Fluorine NMR spectroscopy for biochemical screening

Abstract

High-Throughput Screening (HTS) of large compound libraries is the method of drug-lead discovery. It is now well accepted that for a functional assay, quality is more important than quantity. A biochemical NMR method originally proposed by Percival and Withers (Biochemistry, 1992, 31, 498-505) is extended to the screening of Ser/Thr kinases. The method requires the presence of a CF 3 (or CF) moiety on the substrate and utilizes 19 F NMR spectroscopy for the detection of the starting and enzymatically modified substrates. Experiments can be performed in real time or in an endpoint assay format using protein and substrate concentrations comparable to the ones used by other HTS techniques. Application of this technique to the phosphorylation of a substrate by the protein Ser/Thr kinase AKT1 is presented.

1 .- 8 . (canceled) 
     
     
         9 . A method for characterizing the unknown function of an enzyme comprising:
 a) Labeling a substrate of a known-function enzyme with a CF 3 -moiety;   b) Measuring a  19 F signal shift for said CF 3 -labeled substrate in the presence of the activated known-function enzyme over a period of time whereby said period of time is the time length required for conversion of an unmodified CF 3 -labeled substrate into a modified CF 3 -labeled substrate by said known-function enzyme;   c) Measuring a  19 F signal shift for said CF 3 -labeled substrate in the presence of an activated enzyme of unknown function over a period of time whereby said period of time is the time length required for conversion of an unmodified CF 3 -labeled substrate into a modified CF 3 -labeled substrate by said enzyme of unknown function and   d) Identifying the unknown function of the enzyme by comparing the  19 F signal shifts measured in steps (b) and (c).   
     
     
         10 . The method according to  claim 9 , whereby the known-function enzyme is a kinase, a protease, a phosphatase, or a ligase. 
     
     
         11 . A composition comprising a substrate of an enzyme labeled with a CF 3 -moiety. 
     
     
         12 . The composition of  claim 11 , further comprising said enzyme. 
     
     
         13 . The composition of  claim 12 , wherein said enzyme is selected from the group consisting of a kinase, a protease, a phosphatase, and a ligase. 
     
     
         14 . The composition of  claim 12 , further comprising an inhibitor of said enzyme. 
     
     
         15 . The composition of  claim 14 , wherein said enzyme is a kinase. 
     
     
         16 . The composition of  claim 12 , further comprising an agonist of said enzyme. 
     
     
         17 . The composition of  claim 16 , wherein said enzyme is a kinase. 
     
     
         18 . The composition of  claim 11 , wherein said substrate is a substrate of an known-function enzyme, and wherein said composition further comprises an activated enzyme of unknown function.