Crystallized P38 complexes

Abstract

This invention provides certain crystallized, protein kinase-ligand complexes, in particular P38-ligand complexes, and their structure coordinates. The structure coordinates are based on the structure of a phosphorylated P38γ complex which has now been solved and which reveals new structural information useful for understanding the activated states of other, related kinase proteins as described herein. The key structural features of the proteins, particularly the shape of the substrate binding site, are useful in methods for designing or identifying selective inhibitors of the protein kinases, particularly P38γ and in solving the structures of other proteins with similar features. The structure coordinates may be encoded in a data storage medium for use with a computer for graphical three-dimensional representation of the structure and for computer-aided molecular design of new inhibitors.

We claim:  
     
         1 . A crystalline composition comprising a phosphorylated P38 protein-ligand complex.  
     
     
         2 . The crystalline composition of  claim 1  wherein the complex is capable of being resolved at 2.4 Å resolution, the complex comprising: 
 a) a purified enzyme selected from phosphorylated P38α, phosphorylated P38β, phosphorylated P38δ, phosphorylated P38γ, or a phosphorylated isoform of any of the foregoing;  
 b) a ligand; and  
 c) magnesium ions.  
 
     
     
         3 . The crystalline composition according to  claim 2 , wherein said enzyme is P38γ.  
     
     
         4 . A crystalline protein kinase-ligand complex, said kinase comprising a binding pocket defined by the structure coordinates of the P38γ amino acids Val33, Ala40, Val41, Ala54, Lys56, Ile87, Met109, Pro110, Phe111, Met112, Gly113, Thr114, Asp115, Lys118, Asp153, Lys155, Gly157, Asn158, Ala160, Leu170, Asp171, Gly173, and Leu174 according to FIG. 1, or a homologue of said kinase, wherein said homologue comprises a binding pocket that has a root mean square deviation from the backbone atoms of said amino acids of not more than 1.15 Å.  
     
     
         5 . A crystalline protein kinase-ligand complex, said kinase selected from the proteins listed in Table 1, wherein the ψ angle of the residue corresponding to Met112 of p38γ is in the range of about −60° to 60° and the φ angle of the residue corresponding to Gly113 of p38γ is in the range of about 30° to 150°.  
     
     
         6 . The crystalline protein kinase-ligand complex of  claim 5  wherein the ψ angle is in the range of about −45° to 45°.  
     
     
         7 . The crystalline protein kinase-ligand complex of  claim 6  wherein the ψ angle is in the range of about −30° to 30°.  
     
     
         8 . The crystalline protein kinase-ligand complex of any of  claims 5  to  7  wherein the φ angle is in the range of about 45° to 135°.  
     
     
         9 . The crystalline protein kinase-ligand complex of any of  claims 5  to  7  wherein the φ angle is in the range of about 60° to 120°.  
     
     
         10 . A crystalline protein kinase-ligand complex, said kinase comprising amino acid residues that correspond by functional and/or sequence alignment to the Met112 and Gly113 residues of P38γ or that correspond by functional and/or sequence alignment to the Met112 and Gly113 equivalent residues of one or more proteins listed in Table 1, wherein the ψ angle of the residue corresponding to Met112 is in the range of about −60° to 60° and the φ angle of the residue corresponding to Gly113 is in the range of about 30° to 150°.  
     
     
         11 . A method for evaluating the ability of a chemical entity to associate with a molecule or molecular complex comprising a binding pocket, said method comprising the steps of: 
 a) creating a computer model of the binding pocket using structure coordinates wherein the root mean square deviation between said structure coordinates and the structure coordinates of the P38γ amino acids Val33, Ala40, Val41, Ala54, Lys56, Ile87, Met109, Pro110, Phe111, Met112, Gly113, Thr114, Asp115, Lys118, Asp153, Lys155, Gly157, Asn158, Ala160, Leu170, Asp171, Gly173, and Leu174 according to FIG. 1 is not more than about 1.15 Å;    b) employing computational means to perform a fitting operation between the chemical entity and said computer model of the binding pocket; and    c) analyzing the results of said fitting operation to quantify the association between the chemical entity and the binding pocket model.    
     
     
         12 . The method according to  claim 11 , wherein said binding pocket is further defined by the structure coordinates of P38γ amino acids Pro32, Cys42, Ser43, Val53, Ile55, Lys57, Leu58, Thr59, Arg70, Glu74, Gly88, Leu107, Val108, Leu116, Gly117, Pro156, Leu159, Val161, Lys168, Phe172, Ala175, and Thr188 according to FIG. 1.  
     
     
         13 . The method according to  claim 12  wherein said molecule or molecular complex is defined by the set of structure coordinates for all P38γ amino acids according to FIG. 1.  
     
