HDM2-inhibitor complexes and uses thereof

Abstract

The present invention includes crystallized HDM2 peptides as well as descriptions of the X-ray diffraction patterns of the crystals. The diffraction patterns allow the three dimensional structure of HDM2 to be determined at atomic resolution so that ligand binding sites on HDM2 can be identified and the interactions of ligands with HDM2 amino acid residues can be modeled. Models prepared using such maps permit the design of ligands which can function as active agents which include, but are not limited to, those that function as inhibitors of MDM2 and HDM2 oncoproteins.

We claim:  
     
         1 . A crystal comprising HDM2, or a fragment, or target structural motif or derivative thereof, and a ligand, wherein said ligand is a small molecule inhibitor.  
     
     
         2 . The crystal of  claim 1  wherein said fragment or derivative thereof is a peptide selected from the group consisting of SEQ ID NO: 1 (amino acid sequence of full length HDM2), SEQ ID NO: 2 (amino acid residues 17-111 of SEQ ID NO: 1), SEQ ID NO. 3 (amino acid residues 23-114 of SEQ ID NO: 1) and SEQ ID NO. 4 (Gly 16 -SEQ ID NO: 2).  
     
     
         3 . The crystal of  claim 1  wherein said crystal has a spacegroup selected from the group consisting of a trigonal spacegroup of P3 2 21 and a tetragonal spacegroup of P4 3 2 1 2.  
     
     
         4 . The crystal of  claim 1 , wherein the crystal effectively diffracts X-rays for determination of atomic coordinates to a resolution of at least about 3.0 Å.  
     
     
         5 . The crystal of  claim 1 , wherein the ligand is in crystalline form.  
     
     
         6 . The crystal of  claim 1  wherein said ligand is selected from the group consisting of (4-Chloro-phenyl)-[3-(4-chloro-phenyl)-7-iodo-2,5-dioxo-1,2,3,5-tetrahydro-benzo[e][1,4]diazepin-4-yl]-acetic acid; [8-Chloro-3-(4-chloro-phenyl)-7-iodo-2,5-dioxo-1,2,3,5-tetrahydro-benzo[e][1,4]diazepin-4-yl]-(4-chloro-phenyl)-acetic acid); and derivatives thereof.  
     
     
         7 . The crystal of  claim 1  wherein said HDM2 comprises a peptide having at least 95% sequence identity to SEQ ID NO. 2.  
     
     
         8 . A crystal comprising SEQ ID NO: 2 comprising an atomic structure characterized by the coordinates of Table 1 or Table 2.  
     
     
         9 . The crystal of  claim 1  comprising a unit cell having dimensions selected from the group consisting of: dimensions of about 98.6 Å, 98.6 Å and 74.7 Å, and about alpha=90°, beta=90° and gamma=120°; and, dimensions of about 54.3 Å, 54.3 Å, 83.3 Å and about alpha=90°, beta=90° and gamma=90°.  
     
     
         10 . A computer system comprising: 
 (a) a database containing information on the three dimensional structure of a crystal comprising HDM2, or a fragment or a target structural motif or derivative thereof, and a ligand, wherein said ligand is a small molecule inhibitor, stored on a computer readable storage medium; and,    (b) a user interface to view the information.    
     
     
         11 . A computer system of  claim 10 , wherein the information comprises diffraction data obtained from a crystal comprising SEQ ID NO:2.  
     
     
         12 . A computer system of  claim 10 , wherein the information comprises an electron density map of a crystal form comprising SEQ ID NO:2.  
     
     
         13 . A computer system of  claim 10 , wherein the information comprises the structure coordinates of Table 1 or Table 2 or homologous structure coordinates comprising a root mean square deviation of non-hydrogen atoms of less than about 1.5 Å when superimposed on the non-hydrogen atom positions of the corresponding atomic coordinates of Table 1 or Table 2.  
     
     
         14 . A computer system of  claim 13 , wherein the information comprises structure coordinates for amino acid residues comprising a root mean square deviation of non-hydrogen atoms of less than about 0.75 Å when superimposed on the non-hydrogen atom positions of the corresponding atomic coordinates of Table 1 or Table 2.  
     
     
         15 . A computer system of  claim 10 , wherein the information comprises the structure coordinates for amino acids Ser 17 , Ile 19 , Leu 82  and Arg 97  according to Table 1 or Table 2 or similar structure coordinates for said amino acids comprising a root mean square deviation of non-hydrogen atoms of less than about 1.5 Å when superimposed on the non-hydrogen atom positions of the corresponding atomic coordinates of Table 1 or Table 2.  
     
     
         16 . A computer system of  claim 15 , wherein the information further comprises the structure coordinates for amino acids Val 53 , Leu 54 , Phe 55 , Leu 57 , Gly 58 , Gln 59 , Ile 62 , Met 62 , Tyr 67 , Gln 72 , His 73 , Ile 74 , Val 75 , Phe 86 , Phe 91 , Val 93 , Lys 94 , Glu 95 , His 96 , Ile 99 , Tyr 100 , Ile 103  according to Table 1 or or Table 2 or similar structure coordinates for said amino acids comprising a root mean square deviation of non-hydrogen atoms of less than about 1.5 Å when superimposed on the non-hydrogen atom positions of the corresponding atomic coordinates of Table 1 or Table 2.  
     
