High affinity inhibitors for target validation and uses thereof

Abstract

This invention provides general methods for discovering mutant inhibitors for any class of enzymes as well as the specific inhibitors so identified. More specifically, this invention provides general methods for discovering specific inhibitors for multi-substrate enzymes. Examples of such multi-substrate enzymes include, but are not limited to, kinases and transferases. The mutant inhibitors identified by the methods of this invention can be used to highly selectively disrupt cell functions such as oncogenic transformation. In one particular example, this invention provides a Src protein kinase inhibitor, pharmaceutical compositions thereof and methods of disrupting transformation in a cell that expresses the target v-scr comprising contacting the cell with the protein kinase inhibitor.

What is claimed is:  
     
         1 . An inhibitor that does not inhibit a catalytic activity of a wild-type enzyme but inhibits the same catalytic activity of the corresponding mutant enzyme, wherein the wild-type enzyme and the mutant enzyme are functionally identical.  
     
     
         2 . The inhibitor of  claim 1  that inhibits the catalytic activity of the mutant enzyme with an IC 50  of less than about 200 nM.  
     
     
         3 . A method of inhibiting a catalytic activity of a mutant enzyme comprising contacting the mutant enzyme with an inhibitor of  claim 1 .  
     
     
         4 . An inhibitor that does not inhibit the growth of a cell expressing a wild-type enzyme but inhibits the growth of a cell expressing a mutant form of the wild-type enzyme, wherein the wild-type enzyme and the mutant form of the wild-type enzyme are functionally identical.  
     
     
         5 . The inhibitor of  claim 4 , wherein the inhibitor is selected from the group comprising a protein kinase inhibitor and a methyltransferase inhibitor.  
     
     
         6 . A method of inhibiting the growth of a cell expressing a mutant enzyme comprising contacting the cell with an inhibitor of  claim 4 .  
     
     
         7 . A protein kinase inhibitor represented by the following formula I:  
       
         
           
           
               
               
           
         
       
       wherein R is a 1′-naphthyl, 2′-napthyl; m-phenoxyphenyl; m-benzyloxyphenyl; m-2′, 6′-dichloro, benzyloxyphenyl; 3-piperonylpyrazolo; p-tert-butylphenyl; 1 ′-naphthylmethyl;  1 ′-napthoxymethyl; or 2′-naphthylmethyl.  
     
     
         8 . A protein kinase inhibitor of  claim 7 , wherein R is 1′-naphthyl.  
     
     
         9 . A protein kinase inhibitor of  claim 7 , wherein R is 2′-naphthyl.  
     
     
         10 . A protein kinase inhibitor of  claim 7 , wherein R is 1′-napthylmethyl.  
     
     
         11 . A protein kinase inhibitor of  claim 7 , wherein R is 2′-napthylmethyl.  
     
     
         12 . A composition comprising the protein kinase inhibitor of any of claims  7 - 11 .  
     
     
         13 . A method of disrupting transformation in a cell that expresses a mutant protein kinase of the Src family comprising contacting the cell with the protein kinase inhibitor of  claim 7 .  
     
     
         14 . The method of  claim 13 , wherein the mutant protein kinase is 1338G v-Src.  
     
     
         15 . The method of  claim 13 , wherein the mutant protein kinase is T339G Fyn.  
     
     
         16 . A method of disrupting transformation in a cell that expresses a mutant protein kinase of the Src family comprising contacting the cell with a composition comprising the protein kinase inhibitor of  claim 7 .  
     
     
         17 . The method of  claim 16 , wherein the mutant protein kinase is 1338G v-Src.  
     
     
         18 . The method of  claim 16 , wherein the mutant protein kinase is T339G Fyn.  
     
     
         19 . A method of inhibiting the phosphorylation of a substrate of a mutant protein kinase comprising incubating a protein kinase inhibitor of  claim 7  with a mixture containing the mutant protein kinase and its substrate.  
     
     
         20 . The method of  claim 19 , wherein the mutant protein kinase is a mutant protein kinase of the Src family.  
     
     
         21 . The method of  claim 20 , wherein the mutant protein kinase is a mutant v-Src.  
     
