonding, van der Waals and hydrophobic interactions, and the like.

Second, the compound or binding agent must be able to assume a conformation that aUows it to associate with an IAP molecule. Although certain portions of the compound or binding agent wiU not directly participate in this association, those portions may still influence the overaU conformation of the molecule. This, in turn, may have a significant impact on potency. Such conformational requirements include the overaU three-dimensional structure and orientation of the chemical entity or compound in relation to aU or a portion of the binding site, e.g., active site or accessory binding site of an LAP poiypeptide (e.g., a survivin poiypeptide), or the spacing between functional groups of a compound comprising several chemical entities that directly interact with an LAP.

The potential inhibitory or binding effect of a chemical compound on an IAP may be analyzed prior to its actual synthesis and testing by the use of computer modeling techniques. If the theoretical structure of the given compound suggests insufficient interaction and association between it and an IAP, synthesis and testing of the compound may be obviated. However, if computer modeling indicates a strong interaction, the molecule may then be tested for its abiUty to bind to an LAP. Methods of assaying for LAP activity are known in the art (as identified and discussed herein). Methods for assaying the effect of a potential binding agent can be performed in the presence of a known binding agent of an LAP. For example, the effect of the potential binding agent can be assayed by measuring the abiUty of the potential binding agent to compete with a known binding agent.

An inhibitory or other binding compound of an IAP may be computationally evaluated and designed by means of a series of steps in which chemical entities or fragments are screened and selected for their abiUty to associate with the individual binding pockets or other areas of an LAP.

One skilled in the art may use one of several methods to screen chemical entities or fragments for their abiUty to associate with an IAP and more particularly with the individual binding pockets of a survivin poiypeptide. This process may begin by visual inspection of, for example, the active site on the computer screen based on the survivin coordinates in Table 1. Selected fragments or chemical entities may then be positioned in a variety of orientations, or docked, within an individual binding pocket of an IAP. Docking may be accompUshed using software such as Quanta and Sybyl, foUowed by energy rriinirnization and molecular dynamics with standard molecular mechanics forcefields, such as CHARMM and AMBER.

Specialized computer programs may also assist in the process of selecting fragments or chemical entities. These include:

1. GRID (Goodford, P. J., "A Computational Procedure for Deterrnining EnergeticaUy Favorable Binding Sites on BiologicaUy Important Macromolecules", J. Med. Chem., 28, pp. 849-857 (1985)). GRID is available from Oxford University, Oxford, UK.

2. MCSS (Miranker, A. and M. Karplus, "FunctionaUty Maps of Binding Sites: A Multiple Copy Simultaneous Search Method." Proteins: Structure. Function and Genetics, 11, pp. 29-34 (1991)) . MCSS is avaUable from Molecular Simulations, Burlington, Mass.

3. AUTODOCK (GoodseU, D. S. and A. J. Olsen, "Automated Docking of Substrates to Proteins by Simulated Annealing", Proteins: Structure. Function, and Genetics, 8, pp. 195-202 (1990)). AUTODOCK is available fro Scripps Research Institute, La JoUa, Calif .

4. DOCK (Kuntz, I. D. et al, "A Geometric Approach to Macromolecule-Ligand Interactions", J. Mol. Biol., 161, pp. 269-288 (1982)). DOCK is avaUable from University of California, San Francisco, Calif.

Once suitable chemical entities or fragments have been selected, they can be assembled into a single compound or binding agent (e.g., an inhibitor). Assembly may be performed by visual inspection of the relationship of the fragments to each other on the three-dimensional image displayed on a computer screen in relation to the structure coordinates of the survivin molecule as set forth in Table 1. This would be foUowed by manual model building using software such as Quanta or Sybyl.

Useful programs to aid one of skiU in the art in connecting the individual chemical entities or fragments include:

1. CAVEAT (Bartlett, P. A. et al, "CAVEAT: A Program to FaciUtate the Structure-Derived Design of BiologicaUy Active Molecules". Ln "Molecular Recognition in Chemical and Biological Problems", Special Pub., Royal Chem. Soc, 78, pp. 182-196 (1989)). CAVEAT is available from the University of California, Berkeley, Calif.

2. 3D Database systems such as MACCS-3D (MDL Information Systems, San Leandro, Calif.). This area is reviewed in Martin, Y. C, "3D Database Searching in Drug Design", J. Med. Chem., 35, pp. 2145-2154 (1992)). 3. HOOK (avaUable from Molecular Simulations, BurUngton, Mass.).

In addition to the method of building or identifying an IAP binding agent in a step-wise fashion one fragment or chemical entity at a time as described above, inhibitory or other IAP interaction compounds may be designed as a whole or "de novo" using either an empty active site or optionaUy including some portion(s) of a known inhibitor(s). These methods include:

1. LUDI (Bohm, H.-J., "The Computer Program LUDI: A New Method for the De Novo Design of Enzyme Inhibitors", J. Comp. Aid. Molec. Design, 6, pp. 61-78 (1992)). LUDI is available from Biosym Technologies, San Diego, Calif.

2. LEGEND (Nishibata, Y. and A. Itai, Tetrahedron, 47, p. 8985 (1991)). LEGEND is available from Molecular Simulations, Burlington, Mass.

3. LeapFrog (avaUable from Tripos Associates, St. Louis, Mo.).

Other molecular modeling techniques may also be employed in accordance with this invention. See, e.g., Cohen, N. C. et al, "Molecular Modeling Software and Methods for Medicinal Chemistry", J. Med. Chem., 33, pp. 883-894 (1990). See also, Navia, M. A. and M. A. Murcko, "The Use of Structural Information in Drug Design", Current Opinions in Structural Biology, 2, pp. 202-210 (1992).

Once a compound or binding agent has been designed or selected by the above methods, the efficiency with which that compound may bind to an IAP may be tested and optimized by computational evaluation.

