WO2005113762A1 - CRYSTAL STRUCTURE OF PROTEIN KINASE B-α (AKT-1) AND USES THEREOF - Google Patents

Abstract

The present invention relates to the crystal structure of protein kinase B (Akt-1), and their uses in identifying Akt-1 ligands, including Akt-1 inhibitor compounds.

Description

CRYSTAL STRUCTURE OF PROTEIN KINASE B- α (AKT-1) AND USES THEREOF

FIELD OF THE INVENTION The present invention relates to crystalline compositions of mammalian protein kinase B-α (Akt-1), methods of preparing said compositions, methods of determining the three-dimensional (3-D) X-ray atomic coordinates of said composition, methods of identifying ligands of Akt-1 using structure based drug design, the use of the 3-D crystal structure to design, modify and assess the activity of potential inhibitors, for the treatment of a variety of cancers, such as, for example breast, prostate, pancreas, lung and colorectal cancers. BACKGROUND OF THE INVENTION Protein kinases control many of the intracellular processes that are dysregulated in human cancer, including the suppression of apoptosis and anoikis and the induction of cell cycle progression. (Zinda et ai, Clin Cancer Res. Aug; 7(8):2475-9 (2001)). Those skilled in the art will understand that this critical link between the intracellular signaling pathways and the regulating of cell cycle progression, morphology and the secretion of cellular proteins, such as for example growth hormones and chemokines, has lead to the emergence of protein kinases as a group of molecular targets with potential to be specific to cancer. These kinases have the ability to selectively target cancer cells, thus further having the potential of greatly reducing if not eliminating the cytotoxic side effects that are generally associated with conventional cancer therapies. Protein kinase B/Akts (PKB/Akt) are a group of serine/threonine kinases in the protein kinase family that includes protein kinase A (PKA), protein kinase C (PKC), serum glucocorticoid stimulated kinase (SGK), and the p70 and p90 S6 kinases (Coffer and Woodgett, Eur. J. Biochem, 201 :475- 481 (1991); Jones et ai., Proc. Nat. Acad. Sci, 88:4171 -4175 (1991)). All serine/threonine kinases require phosphorylation of an invariant Ser/Thr residue in the activation segment for activity. Protein kinase and phosphatase regulated phosphorylation and dephosphorylation serve as molecular switches to modulate the extent and duration of cellular signaling cascades. In particular, stimulation of the PKB/Akt's catalytic activity on threonine-308 and serine-473 triggers the stimulation of phosphatidylinositol 3-kinase (P13K), which catalyzes the formation of 3-phosphorylated phosphoinositol lipids, such as phosphatidylinositol- (3,4)-diphosphate (Pl(3,4)-P₂) and phosphatidylinositol-(3,4,5)-triphosphate (Pl(3,4,5)-P₃). (Kumar, et al., Biochimica et Biophysics Ada, 1526:257-268 (2001 )). Mammals have three isoforms of PKB/Akt, designated Akt-1 , Akt-2, and Akt-3 (also known as PKBα, PKBβ, and PKBv, respectively). Akt-2 and Akt-3 show 81 % and 83% amino acid sequence identity, respectively, with Akt-1. (Li, et al., Current Topics in Medicinal Chemistry, 2: 939-971 (2002)). The Akt isoforms share a common domain organization that consists of a pleckstrin homology domain (PH domain) at the N-terminus, a kinase catalytic domain, and a short regulatory region at the C-terminus. Although the Akt isoforms are ubiquititously expressed in mammalian cells, evidence suggests that the relative isoform expression levels differ between tissues. Akt-1 appears to be the predominantly expressed isoform in most tissues, while Akt-2 is highly enriched in insulin target tissues. The primary sequence of Akt-1 is well conserved between species, with greater than 90% identity between the amino acid sequences of chicken, mouse and human. (Kumar, et al. (2001)). Several methods have been used in the past and continue to be used to discover selective inhibitors of biomolecular targets such as Akt-1. The various approaches include ligand-directed drug discovery (LDD), quantitative structure activity relationship (QSAR) analyses; and comparative molecular field analysis (CoMFA). CoMFA is a particular type of QSAR method that uses statistical correlation techniques for the analysis of the quantitative relationship between the biological activity of a set of compounds with a specified alignment, and their three-dimensional electronic and steric properties. Other properties such as hydrophobicity and hydrogen bonding can also be incorporated into the analysis. An invaluable component of these drug discovery approaches is structure based design, which is a design strategy for new chemical entities, or optimization of lead compounds identified by other methods, using the three-dimensional (3D) structure of the biological macromolecule target obtained by for example, X-ray or nuclear magnetic resonance (NMR) studies, or from homology models. Analyzing 3-D structures of proteins provides crucial insights into the behavior and mechanics of drug binding and biological activity. Coupled with computational techniques including modeling and simulation, the study of biomolecular interactions provides details of events that may be difficult to investigate experimentally in the laboratory, and can help reveal topological features important for determining molecular recognition. As those skilled in the art will recognize, this information can, in turn, be used for predicting ligand- receptor complex formation, and for designing ligands and protein mutations that produce desired ligand receptor interactions. To date, high-resolution X-ray structures exist for many classes of protein kinases, both in their active and inactive forms, and in some cases, these structures are co-complexed with an inhibitor and/or substrate mimic. (Kumar, et al., (2001); Li, et al., (2002); Yang, et al., Molecular Cell, 9:1227-1240 (2002); Huang, et al., Structure, 11 :21 -30 (2003). The crystal structure of activated, human Akt-2 was first disclosed by Yang et al., Nature Structural Biology, 9, 940-944 (2002), however, the co-crystallization of activated Akt- 1 has not yet been done. Therefore, since protein kinases, and specifically PKB/Akts, have emerged as potential "cancer- specific" molecular targets, allowing the selective targeting of cancers cells versus normal cells, inhibition represents a significant strategy in drug development design. To that end the quest for specific and potent PKB/Akt inhibitors for use in physiological studies and therapeutic settings continues. Thus, obtaining three-dimensional (3D) structures of PKB/Akts, such as Akt-1 , by for example, X-ray or NMR studies, or from homology models, and analyzing the structures using computational methods facilitates such discovery efforts. SUMMARY OF THE INVENTION The present invention provides crystalline compositions of Akt-1, and specifically of the catalytic region of Akt-1. The invention further provides methods of preparing compositions, methods of determining the 3-D X-ray atomic coordinates of the crystalline compositions, methods of using the atomic coordinates in conjunction with computational methods to identify binding site(s), methods to elucidate the 3-D structure of homologues of Akt-1 , and methods to identify ligands which interact with the binding site(s) to agonize or antagonize the biological activity of Akt-1 , methods for identifying inhibitors of Akt-1 , pharmaceutical compositions of inhibitors, and methods of treatment of cancer using said pharmaceutical compositions. In a preferred embodiment the invention provides crystalline compositions of the catalytic domain of the active form of Akt-1. One aspect of the present invention provides methods for crystallizing an Akt-1 polypeptide. Preferably the methods for crystallizing an Akt-1 polypeptide comprising an amino acid sequence spanning the amino acids 144 to 480 listed in SEQ ID NO:1 comprising the steps of: (a) preparing solutions of the polypeptide, ligand and precipitant; (b) growing a crystal comprising molecules of the polypeptide from the mixture solution; and (c) separating the crystal from the solution. The crystallization growth can be carried out by various techniques known by those skilled in the art, such as for example, batch crystallization, liquid bridge crystallization, or dialysis crystallization. However, the present invention is further intended to include such crystalligation growth techniques which serve equivalent functions and which become known in the area subsequently hereto. Preferably, the crystallization growth is achieved using vapor diffusion techniques. An embodiment of the present invention provides crystalline compositions of Akt-1 comprising a crystalline form of a polypeptide with an amino acid sequence spanning the amino acids Arg144 to Ala480 listed in SEQ ID NO:1, wherein the crystalline composition has a space group P2-i with unit cell dimensions a=42.65, b=55.33, c=90.58 A, α=90.0, β=102.47, γ=90.0°. In a second aspect, the present invention provides vectors useful in methods for preparing a substantially purified C-terminal catalytic domain of Akt-1 comprising the polypeptide with an amino acid sequence spanning amino acids Arg144 to Ala480 listed in SEQ ID NO:1. In a third aspect, the present invention provides methods for determining the X-ray atomic coordinates of the crystalline compositions at a 2.02 A resolution. In a fourth aspect, the present invention provides a molecule or molecular complex crystal, wherein the crystal has substantially similar atomic coordinates to the atomic coordinates listed in FIG.4 or portions thereof, or any scalable variations thereof. In a fifth aspect, the present invention provides a molecule or molecular complex crystal, wherein the crystal comprises a polypeptide with an amino acid sequence spanning the amino acids Arg144 to Ala480 listed in SEQ ID NO:1. A further embodiment of the invention provides a crystal comprising an amino acid sequence that is at least 98% homologous to a polypeptide with an amino acid sequence spanning the amino acids Arg144 to Ala480 listed in SEQ ID NO:1. An even further embodiment of the invention provides a crystal comprising an amino acid sequence that is at least 95% homologous to a polypeptide with an amino acid sequence spanning the amino acids Arg144 to Ala480 listed in SEQ ID NO:1 , and which has the atomic coordinates listed in FIG. 4. In a sixth aspect, the present invention provides a molecule or molecular complex crystal, wherein the crystal comprises a polypeptide, or a portion thereof, with atomic coordinates having a root mean square deviation from the protein backbone atoms (N, Cα, C, and O) listed in FIG.4 of less than 0.2, 0.5, 0.7, 1.0, 1.2, 1.5, 2.0 or 2.5 A. In a seventh aspect, the present invention provides a scalable, or translatable, three dimensional configuration of points derived from atomic coordinates of at least a portion of an Akt-1 molecule or molecular complex comprising a polypeptide with an amino acid sequence spanning the amino acids

