Nmr-solve method for rapid identification of bi-ligand drug candidates

Abstract

Methods for rapidly identifying drug candidates that can bind to an enzyme at both a common ligand site and a specificity ligand site, resulting in high affinity binding. The bi-ligand drug candidates are screened from a focused combinatorial library where the specific points of variation on a core structure are optimized. The optimal points of variation are identified by which atoms of a ligand bound to the common ligand site are identified to be proximal to the specificity ligand site. As a result, the atoms proximal to the specificity ligand site can then be used as a point for variation to generate a focused combinatorial library of high affinity drug candidates that can bind to both the common ligand site and the specificity ligand site. Different candidates in the library can then have high affinity for many related enzymes sharing a similar common ligand site.

1 . A method for identifying an atom of a common ligand mimic that is proximal to an interface region; 
 wherein the enzyme can bind a common ligand (CL) or a common ligand mimic (CL mimic) at a common ligand site (CL site) and can bind a specificity ligand (SL) at an adjacent specificity ligand site (SL site);    wherein an interface region is defined as the atoms of the enzyme between the CL site and SL site, and atoms of an SL if bound to the enzyme;    wherein the enzyme can catalyze a reaction mechanism involving the SL and a reactive atom of the CL; and    wherein a CL reactive region is defined as the reactive atom of the CL and CL atoms immediately adjacent to the reactive atom or CL atoms immediately adjacent to the SL; comprising the steps of    (a) identifying an atom of the interface region, comprising the steps of    (1) binding a CL to the CL site of the enzyme;    (2) perturbing an atom of the CL reactive region; and    (3) identifying an NMR cross-peak corresponding to an atom that is perturbed by the perturbation of the atom of the CL reactive region, thereby identifying an atom of the interface region; then    (b) identifying an atom in the CL mimic that is proximal to the interface region, comprising the steps of    (1) binding a CL mimic to the CL site;    (2) perturbing the interface atom identified in step (a); and    (3) identifying an NMR cross-peak corresponding to an atom of the CL mimic that is perturbed by the perturbation of the interface atom, thereby identifying an atom of the CL mimic that is proximal to the interface region.    
   
   
       2 . The method of  claim 1 , wherein the enzyme has a monomer molecular weight greater than 20 kD.  
   
   
       3 . The method of  claim 2 , wherein the enzyme has a monomer molecular weight greater than 35 kD.  
   
   
       4 . The method of  claim 1 , wherein the enzyme has a complete molecular weight greater than 50 kD.  
   
   
       5 . The method of  claim 4 , wherein the enzyme has a complete molecular weight greater than 100 kD.  
   
   
       6 . The method of  claim 1 , wherein the enzyme is from a human pathogen.  
   
   
       7 . The method of  claim 1 , wherein the enzyme is from bacteria.  
   
   
       8 . The method of  claim 1 , wherein the enzyme is a dehydrogenase.  
   
   
       9 . The method of  claim 1 , wherein the enzyme is a kinase.  
   
   
       10 . The method of  claim 1 , wherein the atom of the interface region in step (b)(2) is an atom of the enzyme.  
   
   
       11 . The method of  claim 1 , wherein the atom of the interface region in step (b)(2) is an atom of an SL bound to the enzyme.  
   
   
       12 . The method of  claim 1 , wherein the CL is a cofactor.  
   
   
       13 . The method of  claim 12 , wherein the CL is SAM (S-adenosyl methionine).  
   
   
       14 . The method of  claim 12 , wherein the cofactor contains a nucleotide.  
   
   
       15 . The method of  claim 14 , wherein the CL is selected from the group consisting of NAD.sup.+, NADH, NADP.sup.+, NADPH, ATP and ADP.  
   
   
       16 . The method of  claim 12 , wherein the CL is selected from the group consisting of farnesyl, geranyl, geranyl-geranyl and ubiquitin.  
   
   
       17 . The method of  claim 1 , wherein the atom of the CL reactive region in step (a)(2) is the reactive atom of the CL.  
   
   
       18 . The method of  claim 1 , wherein the atom of the reactive region in step (a)(2) is a CL atom immediately adjacent to the reactive atom.  
   
   
       19 . The method of  claim 1 , wherein the atom of the reactive region in step (a)(2) is a CL atom immediately adjacent to the SL.  
   
   
       20 . The method of  claim 1 , wherein a perturbing step is achieved by chemically altering an atom.  
   
   
       21 - 58 . (canceled)