A compound designed or selected as an LAP binding agent may be further computationaUy optimized so that in its bound state it would preferably lack repulsive electrostatic interaction with the target site. Such non-complementary (e.g., electrostatic) interactions include repulsive charge-charge, dipole-dipole and charge- dipole interactions. SpecificaUy, the sum of aU electrostatic interactions between the binding agent and the LAP when the binding agent is bound to the LAP, preferably make a neutral or favorable contribution to the enthalpy of binding. Specific computer software is available in the art to evaluate compound deformation energy and electrostatic interaction. Examples of programs designed for such uses include: Gaussian 92, revision C (M. J. Frisch, Gaussian, Lnc, Pittsburgh, Pa., 1992); AMBER, version 4.0 (P. A. KoUman, University of California at San Francisco, 1994); QUANTA/ CHARMM (Molecular Simulations, Inc., Burlington, Mass. 1994); and Insight π/Discover (Biosysm Technologies Lnc, San Diego, Calif., 1994). These programs may be implemented, for example, using a SiUcon Graphics workstation, IRIS 4D/35 or IBM RISC/ 6000 workstation model 550. Other hardware systems and software packages wiU be known to those skiUed in the art of which the speed and capacity are continuaUy modified.

Once an LAP binding agent has been selected or designed, as described above, substitutions may then be made in some of its atoms or side groups in order to improve or modify its binding properties. GeneraUy, initial substitutions are conservative, e.g., the replacement group wiU have approximately the same size, shape, hydrophobicity and charge as the original group. Such substituted chemical compounds may then be analyzed for efficiency of fit to an LAP by the same computer methods described, above.

Conserved regions of the IAP farrύly lend themselves to the methods and compositions of the invention. For example, recognition of mammalian IAP f armly members has provided emergent patterns of protem structure which can be used to design novel diagnostics and therapeutics as described herein. Recognition of patterns in this family aUows for the design of modulators of apoptosis.

Functional fragments of IAP polypeptides such as, for example, fragments of survivin can be designed based on the crystal structure and atomic coordinates described herein. Fragments of a survivin poiypeptide and the corresponding atomic coordinates of such fragments can be used in the modeling described herein. Ln addition, such fragments may be used to inhibit the apoptosis which occurs as part of disease or disorder processes. For example, a survivin fragment may be administered for the treatment of or prevention of apoptosis which occurs as a part of AIDS, neurodegenerative diseases, ischemic injury, toxin-induced Uver disease and myelodysplastic syndromes. In another embodiment of the present invention, the crystal structure and atomic coord ates are employed for the design of novel therapeutics. The apoptosis inhibiting capabUity of IAPs can be defined in an in vitro system known to detect alterations in apoptosis. Mammalian expression constructs carrying IAPs and their truncated forms can be introduced into various cell lines such as CHO, 3T3, HL60, Rat-1, or Jurkart ceUs, for example. In addition, SF21 sect ceUs may be used in which case the IAP gene is preferentiaUy expressed using an insect heat shock promoter. Apoptosis will then be induced in transfected cells and controls employing standard methodologies (e.g., serum withdrawal and staurosporine). A survival index (ratio of surviving transfected ceUs to surviving control ceUs) wUl indicate the strength of each IAP modulating or binding agent to inhibit or activate apoptosis. These experiments can confirm the presence of apoptosis inhibiting or enhancing activity and, can help to determine the minimal functional region of an IAP. Specific examples of apoptosis assays are provided in the foUowing references: Lymphocyte: C. J. Li et al, Science, 268:429-431, 1995; D. GibeUini et al, Br. J.

Haematol. 89:24-33, 1995; S. J. Martin et al, J. Immunol. 152:330-42, 1994; C. Terai et al, J. Clin Invest. 87:1710-5, 1991; J. Dhein et al, Nature 373:438-441, 1995; P. D. Katsikis et al, }. Exp. Med.1815:2029-2036, 1995; Michael O. Westendorp et al, Nature 375:497, 1995; DeRossi et al, Virology 198:234-44, 1994. Fibroblasts: H. Vossbeck et al, Int. J. Cancer 61:92-97, 1995; S. Goruppi et al, Oncogene 9:1537-44, 1994; A.

Fernandez et al, Oncogene 9:2009-17, 1994; E. A. Harrington et al, Embo J. 13:3286- 3295, 1994; N. Itoh et al, J. Biol. Chem. 268:10932-7, 1993. Neuronal CeUs: G. Melino et al, Mol. Cell. Biol. 14:6584-6596, 1994; D. M. Rosenbaum et al, Ann. Neurol. 36:864- 870, 1994; N. Sato et al, J. Neurobiol 25:1227-1234, 1994; G. Ferrari et al, J. Neurosci. 1516:2857-2866, 1995; A. K. TaUey et al, Mol. Cell Biol. 1585:2359-2366, 1995; A. K. TaUey et al, Mol. and Cell. Biol. 15:2359-2366, 1995; G. Walkinshaw et al, J. Clin. Invest. 95:2458-2464, 1995. Insect CeUs: R. J. Clem et al, Science 254:1388-90, 1991; N. E. Crook et al, J. Virol. 67:2168-74, 1993; S. Rabizadeh et al, J. Neurochem. 61:2318-21, 1993; M. J. Birnbaum et al, J. Virol 68:2521-8, 1994; R. J. Clem et al, Mol. CeU. Biol. 14:5212-5222, 1994.

An IAP modulating agent or apoptosis modulating agent may be administered with a pharmaceuticaUy-acceptable cUluent, carrier, or excipient, in unit dosage form. Conventional pharmaceutical practice may be employed to provide suitable formulations or compositions to administer to a subject suffering from or presymptomatic for a IAP-associated carcinoma, for example. Any appropriate route of administration may be employed, for example, parenteral, intravenous, subcutaneous, mtramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, mtracisternal, intraperitoneal, mtranasal, aerosol, oral adrninistration, or the like. Therapeutic formulations may be in the form of Uquid solutions or suspensions; for oral administration, formulations may be in the form of tablets, capsules or the Uke; and for intranasal formulations, in the form of powders, nasal drops, aerosols, or the Uke.

Methods weU known in the art for making formulations are found in, for example, Remington's Pharmaceutical Sciences, 15th ed. Easton: Mack I^bUshing Co., 1405-1412, 1461-1487 (1975) and The National Formulary XIV, 14th ed. Washington: American Pharmaceutical Association (1975), the contents of which are hereby incorporated by reference. Formulations for parenteral adrninistration may, for example, contain excipients, sterile water, or saline, polyalkylene glycols such as polyethylene glycol, oUs of vegetable origin, or hydrogenated naphthalenes. Biocompatible, biodegradable lacti.de polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compounds. Other potentiaUy useful parenteral deUvery systems for IAP modulatory agents include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, Uposomes, and the Uke. Formulations for inhalation may contain excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oUy solutions for administration in the form of nasal drops, or as a gel.

