A [(32)P]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 (tR) 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 tR, 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 tR values of each inhibitor based on percentage drug-target complex recovery following gel filtration. In general, this method showed good agreement with the class

SUBMITTER: Yu W 

PROVIDER: S-EPMC4454744 | biostudies-literature | 2015 Apr

REPOSITORIES: biostudies-literature