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.

1 - 57 . (canceled) 
     
     
         58 . A crystal consisting essentially of HDM2 amino acid residues 17-111 of (SEQ ID NO:1), with a ligand, wherein said ligand is a non-peptide small molecule inhibitor of HDM2, wherein said non-peptide small molecule inhibitor prevents HDM2 from interacting with p53. 
     
     
         59 . 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. 
     
     
         60 . 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. 
     
     
         61 . 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. 
     
     
         62 . An isolated protein fragment comprising a binding pocket or active site defined by atomic structure coordinates of HDM2 amino acid residues 17-111 of SEQ ID NO:1 amino acid residues Ser 17 , Ile 19 , Leu 82  and Arg 97  in Table 1 or Table 2. 
     
     
         63 . An isolated fragment of  claim 62  linked to a solid support. 
     
     
         64 . A kit comprising a protein molecule fragment of  claim 62 . 
     
     
         65 . 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.   
     
     
         66 . The method of  claim 65  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. 
     
     
         67 . The method of  claim 66  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. 
     
     
         68 . The method of  claim 67  wherein said solution further comprises about 1.8-2.4 M (NH 4 ) 2 SO 4  and about 100 mM buffer. 
     
     
         69 . A method for the production of a crystal of claim  1  comprising: complexing a peptide comprising a sequence selected from the group consisting of amino acids 17-111 of SEQ ID NO: 1 with a small molecule; concentrating said complex; and allowing said concentrated complex to crystallize. 
     
     
         70 . A method for the production of a crystal complex comprising:
 mixing 1-2 μl of HDM2 protein consisting of amino acids 17-111 of SEQ ID NO: 1 complexed with a small molecule and concentrated to ca. 10 mg/ml in a 1:1 ratio with well solution (1.8-2.4M (NH4)2SO4, 100 mM buffer pH. 6.5-9.0, 2% PEG 400, 100 mM NaSCN);   placing said mixed solution on a glass cover slip;   inverting the glass cover slip and sealing the glass cover slip over a reservoir of 500-1000 μl of well solution;   incubating the glass cover slip at 4° C. for from about 3 to about 7 days to permit formation of crystals; and   harvesting the crystals.