If desired, treatment with an IAP poiypeptide, fragment thereof, or modulatory compound may be combined with other therapies for the disease such as, for example, surgery, radiation, or chemotherapy for cancers; surgery, steroid therapy, and chemotherapy for autoimmune diseases; antiviral therapies for AIDS; and for example, TPA for ischemic injury. In addition, the binding agents identified by the methods of the invention can be used as a diagnostic in the detection or monitoring of conditions involving apoptosis associated disorders, IAP-associated disorders (e.g., a survivin-associated disorder). Accordingly, a decrease or increase in the level of IAP production may provide an indication of a deleterious condition. Levels of IAP expression may be assayed by any standard technique. For example, binding agents of the invention can be used in immunoassays to detect or monitor IAP protein in a biological sample. IAP-specific polyclonal or monoclonal antibodies (produced by methods known in the art) may be used in any standard immunoassay format (e.g., ELISA, Western blot, or RIA assay) to measure IAP poiypeptide levels; comparisons are made to wUd-type IAP levels, and a decrease in IAP production is indicative of a condition involving increased apoptosis.

The term "agent" as used herein describes any molecule, e.g. protein or pharmaceutical, with the capabiUty of altering or mimicking the physiological function or expression of an LAP or survivin poiypeptide. GeneraUy, a pluraUty of agents are run in paraUel at different concentrations to obtain a differential response to the various concentrations. Typically, one of these concentrations serves as a negative control, i.e. at zero concentration or below the level of detection.

Immunohistochemical techniques may also be utilized for IAP detection. For example, a tissue sample may be obtained from a patient, and a section stained for the presence of IAP using an anti-IAP antibody developed according to the methods of the invention and any standard detection system (e.g., one which includes a secondary antibody conjugated to horseradish peroxidase). General guidance regarding such techniques can be found in, e.g., Bancroft and Stevens (Theory and Practice of Histological Techniques, ChurchiU Livingstone, 1982) and Current Protocols in Molecular Biology, M. Ausubel et al, eds., (Current Protocols, a joint venture between Greene PubUshing Associates, Inc. and John WUey & Sons, Lnc, most recent Supplement).

The IAP diagnostic assays described above may be carried out using any biological sample (for example, any biopsy sample or bodUy fluid or tissue) in which IAP is normaUy expressed. In another embodiment, the invention provides a method for identifying an agent which interacts with or modulates expression or activity of an IAP or survivin poiypeptide. Such method comprises contacting an agent and an LAP or survivin poiypeptide, or a recombinant ceU expressing an LAP or survivin polypepti.de, under conditions sufficient to aUow the agent to interact and deterrnining the effect of the agent on the expression or activity of the poiypeptide. The term "effect", as used herein, encompasses any means by which protein activity can be modulated, and includes measuring the interaction of the agent with the IAP or survivin molecule by physical means including, for example, fluorescence detection of the binding of an agent to the poiypeptide. Such agents can include, for example, polypeptides, peptidomimetics, chemical compounds, smaU molecules and biologic agents. Examples of small molecules include but are not limited to smaU peptides or peptide-Uke molecules.

Contacting or incubating includes conditions which aUow contact between the test agent and an IAP or survivin poiypeptide, a ceU expressing an IAP or survivin poiypeptide or nucleic acid encoding a an LAP or survivin poiypeptide. Contacting includes in solution and in soUd phase. The test agent may optionaUy be a combinatorial Ubrary for screening a plurality of agents. Agents identified in the method of the invention can be further evaluated, detected, cloned, sequenced, and the like, either in solution or after binding to a soUd support, by any method usuaUy appUed to the detection of a specific DNA sequence such as PCR, oUgomer restriction (Sε kietal, Bio/ Technology, 3:1008-1012, 1985), oUgonucleotide Ugation assays (OLAs) (Landegren et al, Science, 241:1077, 1988), and the Like. Molecular techniques for DNA analysis have been reviewed (Landegren et al, Science, 242:229-237, 1988).

Thus, the method of the invention includes combinatorial chemistry methods for identifying chemical agents that bind to or affect an LAP or survivin poiypeptide expression or activity.

As yet another embodiment of the present invention, there are provided therapeutic methods which employ compounds and formulations as described herein. Agents that have been identified using invention methods can further be used to modulate an LAP or survivin poiypeptide function in targeted organisms. Of particular interest are agents that have a low toxicity or a reduced number of side effects for humans. ,

In addition, ceUs or organisms which have a mutations in an IAP or survivin poiypeptide sequence may be used as models to screen for agents which modulate disorders associated with the mutation. A variety of mutations may be generated in critical domains of the survivin molecule, for example, the dimerization domains as described in Example 2. Such mutants create changes in the dimerization potential of survivin, which may also affect survivin function and binding properties. These mutants are also useful in generating alternative crystal structures to further analyze agents that could modulate IAP function or disorders.

The invention provides the first demonstration that the LAP survivin requires dunerization for activity. Accordingly, agents that inhibit dimerization can modulate the activity of survivin. Thus, it is desirable to identify such compounds to modulate the activity of survivin by binding, interacting, or effecting the dimerized form of survivin or can bind to, interact with, or otherwise effect a subunit (e.g., a monomer) to prevent dimerization of the monomers thus preventing formation of survivin and thus modulating survivin activity. Mutants in the dimerization domain can also be used to identify such compounds.

The invention wiU now be described in greater detaU by reference to the foUowing non-limiting examples.