     
         14 . A method of utilizing molecular replacement to obtain structural information about a molecule or a molecular complex of unknown structure, comprising the steps of: 
 a. crystallizing said molecule or molecular complex;    b. generating an X-ray diffraction pattern from said crystallized molecule or molecular complex;    c. applying at least a portion of the structure coordinates set forth in FIG. 1 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 whose structure is unknown.    
     
     
         15 . A computer for producing a three-dimensional representation of a molecule or molecular complex, wherein said computer comprises: 
 a) a machine-readable data storage medium comprising a data storage material encoded with machine-readable data, wherein said machine-readable data comprises the structure coordinates of P38γ amino acids Val33, Ala40, Val41, Ala54, Lys56, Ile87, Met109, Pro110, Phe111, Met112, Gly113, Thr114, Asp115, Lys118, Asp153, Lys155, Gly157, Asn158, Ala160, Leu170, Asp171, Gly173, and Leu174 according to FIG. 1, or structural coordinates having a root mean square deviation from the backbone atoms of said amino acids of not more than 1.15 Å;    b) a working memory for storing instructions for processing said machine-readable data;    c) a central-processing unit coupled to said working memory and to said machine-readable data storage medium, for processing said machine readable data into said three-dimensional representation; and    d) an output hardware coupled to said central-processing unit, for receiving said three-dimensional representation.    
     
     
         16 . A method for identifying a potential agonist or antagonist of a molecule comprising a P38γ-like binding pocket, comprising the steps of: 
 a. using the atomic coordinates of Val33, Ala40, Val41, Ala54, Lys56, Ile87, Met109, Pro110, Phe111, Met112, Gly113, Thr114, Asp115, Lys118, Asp153, Lys155, Gly157, Asn158, Ala160, Leu170, Asp171, Gly173, and Leu174 according to FIG. 1±a root mean square deviation from the backbone atoms of said amino acids of not more than 1.15 Å, to generate a three-dimensional structure of molecule comprising the P38γ-like binding pocket;  
 b. employing said three-dimensional structure to design or select said potential agonist or antagonist;  
 c. synthesizing said agonist or antagonist; and  
 d. contacting said agonist or antagonist with said molecule to determine the ability of said potential agonist or antagonist to interact with said molecule.  
 
     
     
         17 . The method according to  claim 16 , wherein the atomic coordinates of Pro32, Val33, Ala40, Val41, Cys42, Ser43, Val53, Ala54, Ile55, Lys56, Lys57, Leu58, Thr59, Arg70, Glu74, Ile87, Gly88, Leu107, Val108, Met109, Pro110, Phe111, Met112, Gly113, Thr114, Asp115, Leu116, Gly117, Lys118, Asp153, Lys155, Pro156, Gly157, Asn158, Leu159, Ala160, Val161, Lys168, Leu170, Asp171, Phe172, Gly173, Leu174, Ala175, and Thr188 according to FIG. 1±a root mean square deviation from the backbone atoms of said amino acids of not more than 1.15 Å, are used to generate said three-dimensional structure of the molecule comprising a P38γ-like binding pocket.  
     
     
         18 . The method according to  claim 17 , wherein the atomic coordinates of all the amino acids of P38γ according to FIG. 1±a root mean square deviation from the backbone atoms of said amino acids of not more than 1.15 Å, are used to generate a three-dimensional structure of molecule comprising a P38γ-like binding pocket.  
     
     
         19 . A computer for producing a three-dimensional representation of a protein kinase or a protein kinase-ligand complex, or portion thereof, wherein said computer comprises: 
 a) a machine-readable data storage medium comprising a data storage material encoded with machine-readable data, wherein said machine-readable data comprises the structure coordinates of said kinase, or portion thereof, said kinase or portion thereof comprising amino acid residues that correspond by functional and/or sequence alignment to the Met112 and Gly113 residues of P38γ or that correspond by functional and/or sequence alignment to the Met112 and Gly113 equivalent residues of one or more proteins listed in Table 1, wherein the ψ angle of the residue corresponding to Met112 is in the range of about −60° to 60° and the φ angle of the residue corresponding to Gly113 is in the range of about 30° to 150°;    b) a working memory for storing instructions for processing said machine-readable data;    c) a central-processing unit coupled to said working memory and to said machine-readable data storage medium, for processing said machine readable data into said three-dimensional representation; and    d) an output hardware coupled to said central-processing unit, for receiving said three-dimensional representation.    
     
     
         20 . The computer of  claim 19  wherein the ψ angle is in the range of about −45° to 45°.  
     
     
         21 . The computer of  claim 20  wherein the ψ angle is in the range of about −30° to 30°.  
     
     
         22 . The computer of any of  claims 19  to  21  wherein the φ angle is in the range of about 45° to 135°.  
     
     
         23 . The computer of any of  claims 19  to  21  wherein the φ angle is in the range of about 60° to 120°.  
     
     
         24 . The computer of  claim 19  wherein the machine-readable data comprises the structure coordinates of a protein kinase, or portion thereof, said kinase selected from a protein listed in Table 1.  
     
