Crystalline vap-1 and uses thereof

Abstract

The present invention relates to crystalline vascular adhesion protein-1 (VAP-1) and in particular to methods for the use of structural information of crystalline human VAP-1 for ligand and/or inhibitor identification, design and production, as well as screening assays for detections of same. The invention further relates to inhibitors identified by the assays according to the present invention.

1 . Crystalline human vascular adhesion protein 1 (VAP-1), wherein said crystal is defined as a crystal of space group P6522 with two molecules in the asymmetric unit and with unit dimensions of a=b=225.9 Å, c=218.7 Å, a=b=90°, g=120°. 
     
     
         2 . The crystalline VAP-1 according to  claim 1 , or a homolog thereof, comprising domains D2, D3 and D4, characterized by amino acid sequences SEQ ID NO. 3, SEQ ID NO. 4 and SEQ ID NO. 5, respectively. 
     
     
         3 . The crystalline VAP-1 protein according to  claim 2 , comprising an active site cavity which is ˜20 Åט10 Å wide at the surface and ˜15 Å deep. 
     
     
         4 . The crystalline VAP-1 protein according to  claim 3 , wherein said active site cavity comprises amino acids 86-87, 97, 168-173, 176-177, 180, 184, 205-212, 216, 227, 232-234, 236-239, 344, 388-390, 393-397, 415-419, 421, 421-426, 467-470, 647-651 and 758-761 of SEQ ID NO. 2 from one monomer, and amino acids 443-449 and 451 of SEQ ID NO. 2 from the other monomer of human VAP-1. 
     
     
         5 . The crystalline VAP-1 protein according to  claim 4 , wherein said active site further comprises amino acid Leu469 at the top of a narrow ˜4.5 Åט4.5 Å cavity at the bottom of said cavity. 
     
     
         6 . The crystalline VAP-1 protein according to  claim 5 , wherein said bottom part of the active site cavity is lined by amino acid residues Ala370, Tyr384, Asp386, Asn470, Tpq471 and Tyr473. 
     
     
         7 . A composition comprising a crystalline VAP-1 according to  claim 1 . 
     
     
         8 . A method for crystallizing human VAP-1, comprising the steps of:
 a) providing an aqueous, solution comprising human VAP-1 protein;   b) providing a reservoir solution comprising a precipitating agent;   c) mixing a volume of said aqueous solutions with a volume of said reservoir solution forming a mixed solution; and   d) crystallizing at least a portion of said mixed solution.   
     
     
         9 . The method of  claim 8 , wherein said aqueous solution provided in step a) has a concentration of VAP-1 of 1 mg/ml. 
     
     
         10 . The method of  claim 9 , wherein the precipitating agent is K/Na tartrate. 
     
     
         11 . The method of  claim 10 , wherein step d) is performed by vapour diffusion. 
     
     
         12 . A computer readable medium comprising a data storage material en-coded with machine readable data having stored thereon atomic co-ordinate/X-ray diffraction data defining the three-dimensional structure of human VAP-1 protein, capable of displaying a three dimensional representation of a crystal of a molecule comprising a fragment of human VAP-1 protein when read by an appropriate machine and processed by a computer program for determining molecule structures, wherein said data defines the active site cavity of dimeric human VAP-1 protein and said active site cavity is ˜20 Åט10 Å wide at the surface and ˜15 Å deep and further comprises amino acid Leu469 at the top of a narrow ˜4.5ט4.5 cavity at the bottom of the site. 
     
     
         13 . The computer readable medium according to  claim 12 , wherein said active site cavity comprises amino acids 86-87, 97, 168-173, 176-177, 180, 184, 205-212, 216, 227, 232-234, 236-239, 344, 388-390, 393-397, 415-419, 421, 421-426, 467-470, 647-651 and 758-761 from one monomer, and amino acids 443-449 and 451 of the other monomer of human VAP-1. 
     
     
         14 . The computer readable medium according to  claim 13 , wherein said bottom part of the active site cavity is lined by amino acid residues Ala370, Tyr384, Asp386, Asn470, Tpq471 and Tyr473. 
     
     
         15 . A synthetic, small molecule human VAP-1 inhibitor identifiable by a method which comprises the steps of a) providing atomic co-ordinates of said protein on a computer readable medium according to  claim 12 , and b) using a computer to apply molecular modeling techniques to said co-ordinates. 
     
     
         16 . The inhibitor according to  claim 15 , characterized by the formula 
       
         
           
           
               
               
           
         
       
       wherein
 R1 is hydrogen, lower alkyl or an optionally substituted phenyl or heteroaryl group; 
 R2 is hydrogen or lower alkyl; R3-R5 represent each independently hydrogen, lower alkyl, aralkyl, optionally substituted phenyl or a heteroaryl group; 
 R6 is naphtyl, phenyl, substituted phenyl or a heteroaryl group; 
 R7 is hydrogen, lower alkyl or aralkyl; 
 n is 1, 2 or 3; and 
 X═O, S, SO, SO2 or NR2, with the proviso that
 (i) when X is O, R1 to R5 and R7 are hydrogen and n is 1, then R6 is not phenyl or 2-methoxy-phenyl, 
 (ii) when X is O, R1 is hydrogen, R2 is methyl and R3 to R5 and R7 are hydrogen and n is 1, then R6 is not 3-trifluoromethyl-phenyl; and 
 (iii) when X is O, R1 is phenyl, R2 to R5 and R7 are hydrogen and n is 1, then R6 is not 4-hydroxy-phthalazinyl. 
 
 
     
     
         17 . Use of a compound according to  claim 15  for the manufacture of a medicament for use in treating diseases selected from the group comprising; acute and chronic inflammatory conditions or diseases such as chronic arthritis, inflammatory bowel diseases, skin dermatoses and multiple sclerosis; diseases related to carbohydrate metabolism, including diabetes and complications resulting from diabetes, e.g., vasculopathies, such as retinopathy, nephropathy and neuropathy; aberrations in adipocyte differentiation or function, and smooth muscle cell function, in particular atherosclerosis; and various vascular diseases. 
     
     
         18 . A pharmaceutical composition comprising a compound according to  claim 15 .