EXAMPLES

Example 1. Protein Purification

The cDNA of human survivin was amplified by PCR from a HeLa ceU cDNA library. The wUd-type and L54M point mutant (Quickchange, Stratagene) were expressed in E. coli using the pHIS8 expression vector encoding a thrombin cleavable N-terminal octahistidine tag (Jez et al, Biochemistry 39 890-902, 2000). The L54M point mutant has substituted the amino acid at position 54 from leucine to methionine. The foUowing mutants were sirrrilarly expressed, named X#Y, where the amino acid at position # has been substituted from X to Y; mutants with substitutions at more than one amino acid position are designated as X#Y/X#Y: W10A, T34E, H80A, H80A/E76A, T97E, W10A/L98A/F101R/L102S, L6G/W10A/L98A/F101R/L102S, W10A/F93A/L98R, and L6G/W10A/F93A/L98R. The deletion mutant Δ126-142 was constructed by deleting amino acids 126-142 from the wUd type survivin molecule.

Purification from E. coli lysates was accompUshed using Ni²⁺ chelation chromatography by standard procedures (Jez, supra.). The histidine tag was removed by thrombin (Sigma) digestion during dialysis in 50 mM Tris (pH 8.0), 500 mM NaCl, and 20 mM β-mercaptoethanol at 4°C for 24 h. Samples were purified further over a Superdex 20026/60 gel filtration column (Pharmacia) equiUbrated in the dialysis/ thiOmbin cleavage buffer. Peak fractions were coUected and dialyzed against 5 mM HEPES-Na⁺ (pH 7.5) and 1 mM DTT, concentrated to 15 mg/ml using CentriconlOs (Amicon), and stored at -70°C.

Example 2. Oligomer Characterization

The dimeric association of survivin was established using static light scattering (miniDawn, Wyatt Technology, CA). Further quantification was accomplished using equiUbrium sedimentation with the Beckman Optima XL-1 Analytical Ultracentifuge. Analysis was carried out at 20 °C using an An-60 Ti rotor. Survivin samples were diluted in 25 mM HEPES-Na⁺ (pH 7.5), 100 and 500 mM NaCl, and 1 mM DTT. Samples of 0.10, 0.25, and 0.5 mg ml-¹ were monitored by both absorbance at 280 nm and by interference with Rayleigh interference optics whUe being centrifuged at 10, 14, 20, and 28 x 10³ rpm for 20 h to reach equiUbrium. Data were analyzed using the Match7 and Reedit9 software (Jeff Lary, National Analytical Ultracentrifuge Facility).

Determination of the effective reduced molecular weight of survivin was carried out by the method of Johnson et al. (Biophys. J. 36:575-588, 1981) using the program Winnonln. Data were fit to the exponential function A = Ce^σ(r²/2 - r² _m/2), where A is the absorbance at 280 nm, C is the fitting constant, r is the radial position, r_m is the radius of the meniscus, and σ is the effective reduced molecular weight.

Sedimentation velocity runs and hydrodynamic modeling were carried out using wUd type survivin. Briefly, survivin was diluted to 1 mg ml-¹ in 25 mM HEPES-Na+ (pH 7.5), 100 mM NaCl, 1 mM DTT. The sample was centrifuged at 45,000 rpm for 4 h at 20 °C. Sedimentation was monitored by fringe displacement and absorbance at 280 nm. Data sets were coUected every 2 min. Determination of s^* from sedimentation velocity data was accomplished using the method of Philo (Biophys. J. 72:435-444, 1997) and Stafford (Anal. Biochem. 203:295-301, 1992) as incorporated in the programs SVEDBERG and DCDT. The partial specific volume of 0.73 cm³ g-¹ was calculated from the amino acid composition and used in hydrodynamic modeling with the program SEDNTERP.

To probe the relevance of each of the crystaUographically observed dimers in solution, a C-terminal truncation was constructed, purified, and analyzed hydrodynamicaUy. The deletion construct, Δ126-142, lacks aU of the C-terminal hydrophobic patch on α6, which mediates the only other symmetric lattice contact observed in the survivin crystal. Hydrodynamic characterization of Δ126-142 survivin should unequivocaUy delineate which of the two crystaUographicaUy observed dimer interfaces is consistent with the dimer formed in solution. The survivin coU-less mutant spanning residues 1-99 would effectively disrupt both possible interfaces. WUd type, the L54M mutant, and Δ126-142 survivin are dimers in solution (Table 2).

Sedimentation equUibrium analysis of the Δ126-142 survivin truncation suggests that the second crystaUographicaUy observed dimerization interface mediated by residues 126-142 is unlikely to occur in solution. AdditionaUy, sedimentation velocity experiments were used to measure wild type survivin's sedimentation coefficient (s*) of 3.167 ± 0.001 S and its frictional ratio (f/fo) of 1.201. Hydrodynamic modeling using these measurements and survivin's amino acid composition predict a prolate elUpsoid shape with a major axis of 111.2 A and a minor axis of 27.6 A. These values agree favorably with the measured tip-to-tip interheUcal distance of 111 A and the measured distance across the center of the survivin dimer of 26 A.

To probe the other crystaUographicaUy observed dimer interface, further point mutants of survivin were constructed and analyzed. These point mutants were characterized by gel fUtration chromatography, equiUbrium sedimentation analysis, and sedimentation velocity experiments. The average mass of these mutants as determined by sedimentation equiUbrium is shown in Table 2. Survivin molecules with multiple mutations are Usted with a "/" to indicate that more than one amino acid residue has been substituted.

TABLE 2. Average mass of survivin mutants determined by sedimentation equilibrium

Example 3. Crystallography

Crystals of survivin were grown in hanging drops at 4°C by mixing 1.0 μl of survivin with 1.0 μl of a reservoir solution containing 100 mM HEPES-Na⁺ (pH 7.5), 6-8% PEG 8000, 200 mM Li₂SO₄, and 2 mM dithiothreitol (DTT). Crystals were stabiUzed in 20% ethylene glycol, 100 mM HEPES-Na⁺, 10% PEG 8000, 500 mM Li₂SO , and 2 mM DTT and rapidly frozen in a 100 K stream of nitrogen gas. Multiple wavelength anomalous dispersion (MAD) data was coUected around the Zn edge at the Stanford Synchrotron Radiation Laboratory, beamline 9-2 (Table 3).

