FLUORINE NMR SPECTROSCOPY FOR BIOCHEMICAL SCREENING

摘要

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 extend ed to the screening of Ser/Thr kinases. The method requires the presence of a C F3 (or CF) moiety on the substrate and utilizes 19F 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 a nd 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.