Arg144 to Ala480 listed in SEQ ID NO:1. In an embodiment of this aspect, the invention also comprises the structural coordinates of at least a portion of a molecule or a molecular complex that is structurally homologous to an Akt-1 molecule or molecular complex. On a molecular scale, the configuration of points derived from a homologous molecule or molecular complex have a root mean square deviation of less than about 0.2, 0.5, 0.7, 1.0, 1.2, 1.5, 2.0 or 2.5 A. from the atomic coordinates provided in FIG. 4. In an eight aspect, the present invention provides a computer for producing a three-dimensional representation of: a. a molecule or molecular complex comprising a polypeptide with an amino acid sequence spanning amino acids Arg144 to Ala480 listed in SEQ ID NO:1, or a homologue, an analogue, or a variant thereof; b. a molecule or molecular complex, wherein the atoms of the molecule or molecular complex are represented by atomic coordinates that are substantially similar to, or are subsets of the atomic coordinates listed in FIG. 4; c. a molecule or molecular complex, wherein the molecule or molecular complex comprises atomic coordinates having a root mean square deviation of less than 0.2, 0.5, 0.7, 1.0, 1.2, 1.5, 2.0 or even 2.5 A from the atomic coordinates for the protein backbone atoms listed in FIG.4; or d. a molecule or molecular complex, wherein the molecule or molecular complex comprises a binding pocket or site defined by the structure coordinates that are substantially similar to the atomic coordinates listed in FIG. 4, or a subset thereof, or more preferably the structural coordinates in FIG. 4 corresponding to one or more Akt-1 amino acids, conservative replacements, or functional equivalence thereof, in SEQ ID NO:1 selected from Val164, Ala177, Lys179, Glu228, Tyr229, Ala230, Lys276, Met281 and Thr291 ; wherein said computer comprises: (i) a computer-readable data storage medium comprising a data storage medium encoded with computer-readable data, wherein said data comprises the structure coordinates of FIG. 4, or portions thereof, or substantially similar coordinates thereof; (ii) a working memory for storing instructions for processing said computer-readable data; (iii) a central-processing unit coupled to said working memory and to said computer- readable data storage medium for processing said computer-machine readable data into said three- dimensional representation; and (iv) a display coupled to said central-processing unit for displaying said representation. The computer configured according to this aspect of the invention can be used to design and identify potential ligands or inhibitors of Akt-1 by, for example commercially available molecular modeling software in conjunction with structure-based drug design as provided herein. -o- ln a ninth aspect, the present invention provides methods involving molecular replacement to obtain structural information about a molecule or molecular complex of unknown structure. In one embodiment, the method includes crystallizing the molecule or molecular complex, generating an X-ray diffraction pattern from the crystallized molecule or molecular complex, and applying at least a portion of the structure coordinates set forth in FIG. 4 to the X-ray diffraction pattern to generate a three-dimensional electron density map of at least a portion of the molecule or molecular complex. Another aspect of the present invention provides methods for generating 3-D atomic coordinates of a protein homoiogue, an analogue or a variant of Akt-1 using the X-ray coordinates of Akt-1 described in FIG. 4, comprising: a. identifying one or more homologous polypeptide sequences to Akt-1 ; b. aligning said sequences with the sequence of Akt-1 which comprises a polypeptide with an amino acid sequence spanning amino acids Arg144 to Ala480 listed in SEQ ID NO:1 ; c identifying structurally conserved and structurally variable regions between said homologous sequence(s) and Akt-1 ; d. generating 3-D coordinates for structurally conserved residues of the said homologous sequence(s) from those of Akt-1 using the coordinates listed in FIG. 4; e. generating conformations for the loops in the structurally variable regions of said homologous sequence(s); f. building the side-chain conformations for said homologous sequence(s); and g. combining the 3-D coordinates of the conserved residues, loops and side-chain conformations to generate full or partial 3-D coordinates for said homologous sequences. Embodiments of the ninth aspect provide methods which further comprise refining and evaluating the full or partial 3-D coordinates. These methods may thus be used for example to generate 3- dimensional structures for proteins for which 3-dimensional atomic coordinates have not been determined. The newly generated structure can help to elucidate enzymatic mechanisms, or be used in conjunction with other molecular modeling techniques in structure based drug design. In the tenth aspect, the present invention provides methods for identifying ligands, inhibitors, and the like, of Akt-1 by providing the coordinates of a molecule of Akt-1 to a computerized modeling system; identifying chemical entities that are likely to bind to the molecule (e.g., by screening a small molecule library); and, optionally, procuring or synthesizing and assaying the compounds or analogues derived for bioactivity. In yet a further aspect of the present invention relates to methods for identifying potential ligands for Akt-1 or homologues, an analogue or variants thereof comprising the steps of: a. displaying the three dimensional structure of Akt-1 enzyme or homoiogue, an analogue or variant thereof, or portions thereof, as defined by atomic coordinates that are substantially similar to the atomic coordinates listed in FIG. 4 on a computer display screen; b. optionally replacing one or more the enzyme amino acid residues listed in SEQ ID NO:1, or preferably one or more amino acid residues selected from Val164, Ala177, Lys179, Glu228, Tyr229, Ala230, Lys276, Met281 and Thr291 , in said three-dimensional structure with a different naturally occurring amino acid or an unnatural amino acid to display a variant structure; c. optionally conducting ab intio, molecular mechanics or molecular dynamics calculations on the displayed three dimensional structure to generate a modified structure; d. employing said three-dimensional structure, variant structure, or modified structure to design or select said ligand; d. synthesizing or obtaining said ligand; e. contacting said ligand with said enzyme in the presence of one or more substrates; and f. measuring the ability of said ligand to modulate the activity of said enzyme. Those of skill in the art can appreciate that the data obtained by the methods for identifying potential ligands of Akt-1 , as described above, can be used to iteratively refine or modify the structure of an original ligand. Thus, once a ligand is found to modulate the activity of said enzyme, the structural aspects of the ligand may be modified to generate a structural analog of the ligand. This analog can then be used in the above methods to identify ligands. One of ordinary skill in the art will know the various ways a structure may be modified. In embodiments, preferred ligands include selective inhibitor of Akt-1. In embodiments, the methods further comprise computationally modifying the structure of the ligand; computationally determining the fit of the modified ligand using the three-dimensional coordinates described in FIG. 4, or portions thereof; contacting said modified ligand with said enzyme, or homoiogue, an analogue or variant thereof in an in vitro or in vivo setting; and measuring the ability of said ligand to modulate the activity of said enzyme. In an eleventh aspect, the present invention provides compositions, such as, pharmaceutical compositions comprising selective ligands designed according to any of the methods of the present invention. In one embodiment, a composition is provided that includes a selective inhibitor designed or identified by any of the above methods of the present invention. In another embodiment, the composition is a pharmaceutical composition. The twelfth aspect of the present invention are methods for treating cancer comprising administering pharmaceutical compositions, identified by structure based design using the atomic coordinates, or portions thereof, listed in FIG. 4, effective in treating the tumors, of all stages, that over-express Akt-1 (e.g. breast, prostate, pancreas, lung, and colorectal cancers). In yet a further aspect, the present invention provides for a pharmaceutical composition comprising an inhibitor identified from the methods of the present invention and a pharmaceutically acceptable carrier, vehicle or diluent. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is an orthogonal view of the embodiment of the ternary complex of Akt-1 , GSK3-β peptide, and AMP-PNP. Akt-1 and GSK3-β peptide are shown in ribbon representation and AMP-PNP is shown as a ball and stick figure. N- and C-termini of the polypeptide are labeled as well. Figure 2 is another orthogonal view of the embodiment of the ternary complex. Figure 3 is a schematic diagram showing the interactions of AMP-PNP with Akt-1. Figure 4 is a list of the X-ray atomic coordinates of the active form of Akt-1 C-terminal catalytic domain crystal as described in the Examples. DETAILED DESCRIPTION OF THE INVENTION The present invention relates to crystalline compositions of Akt-1 , 3-D X-ray atomic coordinates of said crystalline compositions, methods of preparing said compositions, methods of determining the 3-D X- ray atomic coordinates of said crystalline compositions, and methods of using said atomic coordinates in conjunction with computational methods to identify binding site(s), or identify ligands which interact with said binding site(s) to agonize or antagonize Akt-1. For convenience, certain terms employed in the specification, examples, and appendant claims are collected here. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the arts. The term "affinity" as used herein refers to the tendency of a molecule to associate with another. The affinity of a drug is its ability to bind to its biological target (receptor, enzyme, transport system, etc.). For pharmacological receptors, affinity can be thought of as the frequency with which the drug, when brought into the proximity of a receptor by diffusion, will reside at a position of minimum free energy within the force field of that receptor. The term "agonist" as used herein refers to a drug or compound that can interact with a receptor and initiate a physiological or a pharmacological response characteristic of that receptor (contraction, relaxation, secretion, enzyme activation, etc.). The term "analog" as used herein refers to a drug or chemical compound whose structure is related in some way to that of another drug or chemical compound, but whose chemical and biological properties may be quite different. The term "antagonist" as used herein refers to a drug or a compound that opposes the physiological effects of another. At the receptor level, it is a chemical entity that opposes the receptor- associated responses normally induced by another bioactive agent. "Atomic coordinates", "atomic structural coordinates", "coordinates", "structural coordinates", 3-D coordinates" or "X-ray coordinates" are the Cartesian coordinates corresponding to an atom's spatial relationship to other atoms in a molecule or molecular complex. Structural coordinates may be obtained using X-ray crystallography techniques or NMR techniques, or may be derived using molecular replacement analysis or homology modeling. Various software programs allow for the graphical representation of a set of structural coordinates to obtain a three dimensional representation of a molecule or molecular complex. The structural coordinates of the present invention may be modified from the original sets provided in FIG. 4 by mathematical manipulation, such as by inversion or integer additions or subtractions. As such, it is recognized that the structural coordinates of the present invention are relative, and are in no way specifically limited by the actual x, y, z coordinates of FIG. 4. As used herein the term "binding site" refers to a specific region (or atom) in a molecular entity that is capable of entering into a stabilizing interaction with another molecular entity. In certain embodiments the term also refers to the reactive parts of a macromolecule that directly participate in its specific combination with another molecule. In yet other embodiments, a binding site may be comprised or defined by the three dimensional arrangement of one or more amino acid residues within a folded polypeptide. In further embodiments, the binding site further comprises prosthetic groups, water molecules or metal ions which may interact with one or more amino acid residues. Prosthetic groups, water molecules, or metal ions may be apparent from X-ray crystallographic data, or may be added to an apo protein or enzyme using in silico methods. The term "bioactivity" refers to Akt-1 activity that exhibits a biological property conventionally associated with an Akt-1 agonist or antagonist, such as a property that would allow treatment of one or more of the various diseases such as cancer. The term "catalytic domain" as used herein, refers to the catalytic domain of the Akt-1 class of enzymes, which features a conserved segment of amino acids in the carboxy-terminal portion of the proteins, wherein this segment has been demonstrated to include the catalytic site of these enzymes. This conserved catalytic domain extends approximately from residue Arg144 to Ala480 of the full-length enzyme (SEQ ID NO:1). "To clone" as used herein, means obtaining exact copies of a given polynucleotide molecule using recombinant DNA technology. Furthermore, "to clone into" may be meant as inserting a given first polynucleotide sequence into a second polynucleotide sequence, preferably such that a functional unit combining the functions of the first and the second polynucleotides results. For example, without limitation, a polynucleotide from which a fusion protein may be translationally provided, which fusion protein comprises amino acid sequences encoded by the first and the second polynucleotide sequences. Specifics of molecular cloning can be found in a number of commonly used laboratory protocol books such as Molecular Cloning: A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989). The term "complex" or "co-complex" are used interchangeably and refer to an Akt-1 molecule, or a variant, or homoiogue of Akt-1 in covalent or non-covalent association with a substrate, or ligand. The term "contacting" as used herein applies to in silico, in vitro, and/or in vivo experiments. The term "functional equivalence" refers to the assembly of one or more amino acid residues that form a binding site in an enzyme. These residues may have one or more intervening residues which are distant from the binding site, and therefore may minimally interact with a ligand in the binding sites. In such occurrences, the binding site may be defined for the purpose of structure based drug design as comprising only a handful of amino acid residues. For example in the case of Akt-1 , the ligand binding site can alternatively comprise at least about 80% of the amino acid residues selected from the group consisting of Val164, Ala177, Lys179, Glu228, Tyr229, Ala230, Lys276, Met281 and Thr291 of SEQ ID No:1. In a preferred embodiment, the ligand binding site comprises at least about 90% of the amino acid residues selected from the group consisting of Val164, Ala177, Lys179, Glu228, Tyr229, Ala230, Lys276, Met281 and Thr291 of SEQ ID No:1. Thus any molecular assembly that has a root mean square deviation from the atomic structure coordinates of the protein backbone atoms (N, Cα, C, and O), or the side chain atoms, of one or more of Val164, Ala177, Lys179, Glu228, Tyr229, Ala230, Lys276, Met281 and Thr291 of SEQ ID NO:1 , any conservative substitutions thereof, or any functional equivalence of less than about 2.5, 2.0, 1.5, 1.2, 1.0, 0.7, 0.5, or even 0.2 A when superimposed will be considered substantially similar to the coordinates listed in FIG.4. The functional equivalents may be different from the peptides described herein at one, two, three, four, or more amino acid positions. In the most common instances, such differences will involve conservative amino acid substitutions. As used herein, the terms "gene", "recombinant gene" and "gene construct" refer to a nucleic acid comprising an open reading frame encoding a polypeptide, including both exon and (optionally) intron sequences. The term "intron" refers to a DNA sequence present in a given gene which is not translated into protein and is generally found between exons. The term "high affinity" as used herein means strong binding affinity between molecules with a dissociation constant K, of no greater than 1 mM. In a preferred case, the K, is less than 100 nM, 10 nM, 1 nM, 100 pM, or even 10 pM or less. In a most preferred embodiment, the two molecules can be covalently linked (K, is essentially 0). The term "homoiogue" as used herein means a protein, polypeptide, oligopeptide, or portion thereof, having preferably at least 90% amino acid sequence identity with an Akt-1 enzyme as described in SEQ ID NO:1 or SEQ ID NO:2 or with any catalytic domain described herein, or with any functional or structural domain of lipid binding protein. SEQ ID NO:1 is an amino acid sequence of a wild-type human Akt-1. SEQ ID NO:2 is the amino acid sequence of the S473D mutant of the catalytic domain of human Akt-1 , that was crystallized in the Examples. SEQ ID NO: 3 is the GSK3-β peptide used in the ternary complex. While SEQ ID NO:4 is the wild-type mus musculus (mouse) Akt-1 amino acid sequence, (Bellacosa, A., et al., Ongogene, Vol. 8: 745-754, 1993) SEQ ID NO:5 is the wild-type rattus norvegicus (rat) Akt-1 amino acid sequence (Konishi, H., et al., Biochem. Biophys. Res. Commun., Vol. 205: 817-825, 1994) and SEQ ID NO:6 is the wild-type bovine Akt-1 amino acid sequence, all are at least 90% identical with the Akt-1 enzyme as described in SEQ ID No:1 and, can also be used for crystallization and for the design and identification of potential ligands of Akt-1. Those skilled in the art understand that a set of structure coordinates determined by X-ray crystallography is not without standard error. As used herein, and for the purpose of this invention, the term "substantially similar atomic coordinates" or atomic coordinates that are "substantially similar" refers to any set of structure coordinates of Akt-1 or Akt-1 homologues, or Akt-1 variants, polypeptide fragments, described by atomic coordinates that have a root mean square deviation for the atomic coordinates of protein backbone atoms (N, Cα, C, and O) of less than about 2.5, 2.0, 1.5, 1.2, 1.0, 0.7, 0.5, or even 0.2 A when superimposed using backbone atoms of the structure coordinates listed in FIG. 4. Structures that have substantially similar coordinates as those listed in FIG. 4 shall be considered identical to the coordinates listed in FIG. 4. The term "substantially similar" also applies to an assembly of amino acid residues that may or may not form a contiguous polypeptide chain, but whose three dimensional arrangement of atomic coordinates have a root mean square deviation for the atomic coordinates of protein backbone atoms (N, Cα, C, and O), or the side chain atoms, of less than about 2.5, 2.0,1.5, 1.2, 1.0, 0.7, 0.5, or even 0.2 A when superimposed using backbone atoms, or the side chain atoms of the atomic coordinates of similar or the same amino acids from the coordinates listed in FIG. 4. Those skilled in the art understand that "substantially similar" atomic coordinates are considered identical to the coordinates, or portions thereof, listed in FIG. 4. Those skilled in the art further understand that the coordinates listed in FIG. 4 or portions thereof may be transformed into a different set of coordinates using various mathematical algorithms without departing from the present invention. For example, the coordinates listed in Fig. 4, or portions thereof, may be transformed by algorithms which translate or rotate the atomic coordinates. Alternatively, molecular mechanics, molecular dynamics or ab intio algorithms may modify the atomic coordinates. Atomic coordinates generated from the coordinates listed in FIG. 4, or portions thereof, using any of the aforementioned algorithms shall be considered identical to the coordinates listed in FIG. 4. The term "in silico" as used herein refers to experiments carried out using computer simulations.