Data were processed with DENZO and SCALEPACK (Otwinowski et al, Meth. Enzymol. 276:307-326, 1997). The crystals contain two molecules per asymmetric unit (68% solvent) and belong to the space group C2 (a = 114.040 A, b - 71.45 A, c = 86.63, β = 133.370°). Three wavelength MAD data were scaled to the λ₃ data set usmg SCALEIT (CoUaborative Computational Project, Acta CrystaUogr. D. Biol. CrystaUogr. 50:760-763 ,1994). Both zinc sites were located using SOLVE (TerwiUiger et al, Acta CrystaUogr. D. Biol. CrystaUogr. 55:849-861, 1999) and verified by inspection of both dispersive and anomalous difference Patterson maps usmg XTALVIEW (McRee, J. Mol. Graph. 10:44-46, 1992). MAD phasing was accomplished using SHARP (de La ForteUe et al, Methods Enzymol. 276:472-494, 1997) and solvent flipping was carried out with SOLOMON (Abrahams et al, Acta CrystaUogr. D. Biol. CrystaUogr. 52:30-42, 1996).

The initial model was buUt into experimental electron density maps displayed in O (Jones et al, Acta CrystaUogr. D. Biol. CrystaUogr. 49:148-157, 1993). Two-fold averaging performed with DM (Cowtan et al, Acta CrystaUogr. D. Biol. CrystaUogr. 54:487-493, 1998), using a mask generated from a partial model (residues 10 to 70) with the CCP4 program MAPMASK (Collaborative Computational Project, supra), significantly improved the experimental maps. The model was rebuUt and then positionaUy refined against aU the data using both figure of merit weighted phases from averaging and the observed structure factor ampUtudes. A final round of refinement was accompUshed using the observed amplitudes only. AU refinements utilized the default bulk solvent model in CNS with maximum likelihood targets (Brunger et al, Acta CrystaUogr. D. Biol. CrystaUogr. 54:905-921, 1998).

The current model includes 2 survivin molecules (residues 5 to 140 and 6 to 140), 2 zinc ions, 99 water molecules, and 3 sulphates. PROCHECK (Laskowski et al, J. Appl. CrystaUogr. 26:283-291, 1993) revealed a total of 80.8% of the residues are in the most favored region of the Ramachandran plot, with 18.4% in the additionaUy aUowed regions, and 0.8% in the generously aUowed regions. Main chain and side chain structural parameters were consistently better than average (overaU G value of 0.17). Surface area and dimer contacts were determmed automaticaUy with CNS and then verified manuaUy in O.

Coordmates for the survivin dimer (accession code 1F3H) have been deposited in the Protein Data Bank.

TABLE 3. Data collection and refinement statistics

¹ Number in parenthesis is for highest resolution shell. ² Rs m = Σ I I - <I > I / ∑lh, where <I > is the average intensity over symmetry equivalent reflections.

³ Power of phasing = < | FH(caic) I / I E | >, where FH(caic) is the calculated difference and E is the lack of closure.

⁴ Rcu s = ∑ | E | / ∑ | FPH - FP | . ⁵ R-f actor - ∑ | F₀bs - Fcaic 1 / ∑Fobs, where summation is over the data used for refinement.

6 Rfree _was calculated using 5% of data excluded from refinement. Example 4. Casρase-3 Assay and Potential Binding Interactions

To test whether survivin physically interacts with caspase-3, in vitro binding assays were performed. The K_m (12μM) of caspase-3 for the tetrapeptide substrate, Z-DEVD-AFC, was determined by monitoring the initial enzymatic activity at room temperature in a reaction mixture containing 0.5 nM of recombinant caspase-3

(Calbiochem) and varying concentrations of Z-DEVD-AFC (Calbiochem) in 50 mM HEPES-Na⁺ (pH 7.5), 150 mM KC1, 0.1% CHAPS, 5% glycerol and 5 mM DTT on a PTI Alphascan spectrofluorimeter (Photon Technology Instruments, Santa Clara, CA). Inhibition of caspase-3 by the aldehyde tetrapeptide DEVD-CHO (Calbiochem) was determined by monitoring enzymatic activity at room temperature in a reaction mixture containing 0.5 nM caspase-3, 200 μM Z-DEVD-AFC, and varying concentrations of this inhibitor. The apparent K; value of 10 nM was determined by dividing the IC₅₀ by (1 + [S]/Km) as previously shown, using the reported K_m of caspase-3 (MM et al, J. Biol. Chem. 272:6539-6547, 1997).

Up to 30 μM of survivin exhibited no inhibitory effect on 50-500 pM caspase-

3. An immunoprecipitation assay was used to establish that recombinant survivin and its mutants did not interact with caspase-3 in vitro. Stoichiometric amounts of survivin were mixed and incubated on ice followed by immunoprecipitation using anti-survivin anti-serum. The precipitated proteins and supernatants were subjected to immunoblotting using anti-caspase-3 anti-serum. No interactions between caspase-3 and the various survivin constructs were detected by immunoprecipitation. Moreover, no proteolytic cleavage of survivin occurred over the time course of the experiments or over a 48 h period during which 1.0 μM survivin was incubated with 1.0 nM caspase-3.

Example 5

Survivin expression is regulated in a ceU cycle dependent manner with maximum levels occurring during the G2/M phase (Li et al, 1998, supra). Immunofluorescence and imaging experiments demonstrate co-localization of survivin with γ-tubuUn at the spindle centrioles where survivin forms a sheU with short radiating spokes around the γ-tubulin stained pericentriolar area. Additional studies revealed localization of both caspase-3 and the CDK inhibitor p21 wai/cipi o this same structure. Furthermore, loss of caspase-3 from the spindle centrioles occurred following introduction of survivin antisense DNA into ceUs (Li, F., et al. Nature Cell Bio. 1:461-466, 1999). Survivin's high expression level in maUgnant tissue, including breast, lung, prostate, colon, pancreas, and stomach as weU as neuroblastoma and lymphoma ceUs makes it an ideal target for cancer therapy (Tanaka, K. et al, Clin. Cancer Res. 6:127-134, 2000; Monzo, M. et al, J. Clinic Oncology 7:2100, 1999; Kawasaki, H. et al, Cancer Res. 58:5071-5074, 1998; Lu et al. Cancer Res. 58:1808-1812, 1998; Jaatela etal, Exp. CeU Res. 248:30-43, 1999).

