Compounds and methods used in assessing mono-parp activity

Abstract

Mutant mono ADP-ribose-polymerases (mono-PARP) proteins and small molecule compound substrates specific for the mutant mono-PARP proteins as well as methods of using these compositions to identify protein targets of the mono-PARPs and to screen for antagonists of the mono-PARPs are described.

1 . A small molecule compound (SMC) of formula: 
       
         
           
           
               
               
           
         
       
       wherein R is selected from aryl and alkyl, provided that R is not ethyl. 
     
     
         2 . The compound of  claim 1  wherein R is selected from benzyl, propyl, isobutyl, and methyl. 
     
     
         3 . A recombinant polypeptide comprising a mono-poly-ADP-ribose-polymerases (PARP) catalytic domain selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8 or a polypeptide with 90% identity thereto provided that the amino acid indicated by Xaa is glycine, alanine, serine, cysteine, valine, threonine, or proline and wherein the polypeptide catalyzes the addition of the SMC of  claim 1  to a PARP protein target. 
     
     
         4 . The recombinant polypeptide of  claim 3  comprising a sequence selected from SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, or a polypeptide with 90% identity thereto provided that the polypeptide catalyzes the addition of the SMC of  claim 1  to a PARP protein target. 
     
     
         5 . The recombinant polypeptide of  claim 3  comprising no more than 10% conservative amino acid substitutions. 
     
     
         6 . A method of identifying a protein target of a mono-PARP, the method comprising:
 contacting a SMC of formula:   
       
         
           
           
               
               
           
         
         wherein R is alkyl or aryl with a protein, said protein comprising a polypeptide of  claim 3 , wherein said contacting occurs within a living cell; and 
         subjecting the cell to conditions that result in the protein catalyzing the reaction by which the SMC is covalently attached to one or more cellular proteins; 
         identifying the one or more cellular proteins to which the SMC is covalently attached as protein target(s) of the mono-PARP. 
       
     
     
         7 . The method of  claim 6  wherein the SMC comprises 5-Bn-6-a-NAD + . 
     
     
         8 . The method of  claims 6  further comprising conjugating a label to the SMC. 
     
     
         9 . The method of  claim 8  wherein the label comprises biotin or a fluorescent molecule. 
     
     
         10 . The method of  claims 6  further comprising detecting the one or more cellular proteins to which the SMC is covalently attached via a method that comprises mass spectrometry. 
     
     
         11 . A method of determining if a test compound is an antagonist of a mono-PARP, the method comprising:
 contacting a SMC of formula:   
       
         
           
           
               
               
           
         
         wherein R is alkyl or aryl; 
         with (i) a polypeptide of  claims 3 - 6 , said polypeptide corresponding to the mono-PARP; 
         (ii) a test compound; and 
         (iii) a mono-PARP protein target; 
         said contacting occurring within a mixture;
 subjecting the mixture to conditions that result in the polypeptide catalyzing the reaction by which the SMC is covalently attached to the mono-PARP protein target; 
 wherein a test compound that results in a reliable reduction of covalent attachment of the SMC to the mono-PARP protein target relative to the covalent attachment of the SMC to the mono-PARP protein target in the presence of a negative control is selected as an antagonist of the mono-PARP. 
 
       
     
     
         12 . The method of  claim 11  wherein the SMC comprises 5-Bn-6-a-NAD + . 
     
     
         13 . The method of  claim 11  wherein the mono-PARP protein target was selected using the method of  claim 7 . 
     
     
         14 . The method of  claim 11  wherein the polypeptide comprises SEQ ID NO: 15, SEQ ID NO: 17, or SEQ ID NO: 23. 
     
     
         15 . The method of  claim 14  wherein the mono-PARP protein target comprises at least a fragment of SRPK2 that is mono-ADP-ribosylated by PARP10. 
     
     
         16 . The method of  claim 11  further comprising conjugating a label to the SMC. 
     
     
         17 . The method of  claim 11  wherein the contacting occurs within a cell.