In yet further embodiments, the in silico methods are molecular modeling methods wherein 3-dimensional models of macromolecules or ligands are generated. In other embodiments, the in silico methods comprise computationally assessing ligand binding interactions. The term "ligand" describes any molecule, e.g., protein, peptide, peptidomimetics, oligopeptide, small organic molecule, polysaccharide, polynucleotide, etc., which binds or interacts, generally but not necessarily specifically to or with another molecule. More particularly, a ligand can further be defined as any molecule which is designed or developed with reference to the crystal structure of Akt-1 as represented by the atomic coordinates listed in FIG. 4. In one aspect the ligand is an agonist, whereby the molecule upregulates (i.e., activate or stimulate, e.g., by agonizing or potentiating) activity, while in another aspect of the invention the ligand is an inhibitor or antagonist, whereby the molecule down- regulates (i.e., inhibit or suppress, e.g. by antagonizing, decreasing or inhibiting) the activity. The term "modulate" as used herein refers to both upregulation (i.e., activation or stimulation, e.g., by agonizing or potentiating) and down-regulation (i.e., inhibition or suppression, e.g., by antagonizing, decreasing or inhibiting) of an activity. The term "pharmacophore" as used herein refers to the ensemble of steric and electronic features of a particular structure that is necessary to ensure the optimal supramolecular interactions with a specific biological target structure and to trigger (or to block) its biological response. In certain embodiments, a pharmacophore is an abstract concept that accounts for the common molecular interaction capacities of a group of compounds towards their target structure. In yet another embodiments, the term can be considered as the largest common denominator shared by a set of active molecules. Pharmacophoric descriptors are used to define a pharmacophore, including H-bonding, hydrophobic and electrostatic interaction sites, defined by atoms, ring centers and virtual points. Accordingly, in the context of enzyme ligands, such as for example agonists or antagonists, a pharmacophore may represent an ensemble of steric and electronic factors which are necessary to insure supramolecular interactions with a specific biological target structure. As such, a pharmacophore may represent a template of chemical properties for an active site of a protein/enzyme representing these properties' spatial relationship to one another that theoretically defines a ligand that would bind to that site. The term "precipitant" as used herein is includes any substance that, when added to a solution, causes a precipitate to form or crystals to grow. Examples of suitable precipitants include, but are not limited to, alkali (e.g., Li, Na, or K), or alkaline earth metal (e.g., Mg, or Ca) salts, and transition metal (e.g., Mn, or Zn) salts. Common counterions to the metal ions include, but are not limited to, halides, phosphates, citrates and sulfates. The term "prodrug" as used herein refers to drugs that, once administered, are chemically modified by metabolic processes in order to become pharmaceutically active. In certain embodiments the term also refers to any compound that undergoes biotransformation before exhibiting its pharmacological effects. Prodrugs can thus be viewed as drugs containing specialized non-toxic protective groups used in a transient manner to alter or to eliminate properties, usually undesireable, in the parent molecule. The term "receptor" as used here in refers to a protein or a protein complex in or on a cell that specifically recognizes and binds to a compound acting as a molecular messenger (neurotransmitter, hormone, lymphokine, lectin, drug, etc.). In a broader sense, the term receptor is used interchangeably with any specific (as opposed to non-specific, such as binding to plasma proteins) drug binding site, also including nucleic acids such as DNA. The term "recombinant protein" refers to a polypeptide which is produced by recombinant DNA techniques, wherein generally, DNA encoding a polypeptide is inserted into a suitable expression vector which is, in turn, used to transform a host cell to produce the polypeptide encoded by the DNA. This polypeptide may be one that is naturally expressed by the host cell, or it may be heterologous to the host cell, or the host cell may have been engineered to have lost the capability to express the polypeptide which is otherwise expressed in wild type forms of the host cell. The polypeptide may also be, for example, a fusion polypeptide. Moreover, the phrase "derived from", with respect to a recombinant gene, is meant to include within the meaning of "recombinant protein" those proteins having an amino acid sequence of a native polypeptide, or an amino acid sequence similar thereto which is generated by mutations, including substitutions, deletions and truncation, of a naturally occurring form of the polypeptide. As used herein, the term "selective Akt-1 inhibitor" refers to a substance, such as for example, an organic molecule, that effectively inhibits an enzyme from the Akt-1 family to a greater extent than any other Akt enzyme, particularly any enzyme from the PKB/Akt family. In one embodiment, a selective Akt-1 inhibitor is a substance having a Kj for inhibition of Akt-1 that is less than about one-half, one-fifth, or one- tenth the K| that the substance has for inhibition of any other PKB/Akt enzyme. In other words, the substance inhibits Akt-1 activity to the same degree at a concentration of about one-half, one-fifth, one- tenth or less than the concentration required for any other PKB/Akt enzyme. In general a substance is considered to effectively inhibit Akt-1 if it has an IC₅o or K| of less than or about 10 /M, 1μM, 500nM, 100nM, 50nM, 10nM or 1 nM. As used herein the term "small molecules" refers to preferred drugs as they are orally available (unlike proteins which must be administered by injection or topically). The size of the small molecules is generally under 1000 Daltons, but many estimates seem to range between 300 to 700 Daltons. The term "space group" refers to a group or array of operations consistent with an infinitely extended regularly repeating pattern. It is the symmetry of a three-dimensional structure, or the arrangement of symmetry elements of a crystal. There are 230 space group symmetries possible; however, there are only 65 space group symmetries available for biological structures. The term "substantial portion" of atomic coordinates refers to a plurality of at least twelve atomic coordinates that define or partially define the location of several atoms in the binding protein or ligand. Preferably, a substantial portion is at least 24 coordinates. More preferably, a substantial portion is at least 36 coordinates. The coordinates can be within the standard deviation. By "therapeutically effective" amount is meant that amount which is capable of preventing the progression of the disease, e.g., cancer, treating the disease, or at least partially reversing the symptoms of the disease. A therapeutically effective amount can be determined on an individual basis, and will be based, at least in part, on a consideration of the species of the mammal, the size of the mammal, the type of delivery system used, and the type of administration relative to the progression of the disease. As used herein, the term "transfection" means the introduction of a nucleic acid, e.g., via an expression vector, into a recipient cell by nucleic acid mediated gene transfer. "Transformation" refers to a process in which a cell's genotype is changed as a result of the cellular uptake of exogenous DNA or RNA and, for example, the transformed cell expresses a recombinant form of a polypeptide or, in the case of anti-sense expression from the transferred gene, the expression of a naturally-occurring form of the polypeptide is disrupted. The term "unit cell" refers to a basic parallelepiped shaped block. The entire volume of a crystal may be constructed by regular assembly of such blocks. Each unit cell comprises a complete representation of the unit of pattern, the repetition of which builds up the crystal. The term "variants" in relation to the polypeptide sequence in SEQ ID NO:1 or SEQ ID NO:2 include any substitution of, variation of, modification of, replacement of, deletion of, or addition of one or more amino acids from or to the sequence providing a resultant polypeptide sequence for an enzyme having AKT-1 activity. Preferably the variant, homoiogue, fragment or portion, of SEQ ID NO:1 , or SEQ ID NO:2, comprises a polypeptide sequence of at least 5 contiguous amino acids, preferably at least 10 contiguous amino acids, preferably at least 15 contiguous amino acids, preferably at least 20 contiguous amino acids, preferably at least 25 contiguous amino acids, or preferably at least 30 contiguous amino acids. The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of preferred vector is an episome, i.e., a nucleic acid capable of extra- chromosomal replication. Preferred vectors are those capable of autonomous replication and/or expression of nucleic acids to which they are linked. Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as "expression vectors". In general, expression vectors of utility in recombinant DNA techniques are often in the form of "plasmids" which refer generally to circular double stranded DNA loops which, in their vector form are not bound to the chromosome. In the present specification, "plasmid" and "vector" are used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors which serve equivalent functions and which become known in the art subsequently hereto.