     
         25 . A method for evaluating the ability of a chemical entity to associate with a protein kinase binding pocket, said method comprising the steps of: 
 a) creating a computer model of the binding pocket using structure coordinates wherein: 
 (i) the root mean square deviation between said structure coordinates and the structure coordinates of the P38γ amino acids Val33, Ala40, Val41, Ala54, Lys56, Ile87, Met109, Pro110, Phe111, Met112, Gly113, Thr114, Asp115, Lys118, Asp153, Lys155, Gly157, Asn158, Ala160, Leu170, Asp171, Gly173, and Leu174 according to FIG. 1 is within about 3.0 angstroms,  
 (ii) said binding pocket model depicts amino acid residues that correspond by functional and/or sequence alignment to the Met112 and Gly113 residues of P38γ or that correspond by functional and/or sequence alignment to the Met112 and Gly113 equivalent residues of one or more proteins listed in Table 1, and  
 (iii) said binding pocket model depicts the ψ angle of the residue corresponding to Met112 to be in the range of about −60° to 60° and the φ angle of the residue corresponding to Gly113 to be in the range of about 30° to 150°;  
   b) employing computational means to perform a fitting operation between the chemical entity and the binding pocket model; and    c) analyzing the results of said fitting operation to quantify the association between the chemical entity and the binding pocket model.    
     
     
         26 . The method of  claim 25  wherein the root mean square deviation is within about 2.7 angstroms.  
     
     
         27 . The method of  claim 26  wherein the root mean square deviation is within about 2.5 angstroms.  
     
     
         28 . The method of  claim 25  wherein the ψ angle is in the range of about −45° to 45°.  
     
     
         29 . The method of  claim 26  wherein the ψ angle is in the range of about −45° to 45°.  
     
     
         30 . The method of  claim 27  wherein the ψ angle is in the range of about −45° to 45°.  
     
     
         31 . The method of  claim 25  wherein the ψ angle is in the range of about −30° to 30°.  
     
     
         32 . The method of  claim 26  wherein the ψ angle is in the range of about −30° to 30°.  
     
     
         33 . The method of  claim 27  wherein the ψ angle is in the range of about −30° to 30°.  
     
     
         34 . The method of any of  claims 25  to  33  wherein the φ angle is in the range of about 45° to 135°.  
     
     
         35 . The method of  claim 25  to  33  wherein the φ angle is in the range of about 60° to 120°.  
     
     
         36 . The method of  claim 25  wherein the protein kinase is selected from a Table 1 protein.  
     
     
         37 . A method for identifying a potential agonist or antagonist of a molecule comprising a P38γ-like binding pocket, comprising the steps of: 
 a) creating a computer model of the binding pocket using structure coordinates wherein: 
 (i) the root mean square deviation between said structure coordinates and the structure coordinates of the P38γ amino acids Val33, Ala40, Val41, Ala54, Lys56, Ile87, Met109, Pro110, Phe111, Met112, Gly113, Thr114, Asp115, Lys118, Asp153, Lys155, Gly157, Asn158, Ala160, Leu170, Asp171, Gly173, and Leu174 according to FIG. 1 is within about 3.0 angstroms,  
 (ii) said binding pocket model depicts amino acid residues that correspond by functional and/or sequence alignment to the Met112 and Gly113 residues of P38γ or that correspond by functional and/or sequence alignment to the Met112 and Gly113 equivalent residues of one or more proteins listed in Table 1, and  
 (iii) said binding pocket model depicts the ψ angle of the residue corresponding to Met112 to be in the range of about −60° to 60° and the φ angle of the residue corresponding to Gly113 to be in the range of about 30° to 150°;  
 
 b) employing said model of the binding pocket to design or select said potential agonist or antagonist;  
 c) synthesizing said agonist or antagonist; and  
 d) contacting said agonist or antagonist with said molecule to determine the ability of said potential agonist or antagonist to interact with said molecule.  
 
     
     
         38 . The method of  claim 37  wherein the root mean square deviation is within about 2.7 angstroms.  
     
     
         39 . The method of  claim 38  wherein the root mean square deviation is within about 2.5 angstroms.  
     
     
         40 . The method of  claim 37  wherein the ψ angle is in the range of about −45° to 45°.  
     
     
         41 . The method of  claim 38  wherein the ψ angle is in the range of about −45° to 45°.  
     
     
         42 . The method of  claim 39  wherein the ψ angle is in the range of about −45° to 45°.  
     
     
         43 . The method of any of  claims 37  to  42  wherein the ψ angle is in the range of about −30° to 30°.  
     
     
         44 . The method of any of  claims 37  to  42  wherein the φ angle is in the range of about 45° to 135°.  
     
     
         45 . The method of any of  claims 37  to  42  wherein the φ angle is in the range of about 60° to 120°.