     
         22 . The method of  claim 21 , wherein the mutant v-Src is I338G v-Src.  
     
     
         23 . The method of  claim 19 , wherein the mutant protein kinase is a mutant Fyn.  
     
     
         24 . The method of  claim 23 , wherein the mutant Fyn is T339G Fyn.  
     
     
         25 . The method of  claim 19 , wherein the mutant protein kinase is a mutant c-Abl.  
     
     
         26 . The method of  claim 25 , wherein the mutant c-Abl is T315A Abl.  
     
     
         27 . The method of  claim 19 , wherein the mutant protein kinase is a mutant CAMK IIα.  
     
     
         28 . The method of  claim 27 , wherein the mutant CAMK IIαis F89G CAMK IIα.  
     
     
         29 . The method of  claim 19 , wherein the mutant protein kinase is a mutant CDK2.  
     
     
         30 . The method of  claim 29 , wherein the mutant CDK2 is F80G CDK2.  
     
     
         31 . The method of  claim 19 , wherein the mutant protein kinase is a mutant Cdc28.  
     
     
         32 . The method of  claim 31 , wherein the mutant Cdc28 is Cdc28-as1.  
     
     
         33 . The method of  claim 19 , wherein the mutant protein kinase is a mutant Fus3.  
     
     
         34 . The method of  claim 33 , wherein the mutant Fus3 is Fus-as1.  
     
     
         35 . A method of inhibiting the catalytic activity of a mutant enzyme comprising incubating the mutant enzyme with an inhibitor of  claim 7 .  
     
     
         36 . The method of  claim 35 , wherein the mutant enzyme is a mutant protein kinase of the Src family.  
     
     
         37 . The method of  claim 36 , wherein the mutant protein kinase is a mutant v-Src.  
     
     
         38 . The method of  claim 37 , wherein the mutant v-Src is I338G v-Src.  
     
     
         39 . The method of  claim 35 , wherein the mutant protein kinase is a mutant Fyn.  
     
     
         40 . The method of  claim 39 , wherein the mutant Fyn is T339G Fyn.  
     
     
         41 . The method of  claim 35 , wherein the mutant enzyme is a mutant c-Abl.  
     
     
         42 . The method of  claim 41 , wherein the mutant c-Abl is T315A Abl.  
     
     
         43 . The method of  claim 35 , wherein the mutant enzyme is a mutant CAMK IIα.  
     
     
         44 . The method of  claim 43 , wherein the mutant CAMK IIαis F89G CAMK lIα.  
     
     
         45 . The method of  claim 38 , wherein the mutant enzyme is a mutant CDK2.  
     
     
         46 . The method of  claim 45 , wherein the mutant CDK2 is F80G CDK2.  
     
     
         47 . The method of  claim 35 , wherein the mutant protein kinase is a mutant Cdc28.  
     
     
         48 . The method of  claim 47 , wherein the mutant Cdc28 is Cdc28-as1.  
     
     
         49 . The method of  claim 35 , wherein the mutant enzyme is a mutant Fus3.  
     
     
         50 . The method of  claim 49 , wherein the mutant Fus3 is Fus-as1.  
     
     
         51 . The method of  claim 35 , wherein the mutant enzyme is a mutant methyltransferase.  
     
     
         52 . A method of inhibiting the growth of a cell that expresses a mutant enzyme comprising incubating the cell with a protein kinase inhibitor of  claim 7 .  
     
     
         53 . The method of  claim 52 , wherein the mutant enzyme is a mutant v-Src.  
     
     
         54 . The method of  claim 53 , wherein the mutant v-Src is 1338G v-Src.  
     
     
         55 . The method of  claim 52 , wherein the mutant enzyme is a mutant c-Abl.  
     
     
         56 . The method of  claim 55 , wherein the mutant c-Abl is T315A Abl.  
     
     
         57 . The method of  claim 52 , wherein the mutant enzyme is a mutant CDK2.  
     
     
         58 . The method of  claim 57 , wherein the mutant CDK2 is F80G CDK2.  
     
     
         59 . The method of  claim 52 , wherein the mutant enzyme is a mutant Cdc28  
     
     
         60 . The method of  claim 52 , wherein the mutant Cdc28 is Cdc28-as1.