The following amino acid abbreviations used herein: A = Ala = Alanine T = Thr = Threonine V = Val = Valine C = Cys = Cysteine L = Leu = Leucine Y = Tyr = Tyrosine I = He = Isoleucine N = Asn = Asparagine p = Pro = Proline Q = Gin = Glutamine F = Phe = Phenylalanin D = Asp = Aspartic Acid W = Trp = Tryptophan E = Glu = Glutamic Acid M = Met = Methionine K = Lys = Lysine G = Gly = Glycine R = Arg = Arginine S = Ser = Serine H = His = Histidine

A. Clones and Expressions As would be appreciated by those skilled in the art, the nucleotide sequence coding for an Akt-1 polypeptide, or functional fragment, including the C-terminal peptide fragment of the catalytic domain of Akt-1 protein, derivatives or analogs thereof, including a chimeric protein, thereof, can be inserted into a suitable expression vector, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted protein-coding sequence. Thus, the nucleic acid encoding a Akt-1 polypeptide of the invention or a functional fragment comprising the C-terminal peptide fragment of the catalytic domain of Akt-1 protein, derivatives or analogs thereof, is operationally associated with a promoter in an expression vector of the invention. In preferred embodiments, the expression vector contains the nucleotide sequence coding for the polypeptide comprising the amino acid sequence spanning amino acids Arg144 to Ala480 listed in SEQ ID NO:1. Both cDNA and genomic sequences can be cloned and expressed under the control of such regulatory sequences. An expression vector also preferably includes a replication origin. The necessary transcriptional and translational signals can be provided on a recombinant expression vector. As detailed below, all genetic manipulations described for the Akt-1 gene in this section, may also be employed for genes encoding a functional fragment, including the C-terminal peptide fragment of the catalytic domain of the Akt-1 protein, derivatives or analogs thereof, including a chimeric protein thereof. Suitable host-vector systems include, but are not limited to, mammalian cell systems infected with virus (e.g., vaccinia virus, adenovirus, etc.); insect cell systems infected with virus (e.g., baculovirus); microorganisms such as yeast containing yeast vectors; or bacteria transformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA. The expression elements of vectors vary in their strengths and specificities. As those of skill in the art can appreciate, depending on the host-vector system utilized, any one of a number of suitable transcription and translation elements may be used. A recombinant Akt-1 protein of the invention may be expressed chromosomally, after integration of the coding sequence by recombination. In this regard, any of a number of amplification systems may be used to achieve high levels of stable gene expression. (See Sambrook et al., 1989). A suitable cell for purposes of this invention is one into which the recombinant vector comprising the nucleic acid encoding Akt-1 protein is cultured in an appropriate cell culture medium under conditions that provide for expression of Akt-1 protein by the cell. Any of the methods previously described for the insertion of DNA fragments into a cloning vector may be used to construct expression vectors containing a gene consisting of appropriate transcriptional/translational control signals and the protein coding sequences. These methods may include in vitro recombinant DNA and synthetic techniques, and in vivo recombination (genetic recombination). Expression of Akt-1 protein may be controlled by any promoter/enhancer element known in the art, provided that these regulatory elements must be functional in the host selected for expression, as would be appreciated by those skilled in the art. Vectors containing a nucleic acid encoding an Akt-1 protein of the invention can be identified for example by four general approaches: (1) PCR amplification of the desired plasmid DNA or specific mRNA; (2) nucleic acid hybridization; (3) presence or absence of selection marker gene functions; and (4) expression of inserted sequences. However, the invention is intended to include such other forms of identification of vectors containing a nucleic acid encoding an Akt-1 protein of the present invention, which serve equivalent functions and which become known in the art subsequently hereto. In the first approach, the nucleic acids can be amplified by PCR to provide for detection of the amplified product. In the second approach, the presence of a foreign gene inserted in an expression vector can be detected by nucleic acid hybridization using probes comprising sequences that are homologous to an inserted marker gene. In the third approach, the recombinant vector/host system can be identified and selected based upon the presence or absence of certain "selection marker" gene functions (e.g., beta-galactosidase activity, thymidine kinase activity, resistance to antibiotics, transformation phenotype, occlusion body formation in baculovirus, etc.) caused by the insertion of foreign genes in the vector. In another example, if the nucleic acid encoding Akt-1 protein is inserted within the "selection marker" gene sequence of the vector, recombinant vectors containing the Akt-1 protein insert can be identified by the absence of the Akt-1 protein gene function. In the fourth approach, recombinant expression vectors can be identified by assaying for the activity, biochemical, or immunological characteristics of the gene product expressed by the recombinant vector, provided that the expressed protein assumes a functionally active conformation. A wide variety of host/expression vector combinations may be employed in expressing the DNA sequences of this invention as known by those of skill in the art. Once a particular recombinant DNA molecule is identified and isolated, several methods known in the art may be used to propagate it. Once a suitable host system and growth conditions are established, recombinant expression vectors can be propagated and prepared in quantity. As noted above, the expression vectors which can be used include, but are not limited to, the following vectors or their derivatives: human or animal viruses such as vaccinia virus or adenovirus; insect viruses such as baculovirus; yeast vectors; bacteriophage vectors (e.g., lambda), and plasmid and cosmid DNA vectors, to name but a few. Vectors can be introduced into the desired host cells by methods known in the art, e.g., transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, lipofection (lysosome fusion), use of a gene gun, or a DNA vector transporter (see, e.g., Wu et ai., 1992, J. Biol. Chem. 267:963-967; Wu and Wu, 1988, J. Biol. Chem. 263:14621-14624; Hartmut et al., Canadian Patent Application No. 2,012,311 , filed Mar. 15, 1990).

B. Crystal and Space Groups X-ray structure coordinates define a unique configuration of points in space. Those skilled in the art understand that a set of structure coordinates for a protein or a protein/ligand complex, or a portion thereof, define a relative set of points that, in turn, define a configuration in three dimensions. A similar or identical configuration can be defined by an entirely different set of coordinates, provided the distances and angles between atomic coordinates remain essentially the same. In addition, a scalable configuration of points can be defined by increasing or decreasing the distances between coordinates by a scalar factor while keeping the angles essentially the same. One aspect of the present invention relates to a crystalline composition comprising preferably a polypeptide with an amino acid sequence spanning amino acids Arg144 to Ala480 listed in SEQ ID NO:1. In one embodiment, the present invention discloses a crystalline Akt-1 molecule comprising a polypeptide with an amino acid sequence spanning amino acids Arg144 to Ala480 listed in SEQ ID NO:1 complexed with one or more ligands. In another embodiment, the crystallized complex is characterized by the structural coordinates listed in FIG. 4, or portions thereof. In further embodiments, the atoms of the ligand are within about 4, 7, or 10 angstroms of one or more Akt-1 amino acids in SEQ ID NO: 1 preferably selected from Val164, Ala1 7, Lys179, Glu228, Tyr229, Ala230, Lys276, Met281 and Thr291. One embodiment of the crystallized complex is characterized as belonging to the P2τ space group with unit cell dimensions a=42.65, b=55.33, c=90.58 A, α=90.0, β=102.47, γ=90.0°. This embodiment is encompassed by the structural coordinates of FIG. 4. The ligand may be a small molecule which binds to an Akt-1 catalytic domain defined by SEQ ID NO: 2, or portions thereof, with a K, of less than about 10 μM, 1 μM, 500 nM, 100 nM, 50 nM, or 10 nM. In yet further embodiments of the present invention, the ligand is a substrate or substrate analog of Akt-1. In further embodiments, the ligand(s) may be a competitive or uncompetitive inhibitor of Akt-1. In yet another embodiment, the ligand is a covalent inhibitor of Akt-1. Various computational methods can be used to determine whether a molecule or a binding pocket portion thereof is "structurally equivalent," defined in terms of its three-dimensional structure, to all or part of Akt-1 or its binding pocket(s). Such methods may be carried out in current software applications, such as the molecular similarity application of QUANTA (Accelrys Inc., San Diego, Calif.). The molecular similarity application permits comparisons between different structures, different conformations of the same structure, and different parts of the same structure. The procedure used in molecular similarity to compare structures is divided into four steps: (1) load the structures to be compared; (2) optionally define the atom equivalences in these structures; (3) perform a fitting operation; and (4) analyze the results. Each structure is identified by a name. One structure is identified as the target (i.e., the fixed structure), while all remaining structures are working structures (i.e., moving structures). Since atom equivalency within molecular similarity applications is defined by user input, for the purpose of this invention equivalent atoms are defined as protein backbone atoms (N, Cα, C, and O) for all conserved residues between the two structures being compared. A conserved residue is defined as a residue that is structurally or functionally equivalent (See Table 4 infra). In further embodiments rigid fitting operations are considered. In other embodiments, flexible fitting operations may be considered. When a rigid fitting method is used, the working structure is translated and rotated to obtain an optimum fit with the target structure. The fitting operation uses an algorithm that computes the optimum translation and rotation to be applied to the moving structure, such that the root mean square difference of the fit over the specified pairs of equivalent atoms is an absolute minimum. This number, given in angstroms (A), is reported by the molecular similarity application. Any molecule or molecular complex or binding pocket thereof, or any portion thereof, that has a root mean square deviation of conserved residue backbone atoms (N, Cα, C, and O) of less than about 2.5, 2.0, 1.5 A, 1.0 A, 0.7 A, 0.5 A or even 0.2 A, when superimposed on the relevant backbone atoms described by the reference structure coordinates listed in FIG. 4, is considered "structurally equivalent" to the reference molecule. That is to say, the crystal structures of those portions of the two molecules are substantially identical, within acceptable error. Particularly preferred structurally equivalent molecules or molecular complexes are those that are defined by the entire set of structural coordinates listed in FIG. 4, plus or minus a root mean square deviation from the conserved backbone atoms of those amino acids of not more than 2.5 A. More preferably, the root mean square deviation is less than about 1.0 A. The term "root mean square deviation" means the square root of the arithmetic mean of the squares of the deviations. It is a way to express the deviation or variation from a trend or object. The "root mean square deviation" defines the variation in the backbone of a protein from the backbone of Akt-1 or a binding pocket portion thereof, as defined by the structural coordinates of Akt-1 described herein. The refined X-ray atomic coordinates of the catalytic domain of Akt-1 (amino acids 144 to 480 as listed in SEQ ID NO:1 ), co-crystallized with the non-hyrolyzable ATP analog AMP-PNP and a substrate peptide, preferably GSK3-β, residues 3-12, as shown in SEQ ID NO: 3, 2 Mn²⁺ ions and 76 water molecules are as listed in FIG. 4. Overall, the protein adopts a typical bilobate "kinase-fold", with two alpha-helices (αB and αC, following the naming convention established for PKA-REF, (Knighton, D.R., et al., Science, Vol. 253:407-414, 1991)) and a beta-sheet in the N-terminal lobe (N-lobe) and an all alpha-helical C-terminal lobe (C-lobe). The αC helix adopts the same conformation as seen in other active protein kinases

(Etchebehere, L.C., et al., Eur. J. Biochemistry, Vol. 248: 820-826, 1997) and the C-terminal conserved hydrophobic motif loops over to interact with the N-lobe, acting as an allosteric activator, as also observed in other AGC kinase family members. (Johnson, L.N., et al., Cell, Vol. 85: 149-158, 1996). Consistent with structures of other protein kinases in complex with ATP analogs, AMP-PNP binds in the cleft between the N- and C-terminal lobes of Akt-1 , with a pair of conserved hydrogen bonds formed between the adenine base and the backbone of the kinase linker region. In the ternary complexes of Akt-1 with AMP-PNP and a substrate peptide, the bound peptides appear to help anchor the gamma phosphate of ATP to the enzyme. Of the 318 backbone Cα atoms of the crystal structure of the present invention, 309 match residues with the human Akt-2 crystal structure (Protein Data Bank entry 106k) (Yang, et al., Nature Struct. Biology, Vol. 9: 940-944, 2002), which when overlaid are within a r.m.s. difference of 0.665 A. Two orthogonal views of the molecule are shown in FIG. 1 and FIG. 2, and an illustration of the interactions of the ligand with the protein are shown in FIG. 3. The present invention provides a molecule or molecular complex that includes at least a portion of an Akt-1 and/or substrate binding pocket. In one embodiment, the Akt-1 binding pocket includes the amino acids listed in Table 1 , preferably the amino acids listed in Table 2, and more preferably the amino acids listed in Table 3, the binding pocket being defined by a set of points having a root mean square deviation of less than about 2.5, 2.0, 1.5, 1.2, 1.0, 0.7, 0.5, or even 0.2 A, from points representing the backbone atoms of the amino acids in Tables 1-3. In another embodiment, the Akt-1 substrate binding pocket includes the amino acids selected from VaI164, Ala177, Lys179, Glu228, Tyr229, Ala230, Lys276, Met281 and Thr291 of SEQ ID NO:1. Table 1: Residues near the binding pocket in Akt-1 catalytic domain. Identified residues are 10 A away from the non-hydrolyzable ATP analog, AMP-PNP.

Table 2: Residues near the binding pocket in Akt-1 catalytic domain. Identified residues are 7 A away from the non-hydrolyzable ATP analog, AMP-PNP.

Table 3: Residues near the binding pocket in Akt-1 catalytic domain. Identified residues are 4 A away from the non-hydrolyzable ATP analog, AMP-PNP.

C. Isolated Polypeptides and Variants One embodiment of the invention describes an isolated polypeptide consisting of a portion of Akt- 1 which functions as the binding site when folded in the proper 3-D orientation. One embodiment is an isolated polypeptide comprising a portion of Akt-1 , wherein the portion starts at about amino acid residue Arg144, and ends at about amino acid residue Ala480 as described in SEQ ID NO:1, or a sequence that is at least 95% or 98% homologous to a polypeptide with an amino acid sequence spanning amino acids Arg144 to Ala480 as listed in SEQ ID NO:1 , such as, for example the polypeptide of the wild-type mus musculus (mouse), SEQ ID NO:4, the wild-type rattus norvegicus (rat) Akt-1 amino acid sequence SEQ ID NO:5, and wild-type bovine Akt-1 amino acid sequence, SEQ ID NO:6. Another embodiment comprises crystalline compositions comprising variants of Akt-1. Variants of the present invention may have an amino acid sequence that is different by one or more amino acid substitutions to the sequence disclosed in SEQ ID NO:1 or SED ID NO:2. Embodiments which comprise amino acid deletions and/or additions are also provided. The variant may for example have conservative changes (amino acid similarity), wherein a substituted amino acid has similar structural or chemical properties, for example, the replacement of leucine with isoleucine. As those of skill in the art can appreciate, guidance in determining which and how many amino acid residues may be substituted, inserted, or deleted without adversely affecting biological or proposed pharmacological activity may be reasonably inferred in view of this disclosure, and may further be found using computer programs well known in the art, for example, DNAStar® software (DNAStar Inc. Madison, Wl). As those skilled in the art will understand, amino acid substitutions may be made, for example, on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophiiicity, and/or the amphipathic nature of the residues provided that a biological and/or pharmacological activity of the native molecule is retained. Negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; amino acids, with uncharged polar head groups having similar hydrophiiicity values include leucine, isoleucine, and valine; amino acids with aliphatic head groups include glycine, alanine; asparagine, glutamine, serine; and amino acids with aromatic side chains include threonine, phenylalanine, and tyrosine. Examples of conservative substitutions are set forth in Table 4 as follows: Table 4:

"Homology" is a measure of the identity of nucleotide sequences or amino acid sequences. To characterize the homology, subject sequences are aligned so that the highest percentage homology (match) is obtained, after introducing gaps, if necessary, to achieve maximum percent homology. N- or C- terminal extensions shall not be construed as affecting homology. "Identity" per se has an art-recognized meaning and can be calculated using published techniques. Computer program methods to determine identity between two sequences, for example, include DNAStar® software; the GCG® program package (Devereux, J., et al. Nucleic Acids Research (1984) 12(1): 387); BLASTP, BLASTN, FASTA (Atschul, S.F. ef al., J. Molec Biol (1990) 215: 403). Homology (identity) as defined herein is determined conventionally using the well-known computer program, BESTFIT® (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wl, 53711). When using BESTFIT® or any other sequence alignment program (such as the Clustal algorithm from MegAlign software (DNAStar®)) to determine whether a particular sequence is, for example, about 95% homologous to a reference sequence, according to the present invention, the parameters are set such that the percentage of identity is calculated over the full length of the reference nucleotide sequence or amino acid sequence and that gaps in homology of up to about 95% of the total number of nucleotides in the reference sequence are allowed. Ninety-five percent homology is therefore determined, for example, using the BESTFIT® program with parameters set such that the percentage of identity is calculated over the full length of the reference sequence, e.g., SEQ ID NO:1 , and wherein up to 5% of the amino acids in the reference sequence may be substituted with another amino acid. Percent homologies are likewise determined, for example, to identify preferred species, within the scope of the claims appended hereto, which reside within the range of about 95% to 100% homology to SEQ ID NO: 1 as well as the binding site thereof. As noted above, N- or C-terminal extensions shall not be construed as affecting homology. Thus, when comparing two sequences, the reference sequence is generally the shorter of the two sequences. This means that, for example, if a sequence of 50 nucleotides in length with precise identity to a 50 nucleotide region within a 100 nucleotide polynucleotide is compared, there is 100% homology as opposed to only 50% homology. Although the natural polypeptide of SEQ ID NO: 1 and a variant polypeptide may only possess a certain percentage identity, e.g., 95%, they are actually likely to possess a higher degree of similarity, depending on the number of dissimilar codons that are conservative changes. Conservative amino acid substitutions can frequently be made in a protein without altering either the conformation or function of the protein. Similarity between two sequences includes direct matches as well a conserved amino acid substitutes which possess similar structural or chemical properties, e.g., similar charge as described in Table 4. Percentage similarity (conservative substitutions) between two polypeptides may also be scored by comparing the amino acid sequences of the two polypeptides by using programs well known in the art, including the BESTFIT program, by employing default settings for determining similarity. A further embodiment of the invention is a crystal comprising the coordinates of FIG.4, wherein the amino acid sequence is represented by SEQ ID NO:1. A further embodiment of the invention is a crystal comprising the coordinates of FIG. 4, wherein the amino acid sequence is at least 95% or 98% homologous to the amino acid sequence represented by SEQ ID NO:1. Various methods for obtaining atomic coordinates of structurally homologous molecules and molecular complexes using homology modeling are disclosed in, for example, US Patent No. 6,356,845.

D. Structure Based Drug Design Once the three-dimensional structure of a crystal comprising a Akt-1 protein, a functional domain thereof, homoiogue or variant thereof, is determined, a test compound (antagonist or agonist) may be examined through the use of computer modeling using a docking program such as GRAM, DOCK, or AUTODOCK (See for example, Morris et al., J. Computational Chemistry, 19:1639-1662 (1998)). This procedure can include in silico fitting of potential ligands to the Akt-1 crystal structure to ascertain how well the shape and the chemical structure of the potential ligand will complement or interfere with the catalytic domain of Akt-1. (Bugg et al., Scientific American, December:92-98 (1993); West et al., TIPS, 16:67-74 (1995)). As those skilled in the art will understand, computer programs can also be employed to estimate the attraction, repulsion, and steric hindrance of the ligand to the binding site. Generally the tighter the fit (e.g., the lower the steric hindrance, and/or the greater the attractive force) the more potent the potential drug will be since these properties are consistent with a tighter binding constant. Furthermore, the more specificity in the design of a potential drug the more likely that the drug will not interfere with the properties of other proteins. This will minimize potential side-effects due to unwanted interactions with other proteins. One embodiment of the present invention relates to methods of identifying agents that bind to a binding site on Akt-1 's catalytic domain, wherein the binding site comprises amino acid residues Val164, Ala177, Lys179, Glu228, Tyr229, Ala230, Lys276, Met281 and Thr291 , of SEQ ID NO:1 , comprising: contacting Akt-1 with a test compound under conditions suitable for binding of the test compound to the binding site, and determining whether the test compound binds in the binding site, wherein if binding occurs, the test ligand is an agent that binds in the binding site. In certain embodiments, the testing may be carried out in silico using a variety of molecular modeling software algorithms including, but not limited to, DOCK, ALADDIN, CHARMM simulations, AFFINITY, C2-LIGAND FIT, Catalyst, LUDI, CAVEAT, and CONCORD. (Brooks, ef al. CHARMM: a program for macromolecular energy, minimization, and dynamics calculations. (J Comp.Chem 1983, 4:187-217; E.G. Meng, B.K. Shoichet & I.D. Kuntz. Automated docking with grid-based energy evaluation. J Comp Chem 1992, 13:505-524). In another embodiment, a potential ligand may be obtained by screening a random peptide library produced by a recombinant bacteriophage (Scott and Smith, Science, 249:386-390 (1990); Cwirla et al., Proc. Natl. Acad. Sci., 87:6378-6382 (1990); Devlin et al., Science, 249:404-406 (1990)) or a chemical library, or the like. A ligand selected in this manner can be then be systematically modified by computer modeling programs until one or more promising potential ligands are identified. Such analysis, for example, has been shown to be effective in the development of HIV protease inhibitors. (Lam, et al., Science, 263:380-384 (1994); Wlodawer, et al., Ann. Rev. Biochem., 62:543-585 (1993); Appelt, Perspectives in Drug Discovery and Design, 1 :23-48 (1993); Erickson, Perspectives in Drug Discovery and Design, 1 :109-128 (1993)). Such computer modeling allows the selection of a finite number of rational chemical modifications, as opposed to the countless number of essentially random chemical modifications that could be made, of which any one might lead to a potential compound. Each chemical modification requires additional chemical steps, which while being reasonable for the synthesis of a finite number of compounds, quickly becomes overwhelming if all possible modifications needed to be synthesized are actually synthesized. Thus, through the use of the three-dimensional structure disclosed herein and computer modeling, a large number of these compounds can be rapidly screened on a computer monitor screen, and a few likely candidates can be determined without the laborious synthesis of untold numbers of compounds. Once a potential ligand (agonist or antagonist) is identified, it can be either selected from a library of chemicals as are commercially available from most large chemical companies or alternatively the potential ligand may be synthesized de novo. As mentioned above, the de novo synthesis of one or even a relatively small group of specific compounds is reasonable in the art of drug design. The potential ligand can be placed into any standard binding assay as well known to those skilled in the art to test its effect on Akt-1 activity. When a suitable drug is identified, another crystal can be grown comprising a protein-ligand complex formed between an Akt-1 protein and the drug. Preferably, the crystal diffracts X-rays allowing the determination of the atomic coordinates of the protein-ligand complex to a resolution of less than 5.0 Angstroms, more preferably less than 3.0 Angstroms, and even more preferably less than 2.0 Angstroms. The three-dimensional structure of the supplemental crystal can be determined by Molecular Replacement Analysis. Molecular replacement involves using a known three-dimensional structure as a search model to determine the structure of a closely related molecule or protein-ligand complex in a new crystal form. The measured X-ray diffraction properties of the new crystal are compared with the search model structure to compute the position and orientation of the protein in the new crystal. Computer programs that can be used include: X-PLOR and AMORE (J. Navaza, Acta Crystallographies ASO, 157- 163 (1994)). As those of skill in the art will appreciate, once the position and orientation are known, an electron density map can be calculated using the search model to provide X-ray phases. Thereafter, the electron density is inspected for structural differences, and the search model is modified to conform to the new structure. Using this approach, claimed structure of Akt-1 , can be used to solve the three-dimensional structures of any such Akt-1 complexed with a new ligand. Other suitable computer programs that can be used to solve the structures of such STAT crystals include: QUANTA; CHARMM; INSIGHT; SYBYL; MACROMODEL; and ICM. Suitable in silico methods for screening, designing or selecting ligands are disclosed in, for example, US Patent No. 6,356,845.

E. Ligands In one aspect, the present invention provides ligands which interact with a binding site of the Akt- 1 catalytic domain defined by a set of points having a root mean square deviation of less than about 2.5 A from points representing the backbone atoms of the amino acids represented by the structure coordinates listed in FIG.4. A further embodiment of the present invention comprises binding agents which interact with a binding site of Akt-1 defined by a set of points having a root mean square deviation of less than about 2.5, 2.0, 1.7, 1.5, 1.2, 1.0, 0.7, 0.5, or even 0.2 A from points representing the backbone atoms of the amino acids represented by the structure coordinates listed in FIG. 4. Such embodiments represent variants of the Akt-1 crystal. In another aspect, the present invention provides ligands which bind to a correctly folded polypeptide comprising an amino acid sequence spanning amino acids 144 to 480 listed in SEQ ID NO:1 , or a homoiogue or variant thereof. In other embodiments, the ligand is a competitive or uncompetitive inhibitor of Akt-1. In yet other embodiments, the ligand inhibits Akt-1 with an IC₅₀ or Ki of less than about 10 yM, 1μM, 500nM, 100nM, 50nM, 10nM or even 1 nM. In further embodiments, the ligand inhibits Akt-1 with a K, that is less than about one-half, one-fifth, or one-tenth the Ki that the substance has for inhibition of any other Akt enzyme. More specifically, the substance inhibits Akt-1 activity to the same degree at a concentration of about one-half, one-fifth, one-tenth or less than the concentration required for any other Akt enzyme. One embodiment of the present invention relates to ligands, such as proteins, peptides, peptidomimetics, small organic molecules, etc., designed or developed with reference to the crystal structure of Akt-1 as represented by the coordinates presented herein in FIG. 4, and portions thereof. Such binding agents interact with the binding site of the Akt-1 represented by one or more amino acid residues selected from Val164, Ala177, Lys179, GIu228, Tyr229, Ala230, Lys276, Met281 and Thr291.

