Crystal structure of E.coli GDP-fucose synthetase (and complexes thereof) and methods of identifying agonists and antagonists using same

Abstract

The present invention provides for crystalline GFS. The crystal structure of GFS has also been solved using such material. Models based upon such crystal structure are also provided. Methods of identifying inhibitors of GFS activity using such models are also disclosed.

1 . Crystalline GFS.  
     
     
         2 . The crystalline GFS of  claim 1  wherein said GFS is  E. coli  GFS.  
     
     
         3 . The crystalline GFS of  claim 1  wherein said GFS is recombinant GFS.  
     
     
         4 . The crystalline GFS of  claim 1  wherein said GFS is crystallized with a co-factor selected from the group consisting of NADPH and NADP+.  
     
     
         5 . The crystalline GFS of  claim 1  wherein said GFS comprises the mature sequence of naturally-occurring GFS.  
     
     
         6 . A crystalline composition comprising GFS is association with a second chemical species.  
     
     
         7 . The composition of  claim 6  wherein said second chemical species is selected from the group consisting of NADPH, NADP+ and a potential inhibitor of GFS activity.  
     
     
         8 . A model of the structure of GFS comprising a data set embodying the structure of crystalline GFS of  claim 1 .  
     
     
         9 . The model of  claim 8  wherein said data set was determined by crystallographic analysis of GFS.  
     
     
         10 . The model of  claim 8  wherein said data set was determined by NMR analysis of GFS.  
     
     
         11 . The model of  claim 8  wherein said data set embodies the entire structure of GFS.  
     
     
         12 . The model of  claim 8  wherein said data set embodies a portion of the structure of GFS.  
     
     
         13 . The model of  claim 12  wherein said portion is the active site of GFS.  
     
     
         14 . The model of  claim 8  wherein said GFS is complexed with a second chemical species selected from the group consisting of NADPH, NADP+ and a potential inhibitor of GFS activity.  
     
     
         15 . A computer system comprising computer hardware and the model of  claim 8 .  
     
     
         16 . A method of identifying a species which is an agonist or antagonist of GFS activity or binding comprising: (a) providing the model of  claim 8 , (b) studying the interaction of candidate species with such model, and (c) selecting a species which is predicted to act as said agonist or antagonist.  
     
     
         17 . A species identified in accordance with the method of  claim 16 .  
     
     
         18 . A process of identifying a substance that inhibits GFS activity or binding comprising determining the interaction between a candidate substance and a model of  claim 8 .  
     
     
         19 . A method of identifying inhibitors of GFS activity by rational drug design comprising: 
 (a) designing a potential inhibitor that will form non-covalent bonds with one or more amino acids in the GFS sequence based upon the crystal structure co-ordinates of crystalline GFS of  claim 1;     (b) synthesizing the inhibitor; and    (c) determining whether the potential inhibitor inhibits the activity of GFS.    
     
     
         20 . The method of  claim 19  wherein said inhibitor is designed to interact with one or more amino acids in the GFS sequence selected from the group consisting of Arg12, Met14, Val15, Arg36, Asn40, Leu41, Ala63, Ile86, Gly106, Ser107, Ser108, Cys109, Tyr136, Lys140, Asn165, Leu166, His179, Val180, Leu184, Val201, Trp202, Arg209, and Lys283.  
     
     
         21 . An agonist or antagonist identified by the method of  claim 19 .  
     
     
         22 . A substance identified by the method of  claim 18 .  
     
     
         23 . A method of identifying a species which is an agonist or antagonist of human FX protein or binding comprising: (a) providing the model of  claim 8 , (b) studying the interaction of candidate species with such model, and (c) selecting a species which is predicted to act as said agonist or antagonist.  
     
     
         24 . A species identified in accordance with the method of  claim 23 .  
     
     
         25 . A process of identifying a substance that inhibits human FX protein activity or binding comprising determining the interaction between a candidate substance and a model of  claim 8 .  
     
     
         26 . A method of identifying inhibitors of human FX protein activity by rational drug design comprising: 
 (a) designing a potential inhibitor that will form non-covalent bonds with one or more amino acids in the GFS sequence based upon the crystal structure co-ordinates of crystalline GFS of  claim 1;     (b) synthesizing the inhibitor; and    (c) determining whether the potential inhibitor inhibits the activity of human FX protein.    
     
     
         27 . The method of  claim 26  wherein said inhibitor is designed to interact with one or L-=more amino acids in the GFS sequence selected from the group consisting of Arg12, Met14, Val15, Arg36, Asn40, Leu41, Ala63, Ile86, Gly106, Ser107, Ser108, Cys109, Tyr136, Lys140, Asn165, Leu166, His179, Val180, Leu184, Val201, Trp202, Arg209, and Lys283.  
     
     
         28 . An agonist or antagonist identified by the method of  claim 26 .  
     
     
         29 . A substance identified by the method of  claim 25.