BacteriaUy expressed fuU length human survivin and the L54M mutant form a 35-kDa dimer in solution. The L54M point mutant was initiaUy constructed to aid in MAD phasing using selenornetmonrne-substituted protein, however, selenomethionine substitution hindered crystal growth. Nevertheless, me unsubstituted L54M mutant crystalUzes isomorphously with wUd type survivin. Moreover, crystal quality was significantly higher yielding measurably better data. Characterization of the oUgomeric form of survivin was accompUshed using gel filtration chromatography, static Ught scattering, and equiUbrium sedimentation by analytical ultracentrUugation.

Examination of survivin's crystalline lattice reveals two distinct dimerization interfaces. One fairly Umited contact surface comprises the C-terminal haU of α6 spanning residues 126-142. This region constitutes the C-terminal hydrophobic patch likely to mediate localization to the spindle centrosome. The other crystaUographicaUy observed dimerization interface, utilizing residues 6 to 10 in the N-terminal portion of the BIR domain and a 14 amino acid region encompassing residues 89 to 102 located just after the BIR domain, is significantly more extensive (Figs. la-f). The chemical features of this protein-protein interaction and the 1000 A² of buried surface area of the dimerization interface bolsters the functional significance of this particular symmetric arrangement of monomers. The interfacial contacts are extensive considering the size of the survivin monomer, with residues 94 to 99 forming an intermolecular anti-paraUel β-sheet at the dimer juncture (Figs, la-b and le). Hydrophobic contacts dominate the interaction surface with Leu 98 protruding from one monomer and extending into a hydrophobic pocket formed by Leu 6, Trp 10, Phe 93, Phe 101, and Leu 102 on the neighboring molecule (Fig. If). Sequence alignments of these residues with other BIR domain sequences show that the murine homologue of survivin should form an analogous dimer (Fig. 2).

Survivin's BIR domain is composed of a three-stranded anti-paraUel β-sheet (residues 15 to 89) surrounded by four smaU α-heUces (Figs. la-b). The tertiary fold of survivin's BIR domain closely resembles the reported NMR structure of the BIR2 domain of XIAP with a larger central β-sheet architecture (Sun et al, Nature 401:818- 822, 1999). A zinc ion tetrahedraUy coordinated by Cys 57, Cys 60, His 77, and Cys 84 bridges the core β-sheet with α4 and α.5 (Fig. 3a). One of survivin's most striking features is its 65 A long C-terminal heUx, α6, comprising residues 100 to 140 (Fig. la). Both hydrogen bonding and hydrophobic contacts between the BIR domain and residues in the first few turns of α6 stabilize and fix the direction of this helical rod. The remaining seven helical turns extend out and away from the BIR domain. The two α6 heUces of the dimer form an approximate 110° angle while n amtaining a tip- to-tip interheUcal distance of 111 A (Fig. lb). This structural arrangement creates a curved and extended interface on one side of the survivin dimer.

OveraU, survivin spatiaUy organizes three separate and chemicaUy distinct surfaces including acidic and basic patches on the BIR domain and a hydrophobic helical surface on α6. The BIR domain structure assembles a contiguous acidic surface made up primarily of residues in the core β-sheet. Residues from β2 (Asp 53), β3 (Glu 63, Glu 65), α4 (Glu 76), and the α3-α4 connecting loop (Glu 68, Asp 70, Asp 71, Asp 72) contribute to this highly charged and extensive surface (Figs. lc-d). Residues 48 to 52 are unique to survivin and they form an acidic knuckle which protrudes from this patch. Given the number of potential survivin binding partners, this acidic region may represent one of the structural determinants that mediate electrostatic interactions between survivin's BIR domain and other proteins.

A second surface on the BIR domain together with the segment linking the

BIR domain and α6 form an extensive basic patch (Figs. lc-d). In addition, Lys 103, Arg 106 and Lys 110, which form part of this basic cluster, sequester a sulfate ion derived from the crystaUization solution on each survivin monomer in the asymmetric unit (Fig. 3b). While this crystaUographic arrangement may simply reflect charge neutralization of this positive surface, sulphates sequestered in this manner often structuraUy correlate with regions likely to mediate phosphorylation dependent interactions. This suUate-sequestering region precedes two putative phosphorylation sites as well as a C-terminal hydrophobic patch (Fig. 2). Phe 124, Ala 128, Val 131, Ala 134, lie 135, and Leu 138 form a slightly twisted hydrophobic surface around the last haU of α6 (Fig. 3c). Removal of this heUcal region results in a loss of survivin's localization to the spindle centrosomes with γ-tubulin and its abiUty to co-sediment with polymerized microtubules (Li et al, Nature 396:580-583, 1998). The surface and residue composition of this heUcal hydrophobic cluster, its proximity to putative phosphate-binding and phosphorylation sites, along with the findings that the α6 helix mediates survivin locaUzation, suggest a regulatory mechanism in which phosphorylation of survivin or its binding partners can aboUsh or potentiate a protein-protein interaction.

Zinc chelation appears essential for survivin function as mutation of Cys 84 to alanine aboUshes survivin's abiUty to block apoptosis by acting as dominant negative mutant (Li, supra.). One explanation for this result may be due to the disruption of the BIR domain architecture necessary for a survivin-caspase-3 interaction. However, survivin's role, as a direct, physiologicaUy relevant, caspase regulator remains controversial. Mutational analysis of XIAP reveals that the most important residues for caspase-3 inhibition reside in a loop N-terminal to XIAP's BIR2 domain (Sun, supra.) (Fig. 2) . Survivin lacks this N-terminal extension, and would be predicted to act as an inefficient caspase inhibitor. Furthermore, inhibition of expression of the C. elegans survivin homologue bir-1 in embryos results in a cytokinesis defect rather than an apoptotic event (Fraser et al, Current Biol. 9:292-301 (1999)). This is not entirely unexpected, as BIR domain containing proteins have been found in organisms with no known caspases, including S. cerevisiae and S. pombe (Uren et al, Proc. Natl. Acad. Sci. 96:10170-10175, 1999). Disruption of these genes results in a variety of meiotic and mitotic defects, including f aUure to elongate the mitotic spindle in fission yeast (Xu et al, Mol. CeU 3:389-395, 1999). In order to investigate the caspase inhibitory functions of properly folded, fuU length survivin, both recombinant wUd type survivin and the L54M point mutant were each tested for their abiUty to block caspase-3 activity. Neither of the samples tested affected caspase-3 proteolytic cleavage of the fluorogenic peptide Z-DEVD- AFC. In contrast, the reversible caspase inhibitor DEVD-CHO displayed potent caspase-3 inhibition (Ki =10 nM). AdditionaUy, assays performed with the BIR2- containing full-length cIAPl/hMIHB or with cIAPl/hMIHB lacking its BIR1 domain resulted in significant levels of caspase-3 inhibition (Kj =40 nM) sirrular to those previously reported (Roy et al, EMBO J. 16:6914-6925, 1997). The current work suggests that human survivin is not a direct caspase inhibitor and supports the recent proposal that survivin's anti-apoptotic function results from an indirect inhibitory role of caspase-3 by promoting a pro-caspase-3/p21 complex (Suzuki et al, Oncogene 19:1346-1353 2000). This does not exclude the possibiUty that survivin and caspase-3 interact nor that survivin promotes inhibition of caspase activity during mitosis (Tamm et al, Cancer Res. 58:5315-5320, 1998).

In summary, recombinantly expressed, full length human survivin forms a symmetric dimer with two BIR domains and two extended heUces. Survivin's BIR domain lacks the region previously mapped as necessary for caspase inhibition and is incapable of blocking proteolytic cleavage of the preferred caspase-3 peptide substrate DEVD. The orientation and length of the dimer's two C-terminal heUces suggest that survivin could bridge multiple γ-tubulin molecules. Survivin may act as a structural adapter spanning γ-tubulin monomers or a gamma-tubulin complex protein (GCP) during formation of the microtubule nucleation complex. This structural role mediated by α.6, would locaUze survivin's BIR domains to the same site possibly resulting in the recruitment of other proteins to the MTOC, including p21, caspase-3, and CDK4 (Suzuki, supra.). Survivin's ability to block apoptosis may be due to increased local concentrations of cell death proteins and anti-apoptotic factors near the MTOC, thereby, elevating caspase inhibition and protecting the MTOC from proteolysis. The functional consequences of these events are directly dependent on survivin's three-dimensional structure. Solvent-exposed hydrophobic patches and to a lesser extent consteUations of identicaUy charged amino acid side chains are generally energetically disfavored when not engaged with binding partners. These regions, aU of which are found in survivin, are often maintained for functionally important interactions. As such, the work described herein provides specific structural targets for functional experiments.

WhUe the invention has been described in detaU with reference to certain preferred embodiments thereof, it wiU be understood that modifications and variations are within the spuϊt and scope of that which is described and claimed.

Claims

What is claimed is:

1. A method of predicting a binding agent for an inhibitor of apoptosis protein (LAP), said method comprising: (a) modeling a potential binding agent that interacts with one or more domains of a survivin poiypeptide or fragment thereof, defined by a pluraUty of atomic coordinates of the survivin poiypeptide or fragment thereof; and

(b) deterrnining the abiUty of said potential bmding agent to modulate a survivin biological function, thereby predicting an LAP binding agent.

2. The method of claim 1, wherein the survivin poiypeptide or fragment thereof is a vertebrate survivin poiypeptide.

3. The method of claim 2, wherein the survivin poiypeptide or fragment thereof is a mammalian poiypeptide.

4. The method of claim 3, wherein the survivin poiypeptide or fragment thereof is a mouse or human poiypeptide.

5. The method of claim 1, wherein the survivin poiypeptide or fragment thereof has a sequence selected from the group consisting of:

(a) SEQ ID NO: 3; (b) conservative substitutions of (a);

(c) variants of (a);

(d) mutants of (a), (b), or (c); and

(e) fragments of (a), (b), (c), or (d).

6. The me od of claim 5, wherein the mutant survivin is more hydrophiUc than wUd-type survivin.

7. The method of claim 6, wherein the mutant survivin has a sequence selected from the group consisting of:

(a) SEQ ID NO: 4;

(b) conservative substitutions of (a); (c) variants of (a); and

(d) fragments of (a), (b), or (c).

8. The method of claim 5, wherein the mutant survivin is selected from the group consisting of W10A, T34E, L54M, H80A, H80A/E76A, T97E, W10A/L98A/F101R/L102S, L6G/ W10A/L98A/F101R/L102S, W10A/F93A/L98R, L6G/ W10A/F93A/L98R and Δ126-142.

9. The method of claim 8, wherein a conservative substitution, variant or fragment of the selected mutant survivin is used.

10. The method of claim 1, wherein the pluraUty of atomic coordinates are as set forth in Table 1.

11. The method of claim 1, wherein the potential binding agent is selected from the group consisting of a peptide, an antibody, a peptidomimetic, and a small molecule.

12. The mettiod of claim 1, wherein the biological activity of survivin is inhibited by said binding agent.

13. The method of claim 1, wherein the biological activity of survivin is increased by said binding agent.

14. The method of claim 1, wherein the domain is a baculovirus LAP repeat (BIR) domain of survivin.

15. The method of claim 12, wherein the binding agent binds to the N- termrnal portion of the BIR domain.

16. The method of claim 1, wherein the domain is a C-terminal helix of survivin.

17. The method of claim 16, wherein the C-terminal helix comprises residues 100 to 140 of SEQ ID NOs: 3 or 4, conservative substitutions thereof, variants thereof, or mutants thereof.

18. The method of claim 1, wherein the bmding agent is modeled to bind to amino acid residues 89-102 of SEQ ID NOs: 3 or 4, conservative substitutions thereof, variants thereof, or mutants thereof.

19. The method of claim 1, wherein the binding agent is modeled to bind to amino acid 48-52 of SEQ ID NOs: 3 or 4, conservative substitutions thereof, variants thereof, or mutants thereof.

20. The method of claim 1, wherein said potential binding agent is designed de novo.

21. The method of claim 1, wherein the binding agent is designed from a known binding agent.

22. The method of claim 1, wherein the binding agent is identified using a computer algorithm to predict a three-dimensional representation of the potential binding agent based upon a three-dimensional representation of the survivin poiypeptide or fragment thereof.

23. The method of claim 1, wherein the biological function is dimerization activity; tubutin interaction activity; p21, caspase-3 and CDK4 requirement activity; or zinc chelation activity.