F. Machine Readable Storage Media Transformation of the structure coordinates for all or a portion of Akt-1 , or the Akt-1 /ligand complex or one of its binding pockets, for structurally homologous molecules as defined below, or for the structural equivalents of any of these molecules or molecular complexes as defined above, into three- dimensional graphical representations of the molecule or complex can be conveniently achieved through the use of commercially-available software. The invention thus further provides a machine-readable storage medium comprising a data storage material encoded with machine readable data which, when using a machine programmed with instructions for using said data, is capable of displaying a graphical three-dimensional representation of any of the molecule or molecular complexes of this invention that have been described above. In a preferred embodiment, the machine-readable data storage medium comprises a data storage material encoded with machine readable data which, when using a machine programmed with instructions for using said data, is capable of displaying a graphical three-dimensional representation of a molecule or molecular complex comprising all or any parts of a Akt-1 C-terminal catalytic domain or binding pocket, as defined above. In another preferred embodiment, the machine-readable data storage medium is capable of displaying a graphical three-dimensional representation of a molecule or molecular complex defined by the structure coordinates of the amino acids listed in FIG. 4, plus or minus a root mean square deviation from the backbone atoms of said amino acids of not more than 2.5 A. In an alternative embodiment, the machine-readable data storage medium comprises a data storage material encoded with a first set of machine readable data which comprises the Fourier transform of the structural coordinates set forth in FIG. 4, and which, when using a machine programmed with instructions for using said data, can be combined with a second set of machine readable data comprising the X-ray diffraction pattern of a molecule or molecular complex to determine at least a portion of the structural coordinates corresponding to the second set of machine readable data. For example, a system for reading a data storage medium may include a computer comprising a central processing unit ("CPU"), a working memory which may be, e.g., RAM (random access memory) or "core" memory, mass storage memory (such as one or more disk drives or CD-ROM drives), one or more display devices (e.g., cathode-ray tube ("CRT") displays, light emitting diode ("LED") displays, liquid crystal displays ("LCDs"), electroluminescent displays, vacuum fluorescent displays, field emission displays ("FEDs"), plasma displays, projection panels, etc.), one or more user input devices (e.g., keyboards, microphones, mice, touch screens, etc.), one or more input lines, and one or more output lines, all of which are interconnected by a conventional bidirectional system bus. The system may be a stand-alone computer, or may be networked (e.g., through local area networks, wide area networks, intranets, extranets, or the internet) to other systems (e.g., computers, hosts, servers, etc.). The system may also include additional computer controlled devices such as consumer electronics and appliances. Input hardware may be coupled to the computer by input lines and may be implemented in a variety of ways. Machine-readable data of this invention may be inputted via the use of a modem or modems connected by a telephone line or dedicated data line. Alternatively or additionally, the input hardware may comprise CD-ROM drives or disk drives. In conjunction with a display terminal, a keyboard may also be used as an input device. Output hardware may be coupled to the computer by output lines and may similarly be implemented by conventional devices. By way of example, the output hardware may include a display device for displaying a graphical representation of a binding pocket of this invention using a program such as QUANTA as described herein. Output hardware might also include a printer, so that hard copy output may be produced, or a disk drive, to store system output for later use. In operation, a CPU coordinates the use of the various input and output devices, coordinates data accesses from mass storage devices, accesses to and from working memory, and determines the sequence of data processing steps. A number of programs may be used to process the machine-readable data of this invention. Such programs are discussed in reference to the computational methods of drug discovery as described herein. References to components of the hardware system are included as appropriate throughout the following description of the data storage medium. Machine-readable storage devices useful in the present invention include, but are not limited to, magnetic devices, electrical devices, optical devices, and combinations thereof. Examples of such data storage devices include, but are not limited to, hard disk devices, CD devices, digital video disk devices, floppy disk devices, removable hard disk devices, magneto-optic disk devices, magnetic tape devices, flash memory devices, bubble memory devices, holographic storage devices, and any other mass storage peripheral device. It should be understood that these storage devices include necessary hardware (e.g., drives, controllers, power supplies, etc.) as well as any necessary media (e.g., disks, flash cards, etc.) to enable the storage of data. G. Pharmaceutical Compositions The present invention provides methods for treating certain diseases in a mammal, preferably a human beings, in need of such treatment using the ligands, and preferably the inhibitors, as described herein. The ligand can be advantageously formulated into pharmaceutical compositions comprising a therapeutically effective amount of the ligand, a pharmaceutically acceptable carrier and other compatible ingredients, such as adjuvants, Freund's complete or incomplete adjuvant, suitable for formulating such pharmaceutical compositions as is known to those skilled in the art. Pharmaceutical compositions containing the ligand can be used for the treatment of a a variety of cancers, such as, for example breast, prostate, colon and lung. Examples of cancers that can be treated according to the methods of the present invention include, but are not limited to, breast, prostate, pancreas, lung, and colorectal cancers. The pharmaceutical composition is administered to the mammal in a therapeutically effective amount such that treatment of the disease occurs. The present invention is further illustrated by the following examples, which should not be construed as limiting in any way. The contents of all cited references (including literature references, issued patents, published patent applications as cited throughout this application) are hereby expressly incorporated by reference in their entireties. The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, microbiology and recombinant DNA, X-ray crystallography, and molecular modeling which are within the skill of the art. As those of skill in the art will appreciate, such techniques are explained fully in the literature. See, for example, Molecular Cloning: A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al. U.S. Patent No: 4,683,195; Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames & S. J. Higgins eds. 1984); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds.), Crystallography Made Clear: A Guide For Users Of Macromolecular Models (Gales Rhodes, 2^nd Ed. San Diego: Academic Press, 2000). EXAMPLES Example 1 : Construction and expression of human Akt-1 wild type catalytic domain

A. Construction of hAkt-1 (S473D) in pFastbac Hta: The DNA fragment encoding residues 144-480 of human Akt-1 (SEQ ID NO:1) was amplified by PCR using the following primers: (1 ) Akt1 -R144F (5'-AATATGGATCCGCGCGTGACCATGAACGAG-3'), which introduces a Bam HI site at the N-terminus, and (2) (2) Akt1-A480R (5'- AATTC CGAGTTATCAGGCCGTGCTGCTGGCCGAGTAGG-3'), which introduces a Xho I site at the C-terminus.

The high-fidelity PCR core reagent kit (Stratagene, La Jolla, CA, USA) and the PTC-200 DNA Engine (96- well format; MJ Research, Watertown, MA, USA) were used for PCR using the following protocol: 1 cycle of 2 min at 92°C and then 35 cycles of 40 s at 92°C, 50 s at 55°C, 2 min at 68°C. The PCR product (1014 bp) was column (Roche Applied Sciences) purified, digested with BamYW and Xho\ restriction enzymes and sub- cloned into the pFastbac HTa baculovirus transfer vector (Gibco BRL), according to manufacturer's protocol (Gibco BRL). Site-specific substitution of Akt-1 : Ser 473 to Asp (S473D) was performed using a QuickChange XL Site-Directed mutation kit (Stratagene) as described by the manufacturer, and resulted in the A473D human Akt mutant construct. The final construct that encodes the hAkt-1 , (residues 144-480 and S473D), in pFastbac HTa is referred to as Akt1 -Construct #7. Oligonucleotides were synthesized by Invitrogen Life Technologies (Frederick, MD).

B. Production of recombinant baculovirus: Cloning steps were monitored by restriction endonuclease mapping and sequencing analysis. E. coli clones with recombinant bacmid were obtained after transformation of E. coli DHIOBac cells (Invitrogen Corp.) with 5 ng of Akt1 -Construct #7 plasmid DNA and blue/white-screening according to manufacturer's protocol (Invitrogen). Monolayers of Sfθ cells (20 X 10⁶ cells in a 162 cm² culture flask) were transfected by overlaying 20 ml of transfection mixture containing 100 μl of mini-prep bacmid DNA and 100 μl of CellFECTIN reagent (Gibco BRL) in Sf-900 II Serum Free Medium (SFM). The transfection mixture was removed after 5 h of incubation (27^SC) and the cells were overlaid with 25 ml of Sf-900 II SFM. The recombinant virus (P0) were harvested at 72h post-transfection, and further amplification of the virus was achieved by infecting 1 L of Sf-9 cells (1.2 X 10⁶ cells/ml) with 20 ml of the P0 virus for 65-72 h. Baculo Infected Insect Cells (BMC) stocks were prepared as follows: when the cell diameter increased by 2-4 μm above the baseline (usually 65-72h) and while the cell viability was > 80%, the BIIC's were gently spun down and re-suspended in a freezing medium (90% SF900 II, 1% w/v BSA, 10% v/v DMSO) at 1 X 10⁷ viable cells/ml. The BIIC's were frozen down as 1 ml aliquots using cryopreservation methods know in the art, and stored in -70°C in liquid nitrogen for long-term storage. C. Expression of recombinant Akt-1 Construct: Quickly thaw one vial (1 ml) of BIIC in a 37°C water bath and transfer the cell suspension to 100 ml of SF900 II media (1 :100 dilution). A 3L erienmeyer flask containing 1 L of Sf9 cells (1.2 X 10⁶ cells/ml) in SF900 II media was infected with 10 ml of 1 :100 diluted BIIC cell suspension. Culture was incubated at 27°C at a constant stirring speed of 67 rpm. Cells were harvested by centrifugation (4000 rpm for 10 min) when the cell diameter increased by 2-4 μm above the baseline (usually 65-72h) and while the cell viability was > 80%. Example 2: Purification of Akt-1 wild type catalytic domain Baculovirus cell paste, from a 10L cell culture, containing the overexpressed Akt-1 recombinant protein was resuspended in 3 volumes (3.0ml_/g) Buffer A (50mM Tris pH 7.5, 300mM NaCl (sodium chloride), 0.5mM TCEP (Tri(2-carboxyethyl) phosphine hydrochloride)) and Complete™ protease inhibitor cocktail tablets (Roche). The cells were lysed with one pass on a microfluidizer and the cell debris was removed by centrifugation at 4°C for 35 minutes at 14,000 rpm in a Sorval SLA-1500 rotor. The supernatant was transferred to a clean tube and 10ml of TALON Metal Affinity Resin (BD-Clonetech) was added. The suspension was incubated with gentle rocking at 4°C for 1 hour and then subjected to centrifugation at 700 x g in a swinging bucket rotor. The supernatant was discarded and the resin was resuspended in 20ml Buffer A and transferred to an XK-16 column (Amersham Biosciences (UK)) connected to an FPLC™. The resin was washed with 10 column volumes of Buffer A, or until, absorption reading at a wavelength of 280 was < 0.1. Following the wash step, the column containing the bound resin was connected upstream to a XK-26/30 Fast Desalt column (Pharmacia) previously equilibrated with Buffer A. The Akt-1 was eluted from the TALON resin with a step gradient of Buffer A pius120mM Imidizole (pH 7.5). The eluted fractions were pooled and directly loaded onto a HiPrep 26/10 desalting column (Amersham Biotech), which was previously equilibrated with Buffer B (25mM Tris pH 7.5, 200mM NaCl, 0.5mM TCEP (tri(2-carboxyethyl) phosphine hydrochloride). The eluted fractions were digested with 2000U rTEV protease (Invitrogen Frederick, MD) overnight at 17°C. Completeness of cleavage was checked using SDS-Page or LC/MS. The digested Akt- 1 protein was loaded onto a TALON column, which was equilibrated with Buffer B. The flow through fractions were collected, pooled and concentrated using Vivaspin-20, 10K MWCO at 4°C. The concentrated fractions where next loaded onto a Superdex 75 HiLoad 16/60 prep grade column (Pharmacia) equilibrated with Buffer C (25mM Tris pH 7.5, 150mM NaCl, and 1mM TCEP). The protein eluted between 55-65ml. The eluted fraction was concentrated to 5.7 mg/ml. Example 3: Activation of Akt-1 with (PDK-1) PDK1 -active his-myc protein was added to the purified Akt-1 recombinant protein from Example 2, at a ratio of 1.0mgPDk/30.0mg Akt-1. (PDK-1 was produced by recombinant expression in an insect cell system using standard cloning and purification procedures known in the art). 1.0 M of MgCI₂ was added making a final concentration of 5.0 mM, and 0.1M ATP was also added to a final concentration of 1.0 mm in Buffer C (25 mM Tris pH 7.5, 150 mM Na and 1.0 mM TCEP) forming a reaction. The reaction was incubated reaction at room temperature for 90 minutes, thereafter loaded reaction onto a 1.0 mL HiTrap Heparin HP column (Amersham Biotech) equilibrated with Buffer C. Collected the flow through fractions. Performed a buffer exchange on the flow through fractions into Buffer C using a HiPrep 26/10 desalting column (Amersham Biotech) to remove ATP. Aliquots of 0.2 ml fractions were collected and frozen down using normal cryopreservation methods, and stored at -80 °C or liquid nitrogen for long-term storage. Example 4: Crystallization of the ternary complex of Akt-1 wild-type catalytic domain with AMP-

