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

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