     
         17 . A method of evaluating the potential of an agent to associate with HDM2 comprising: 
 (a) exposing HDM2 to the agent; and    (b) detecting the association of said agent to HDM2 amino acid residues Ser 17 , Ile 19 , Leu82 and Arg 97  thereby evaluating the potential.    
     
     
         18 . A method of  claim 17 , wherein the agent is a virtual compound.  
     
     
         19 . A method of evaluating the potential of an agent to associate with the peptide having aa 16  -SEQ ID NO: 2, comprising: 
 (a) exposing aa 16 -SEQ ID NO: 2 to the agent; and    (b) detecting the level of association of the agent to aa 16 -SEQ ID NO: 2, thereby evaluating the potential.    
     
     
         20 . A method of  claim 19 , wherein the agent is a virtual compound.  
     
     
         21 . A method of  claim 17  wherein step (a) comprises comparing the atomic structure of the compound to the three dimensional structure of HDM2.  
     
     
         22 . A method of  claim 17 , wherein the comparing comprises employing a computational means to perform a fitting operation between the compound and at least one binding site of HDM2.  
     
     
         23 . A method of  claim 22 , wherein the binding site is defined by structure coordinates for amino acids Ser 17 , Ile 19 , Leu 82  and Arg 97  according to Table 1 or Table 2 or similar structure coordinates for said amino acids comprising a root mean square deviation of non-hydrogen atoms of less than about 1.5 Å when superimposed on the non-hydrogen atom positions of the corresponding atomic coordinates of Table 1 or Table 2.  
     
     
         24 . A method of  claim 23 , wherein the binding site is further defined by structure coordinates for amino acids Val 53 , Leu 54 , Phe 55 , Leu 57 , Gly 58 , Gln 59 , Ile 61 , Met 62 , Tyr 67 , Gln 72 , His 73 , Ile 74 , Val 75 , Phe 86 , Phe 91 , Val 93 , Lys 94 , Glu 95 , His 96 , Ile 99 , Tyr 100 , Ile 103  according to Table 1 or Table 2 or similar structure coordinates for said amino acids comprising a root mean square deviation of non-hydrogen atoms of less than about 1.5 Å when superimposed on the non-hydrogen atom positions of the corresponding atomic coordinates of Table 1 or Table 2.  
     
     
         25 . A method of  claim 17 , wherein the agent is exposed to crystalline SEQ ID NO:2 and the detecting of step (b) comprises determining the three dimensional structure of the agent-SEQ ID NO: 2 complex.  
     
     
         26 . A method of identifying a potential agonist or antagonist against HDM2 comprising: 
 (a) employing the three dimensional structure of HDM2 cocrystallized with a small molecule inhibitor to design or select said potential agonist or antagonist.    
     
     
         27 . A method of  claim 26 , wherein the three dimensional structure corresponds to the atomic structure characterized by the coordinates of Table 1 or Table 2 or similar structure coordinates comprising a root mean square deviation of non-hydrogen atoms of less than about 1.5 Å when superimposed on the non-hydrogen atom positions of the corresponding atomic coordinates of Table 1 or Table 2.  
     
     
         28 . A method of  claim 26 , further comprising the steps of: (b) synthesizing the potential agonist or antagonist; and (c) contacting the potential agonist or antagonist with HDM2.  
     
     
         29 . A method of locating the attachment site of an inhibitor to HDM2, comprising: 
 (a) obtaining X-ray diffraction data for a crystal of HDM2;    (b) obtaining X-ray diffraction data for a complex of HDM2 and an inhibitor;    (c) subtracting the X-ray diffraction data obtained in step (a) from the X-ray diffraction data obtained in step (b) to obtain the difference in the X-ray diffraction data;    (d) obtaining phases that correspond to X-ray diffraction data obtained in step (a);    (e) utilizing the phases obtained in step (d) and the difference in the X-ray diffraction data obtained in step (c) to compute a difference Fourier image of the inhibitor; and,    (f) locating the attachment site of the inhibitor to HDM2 based on the computations obtained in step (e).    
     
     
         30 . A method of obtaining a modified inhibitor comprising: 
 (a) obtaining a crystal comprising HDM2 and an inhibitor;    (b) obtaining the atomic coordinates of the crystal;    (c) using the atomic coordinates and one or more molecular modeling techniques to determine how to modify the interaction of the inhibitor with HDM2; and    (d) modifying the inhibitor based on the determinations obtained in step (c) to produce a modified inhibitor.    
     
     
         31 . The method of  claim 30  wherein said crystal comprises a peptide selected from the group consisting of: a peptide having SEQ ID NO: 2; a peptide having SEQ ID NO: 3 and a peptide having SEQ ID NO:4.  
     