PNP with the substrate peptide GSK3-β

Crystallization screens were setup using the sitting drop/vapor diffusion method in 96-well Greiner plates. A mixture of activated Akt-1 protein was pre-incubated for 1 hour at a concentration of 2 mg/ml with the following mixture: 10OmM MnCI₂ (manganese chloride) to a final concentration of 2.5mM; 2mM of the substrate peptide GSK3-β to a final concentration of 0.3mM; and 20mM AMP-PNP to a final concentration of 2.5mM. The protein mixture sample was at a final concentration of 5-6 mg/ml. The protein mixture was next combined (at a ratio of 1 :1) with the precipitate containing 20% PEG 10K (polyethylene glycol-10,000), and 0.1 M HEPES (4-(2-hydroxyethyl)-1 -piperazineethanesulfonic acid) pH 7.5. Conditions were screened using the Hampton Research Crystal Screen HT and Emerald Biosciences Wizard I & II screens. Crystals were obtained under condition B5 of the Crystal Screen HT containing 20% PEG-4000 and 1 M HEPES pH 7.5. Crystals grew as long clustered rods, and rods were broken apart into smaller fragments measuring 150 x 40 x 20 μm. Example 5: X-ray data collection, structure determination and refinement of active Akt-1 :AMP- PNP:GSK3-β complex The crystals prepared in Example 4 were transferred to a cryoprotectant solution, made up of the reservoir solution, with 15% ethylene glycol, and then flash-frozen in a stream of cold nitrogen gas at 100K. X-ray diffraction data were collected on a Rigaku HTC detector, mounted on a Rigaku FR-E X-ray generator operated at 45kV, 45mA. Data were processed using the d^*trek suite of software (Molecular Structure Corporation), and data collection statistics are summarized in Table 5a. The crystal belongs to the monoclinic space group P2_L with unit cell dimensions a=42.65, b=55.33, c=90.58A, oc=90.0, β=102.47, γ=90.0^s. The structure was solved by the method of molecular replacement, using the program AmoRe (Navaza, J., (1994). Ada Cryst AδO 157-163). A homology model of Akt-1 was used as the starting search model. The final model was built with a combination of automatic fitting in the program arpWarp (http://www. arp-warp.orq) , and manual rebuilding on the graphics screen using the program O. Partial structure factors from a bulk-solvent model and anisotropic B-factor correction were supplied throughout the refinement. The R-factor for the current model is 0.215 (free R-factor, 5% of the data, 0.267). The refinement statistics are summarized in Table 1 b.

The final refined model contains 318 out of 342 amino acid residues of the Akt1 protein construct. Clear electron density is seen for the substrate peptide, AMP-PNP and two putative Mn²⁺ ions. Table 5a -Data statistics

¹ Numbers in parentheses refer to the highest resolution range (2.02-2.09A). ² R_sym = Σ(l-<l>)/Σ <l>. Table 5b- Refinement statistics

^d R = ∑||F₀bs Fcalc||/∑|Fobs| Equivalents While specific embodiments of the subject invention have been discussed, the above specification is illustrative and not restrictive. Many variations of the invention will become apparent to those skilled in the art upon review of this specification. The appended claims should be interpreted by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations. AH publications and patents mentioned herein are hereby incorporated by reference in their entireties. In case of conflict any definitions herein will control.

Claims

What is claimed is: I . A crystal composition of the catalytic domain of the active form of protein kinase B (Akt-1).

2. The Akt-1 crystal of claim 1 having the atomic structure coordinates of Fig. 4, a derivative or variant thereof.

3. The Akt-1 crystal of claim 1 having a resolution of about 2.02 Angstroms.

4. The Akt-1 crystal of claim 1 having a space group of P2-ι so as to form a unit cell of dimensions of about a=42.65 A b= 55.33A, and c= 90.58A.

5. The Akt-1 crystal of claim 1 comprising a binding site defined by the spatial orientation of the amino acid residues of one of Table 1 , Table 2 or Table 3.

6. An AKT-1 crystal comprising a polypeptide with an amino acid sequence spanning amino acids Arg144 to Ala480 as listed in SEQ ID NO: 2, a homoiogue, an analogue or a variant thereof.

7. The crystal of claim 6, wherein said homoiogue, functional equivalent, an analogue or a variant thereof has an amino acid identity of at least 95% with a polypeptide having an amino acid sequence spanning amino acids Arg144 to Ala480 as listed in SEQ ID NO:2.

8. The crystal of claim 6, wherein the homoiogue or variant thereof has a protein backbone comprising the atomic structure coordinates, or portions thereof, that are within a root mean square of +/- 2.5, 2.0, 1.5, 1.2, 1.0, 0.7, 0.5, or even 0.2 A of the atomic structure coordinates, or portions thereof, listed in FIG. 4.

9. A crystal of a protein-ligand molecule or molecular complex comprising: (a) a polypeptide with an amino acid sequence from Arg144 to Ala480 listed in SEQ ID NO:1 , or a homoiogue, an analogue or a variant thereof; (b) a ligand; (c) and the crystal effectively diffracts X-rays for the determination of atomic structure coordinates of the protein-ligand complex to a resolution of greater than 2.02 Angstroms.

10. The crystal of claim 9 having a space group of P2-i so as to form a unit ceil of dimensions of about a=42.65 A b= 55.33A, and c= 90.58A.

I I . The binding site of Akt-1 represented by one or more amino acid residues from Val164, Ala177, Lys179, Glu228, Tyr229, Ala230, Lys276, Met281 and Thr291.

12. A method of designing a compound that binds to Akt-1 using the crystal of claim 1 , comprising selecting a compound by performing structure-based drug design with the atomic structure coordinated determined for the crystal, wherein said selecting is performed in conjunction with computer modeling.

13. A computer for producing a three-dimensional representation of a polypeptide with an amino acid sequence spanning amino acids Arg144 to Ala480 listed in SEQ ID NO:1 , or a homoiogue, or a variant thereof, comprising: a computer-readable data storage medium comprising a data storage material encoded with computer-readable data, wherein said data comprises the structure coordinates of FIG. 4, or portions thereof; a working memory for storing instructions for processing said computer-readable data; a central-processing unit coupled to said working memory and to said computer-readable data storage medium for processing said computer-machine readable data into said three-dimensional representation; and a display coupled to said central- processing unit for displaying said representation.

14. A computer for producing a three-dimensional representation of a molecule or molecular complex comprising: a binding site defined by the structure coordinates in FIG. 4, or a the structural coordinates of a portion of the residues in FIG. 4, or the structural coordinates of one or more Akt-1 amino acids in SEQ ID NO: 1 selected from Vah 64, Ala1 7, Lys179, Glu228, Tyr229, Ala230,

Lys276, Met281 and Thr291 , wherein said computer comprises; a computer-readable data storage medium comprising a data storage material encoded with computer-readable data, wherein said data comprises the structure coordinates of FIG. 4, or portions thereof; a working memory for storing instructions for processing said computer-readable data; a central-processing unit coupled to said working memory and to said computer-readable data storage medium for processing said computer- machine readable data into said three-dimensional representation; and a display coupled to said central-processing unit for displaying said representation.

15. A method for identifying potential ligands for Akt-1 , or homologues, an analogues or a variants thereof, comprising the steps of: displaying three dimensional structure of Akt-1 enzyme, or portions thereof, as defined by atomic structure coordinates in FIG. 4, on a computer display screen; optionally replacing one or more Akt-1 enzyme amino acid residues listed in SEQ ID NO:1 , or one or more of the amino acids listed in Tables 1 -3, or one or more amino acid residues selected from Val164, Ala177, Lys179, Glu228, Tyr229, Ala230, Lys276, Met281 and Thr291, in said three- dimensional structure with a different naturally occurring amino acid or an unnatural amino acid; employing said three-dimensional structure to design or select said ligand; contacting said ligand with Akt-1 , or variant thereof, in the presence of one or more substrates; and measuring the ability of said ligand to modulate the activity of Akt-1.