The use of biochemical and biophysical tools for triage of high-throughput screening hits - A case study with Escherichia coli phosphopantetheine adenylyltransferase.

摘要: High-throughput screening is utilized by pharmaceutical researchers and, increasingly, academic investigators to identify agents that act upon enzymes, receptors, and cellular processes. Screening hits include molecules specifically bind the target a greater number of non-specific compounds. It necessary ‘triage’ these subset worthy further exploration. As part our antibacterial drug discovery effort, we applied suite biochemical biophysical tools accelerate triage process. We describe application series 9-oxo-4,9-dihydropyrazolo[5,1-b]quinazoline-2-carboxylic acids (PQ) from screen Escherichia coli phosphopantetheine adenylyltransferase (PPAT). Initial confirmation specific binding was obtained using tools, including novel orthogonal assay, isothermal titration calorimetry, saturation transfer difference NMR. To sub-site bound inhibitors, two techniques were utilized: steady-state enzyme kinetics 19F NMR method in which fluorine-containing fragments ATP and/or sites serve as competitive reporter probes. These data are consistent with PQs sub-site. In addition identification PPAT described hit process broadly applicable other targets milligram quantities purified protein available.