A general NMR method for rapid, efficient, and reliable biochemical screening |
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Authors: | Dalvit Claudio Ardini Elena Flocco Maria Fogliatto Gian Paolo Mongelli Nicola Veronesi Marina |
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Institution: | Chemistry and Biology Departments, Pharmacia, Viale Pasteur 10, 20014 Nerviano (MI), Italy. claudio.dalvit@pharmacia.com |
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Abstract: | High-throughput screening is usually the method of drug-lead discovery. It is now well accepted that, for a functional assay, quality is more important than quantity. The ligand-based or protein-based NMR screening methodologies for detecting compounds binding to the macromolecular target of interest are now well established. A novel and sensitive NMR method for rapid, efficient, and reliable biochemical screening is presented. The method named 3-FABS (three fluorine atoms for biochemical screening) requires the labeling of the substrate with a CF(3) moiety and utilizes (19)F NMR spectroscopy for the detection of the starting and enzymatically modified substrates. The method allows for high-quality screening of large compound or natural product extract collections and for measuring their IC(50) values. Applications of this technique to the screening of inhibitors of the Ser/Thr kinase AKT1 and the protease trypsin are presented. In addition, an interesting application of 3-FABS to functional genomics is also presented. |
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