A [32P]NAD+-based method to identify and quantitate long residence time enoyl-acyl carrier protein reductase inhibitors

摘要: Abstract The classical methods for quantifying drug–target residence time (tR) use loss or regain of enzyme activity in progress curve kinetic assays. However, such become imprecise at very long times, mitigating the alternative strategies. Using NAD(P)H-dependent FabI enoyl-acyl carrier protein (enoyl-ACP) reductase as a model system, we developed Penefsky column-based method direct measurement tR, where off-rate drug was determined with radiolabeled [adenylate-32P]NAD(P+) cofactor. In total, 23 inhibitors were analyzed, and mathematical used to estimate limits tR values each inhibitor based on percentage complex recovery following gel filtration. general, this showed good agreement steady-state compounds 10 100 min. addition, able identify seven (100–1500 min) accurately determine their values. then measure function temperature, an analysis not previously possible using standard approach due decreased NAD(P)H stability elevated temperatures. 4-fold difference observed when temperature increased from 25 37 °C.