24. A method of identifying an inhibitor of apoptosis protein (LAP) binding agent, said method comprising:

(a) defining a survivin poiypeptide or fragment thereof based on a pluraUty of atomic coordinates of the survivin poiypeptide; (b) modeling a potential binding agent that interacts with a domain of the survivin poiypeptide;

(c) contacting the potential binding agent with the survivin poiypeptide; and

(d) deterrnining the abiUty of said potential binding agent to modulate a survivin biological function, thereby identifying a survivin binding agent. ,

25. An IAP binding agent identified by the method of claim 1.

26. A method for increasing apoptosis in a ceU with a ceU proliferative disorder, said method comprising contacting the ceU with the binding agent of claim 22 in an amount effective to inhibit LAP activity.

27. The method of claim 26, wherein the brndrng agent is an antibody.

28. The method of claim 26, wherein the b ding agent is selected from the group consisting of a peptide, a peptidomimetic, and a small molecule.

29. The method of claim 26, wherein the ceU proliferative disorder is cancer.

30. The method of claim 26, wherein the ceU is derived from a tissue selected from the group consisting of ovary, breast, pancreas, lymph node, skin, blood, lung, brain, kidney, Uver, nasopharyngeal cavity, thyroid, central nervous system, prostate, colon, rectum, cervix, and endometrium.

31. The method of claim 26, wherein the LAP activity is survivin activity.

32. A method for treating a mammal diagnosed as having a ceU proliferative disorder, said method comprising contacting the mammal with the binding agent of claim 22 in an amount effective to inhibit LAP activity.

33. The method of claim 32, wherein the b ding agent is an antibody.

34. The method of claim 32, wherein the binding agent is selected from the group consisting of a peptide, a peptidomimetic, and a smaU molecule.

35. The method of claim 32, wherein the ceU proliferative disorder is cancer.

36. The method of claim 32, wherein the ceU is derived from a tissue selected from the group consisting of ovary, breast, pancreas, lymph node, skin, blood, lung, brain, kidney, Uver, nasopharyngeal cavity, thyroid, central nervous system, prostate, colon, rectum, cervix, and endometrium.

37. A method of detecting survivin in a sample, said method comprising contacting the sample with the binding agent of claim 25 and detecting the binding of the agent to survivin.

38. A method for identifying an agent that enhances apoptosis, said method comprising:

(a) modeling a potential apoptosis enhancing agent that interacts with one or more domains of a survivin poiypeptide or fragment thereof, defined by a pluraUty of atomic coordinates of the survivin poiypeptide; and

(b) deterrnining the abiUty of said potential apoptosis enhancing agent to modulate apoptosis, thereby identifying an apoptosis enhancing agent.

39. A computer program on a computer readable medium, said computer program comprising instructions to cause a computer to:

(a) define a survivin poiypeptide or fragment thereof based on a pluraUty of atomic coordinates of the survivin poiypeptide; and (b) model a potential survivin activity modulating agent that interacts with the survivin poiypeptide.

40. The computer program of claim 39, wherein the pluraUty of atomic coordinates are as set forth in Table 1.

41. An isolated crystaUine survivin poiypeptide.

42. The crystaUine survivin poiypeptide of claim 41, wherein the survivin poiypeptide has a sequence selected from the group consisting of: (a) SEQ ID NO: 3;

(b) conservative substitutions of (a);

(c) variants of (a);

(d) mutants of (a), (b), or (c); and

(e) fragments of (a), (b), (c), or (d).

43. The method of claim 42, wherein the mutant survivin is more hydrophiUc than wUd type survivin.

44. The method of claim 43, wherein the mutant survivin has a sequence selected from the group consisting of:

(a) SEQ ID NO: 4;

(b) conservative substitutions of (a);

(c) variants of (a); and

(d) fragments of (a), (b), or (c).

45. The method of clai 42, wherein the mutant survivin is selected from the group consisting of W10A, T34E, L54M, H80A, H80A/E76A, T97E, W10A/L98A/F101R/L102S, L6G/W10A/L98A/F101R/L102S, W10A/F93A/L98R, L6G/W10A/F93A/L98R and Δ126-142.

46. The method of claim 45, wherein a conservative substitution, variant or fragment of the selected mutant survivin is used.

47. The crystaUine survivin poiypeptide of claim 41, wherein the atomic coordinates of the atoms of the survivin poiypeptide are as set forth in Table 1.

48. A method for identifying an agent which inhibits dirnerization of a survivin poiypeptide, the method comprising:

(a) contacting the agent and a survivin poiypeptide under conditions sufficient to allow the agent and survivin to interact; and

(b) determining the effect of the agent on the ability of survivin poiypeptide to demerit.

49. The method of claim 48, wherein the agent is a peptide.

50. The method of claim 48, wherein the agent is a peptidomimetic.

51. The method of claim 48, wherein the survivin poiypeptide is expressed in a ceU.

52. The method of claim 48, wherein the abUity of the agent to modulate dimerization is determined by detection of a change in apoptosis.

53. The method of claim 48, wherein the survivin poiypeptide has a sequence selected from the group consisting of:

(a) SEQ ID NO: 3; (b) conservative substitutions of (a);

(c) variants of (a);

(d) mutants of (a), (b), or (c); and

(e) fragments of (a), (b), (c), or (d).

54. The method of claim 53, wherein the mutant survivin is selected from the group consisting of WlOA, T34E, L54M, H80A, H80A/E76A, T97E, W10A/L98A/F101R/L102S, L6G/W10A/L98A/F101R/L102S, W10A/F93A/L98R, L6G/W10A/F93A/L98R and Δ126-142.

55. The method of claim 54, wherein a conservative substitution, variant or fragment of the selected mutant survivin is used.

56. A method for identifying an agent that enhances apoptosis, said method comprising: (a) defining a survivin poiypeptide or fragment thereof based on a pluraUty of atomic coordinates of the survivin poiypeptide;

(b) modeling a potential apoptosis enhancing agent that interacts with the survivin poiypeptide;

(c) contacting the potential apoptosis enhancing agent with the survivin poiypeptide; and

(d) determining the abiUty of the agent to enhance apoptosis of a ceU, thereby identifying the apoptosis enhancing agent.