     
         32 . A method of  claim 30 , wherein the one or more molecular modeling techniques are selected from the group consisting of graphic molecular modeling and computational chemistry.  
     
     
         33 . A method of  claim 30 , wherein step (a) comprises detecting the interaction of the inhibitor to HDM2 amino acid residues Ser 17 , Ile 19 , Leu 82  and Arg 97 .  
     
     
         34 . An HDM2 inhibitor identified by the method of  claim 30 .  
     
     
         35 . An isolated protein fragment comprising a binding pocket or active site defined by structure coordinates of HDM2 amino acid residues Ser 17 , Ile 19 , Leu 82  and Arg 97 .  
     
     
         36 . An isolated fragment of  claim 35  linked to a solid support.  
     
     
         37 . An isolated nucleic acid molecule encoding the fragment of  claim 35 .  
     
     
         38 . A vector comprising a nucleic acid molecule of  claim 37 .  
     
     
         39 . A host cell comprising the vector of  claim 38 .  
     
     
         40 . A method of producing a protein fragment, comprising culturing the host cell of  claim 39  under conditions in which the fragment is expressed.  
     
     
         41 . A method of screening for an agent that associates with HDM2, comprising: 
 (a) exposing a protein molecule fragment of  claim 35  to the agent; and    (b) detecting the level of association of the agent to the fragment.    
     
     
         42 . A kit comprising a protein molecule fragment of  claim 35 .  
     
     
         43 . A method for the production of a crystal complex comprising an HDM2 polypeptide-ligand comprising: 
 (a) contacting the HDM2 polypeptide with said ligand in a suitable solution comprising PEG and NaSCN; and,    b) crystallizing said resulting complex of HDM2 polypeptide-ligand from said solution.    
     
     
         44 . The method of  claim 43  wherein said HDM2 polypeptide is a polypeptide having SEQ ID NO: 2.  
     
     
         45 . The method of  claim 43  wherein said PEG has an average molecular weight range from 100 to 1000, wherein said PEG is present in solution at a range from about 0.5% w/v to about 10% w/v and said NaSCN is present in solution at a range of from about 50 mM to about 150 mM.  
     
     
         46 . The method of  claim 45  wherein said PEG has an average molecular weight of about 400 and is present in solution at about 2% w/v and said NaSCN is present in solution at about 100 mM.  
     
     
         47 . The method of  claim 46  wherein said solution further comprises about 1.8-2.4 M (NH 4 ) 2 SO 4  and about 100 mM buffer.  
     
     
         48 . A method for the production of a crystal of  claim 1  comprising crystallizing a peptide comprising a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO:4 with a potential inhibitor.  
     
     
         49 . A method for identifying a potential inhibitor of HDM2 comprising: 
 a) using a three dimensional structure of HDM2 as defined by atomic coordinates according to Table 1 or Table 2;    b) replacing one or more HDM2 amino acids selected from Ser 17 , Ile 19 , Leu 82  and Arg 97  in said three-dimensional structure with a different amino acid to produce a modified HDM2;    c) using said three-dimensional structure to design or select said potential inhibitor;    d) synthesizing said potential inhibitor; and,    e) contacting said potential inhibitor with said modified HDM2 in the presence of a substrate to test the ability of said potential inhibitor to inhibit HDM2 or said modified HDM2.    
     
     
         50 . The method of  claim 49  wherein said replacing one or more amino acid residues further comprises replacing SEQ ID NO: 2 amino acids selected from the group consisting of Val 53 , Leu 54 , Phe 55 , Leu 57 , Gly 58 , Gln 59 , Ile 61 , Met 62 , Tyr 67 , Gln 72 , His 73 , Ile 74 , Val 75 , Phe 86 , Phe 91 , Val 93 , Lys 94 , Glu 95 , His 96 , Ile 99 , Tyr 100 , and Ile 103 .  
     
     
         51 . The method of  claim 49  wherein said potential inhibitor is selected from a database.  
     
     
         52 . The method of  claim 49  wherein said potential inhibitor is designed de novo.  
     
     
         53 . The method of  claim 49  wherein said potential inhibitor is designed from a known inhibitor.  
     
     
         54 . The method of  claim 49 , wherein said step of employing said three-dimensional structure to design or select said potential inhibitor comprises the steps of: 
 a) identifying chemical entities or fragments capable of associating with modified HDM2; and    b) assembling the identified chemical entities or fragments into a single molecule to provide the structure of said potential inhibitor.    
     
     
         55 . The method of  claim 49 , wherein the potential inhibitor is a competitive inhibitor of SEQ ID NO:4 (Gly 16 -SEQ ID NO: 2).  
     
     
         56 . The method of  claim 49 , wherein said potential inhibitor is a non-competitive or uncompetitive inhibitor of SEQ ID NO:4 (Gly 16 -SEQ ID NO: 2).  
     
     
         57 . The inhibitor identified by the method of  claim 49.