A general NMR method for rapid, efficient, and reliable biochemical screening.
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.
Dalvit
Claudio
C
Chemistry and Biology Departments, Pharmacia, Viale Pasteur 10, 20014 Nerviano (MI), Italy. claudio.dalvit@pharmacia.com
Ardini
Elena
E
Flocco
Maria
M
Fogliatto
Gian Paolo
GP
Mongelli
Nicola
N
Veronesi
Marina
M
D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology; D005461:Fluorine; D023281:Genomics / Q000379:methods; D006801:Humans; D007700:Kinetics; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D009842:Oligopeptides / Q000378:metabolism / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D012680:Sensitivity and Specificity; D012709:Serum Albumin / Q000737:chemistry; D014361:Trypsin Inhibitors / Q000378:metabolism / Q000494:pharmacology
D004791:Enzyme Inhibitors; D009842:Oligopeptides; D011518:Proto-Oncogene Proteins; D012709:Serum Albumin; D014361:Trypsin Inhibitors; D005461:Fluorine; C494918:AKT1 protein, human; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt