title: 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 classical steady-state kinetic methods for compounds with tR values of 10 to 100 min. In addition, we were able to identify seven long tR inhibitors (100-1500 min) and to accurately determine their tR values. The method was then used to measure tR 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 tR was observed when the temperature was increased from 25 to 37 °C.
authors: Institute of Chemical Biology and Drug Discovery, Department of Chemistry, Stony Brook University, Stony Brook, NY 11790, USA.
authors: Weixuan
authors: W
authors: Yu
authors: Institute of Chemical Biology and Drug Discovery, Department of Chemistry, Stony Brook University, Stony Brook, NY 11790, USA.
authors: Carla
authors: C
authors: Neckles
authors: Institute of Chemical Biology and Drug Discovery, Department of Chemistry, Stony Brook University, Stony Brook, NY 11790, USA.
authors: Andrew
authors: A
authors: Chang
authors: Institute of Chemical Biology and Drug Discovery, Department of Chemistry, Stony Brook University, Stony Brook, NY 11790, USA.
authors: Gopal Reddy
authors: GR
authors: Bommineni
authors: Institute of Chemical Biology and Drug Discovery, Department of Chemistry, Stony Brook University, Stony Brook, NY 11790, USA.
authors: Lauren
authors: L
authors: Spagnuolo
authors: Institute of Chemical Biology and Drug Discovery, Department of Chemistry, Stony Brook University, Stony Brook, NY 11790, USA.
authors: Zhuo
authors: Z
authors: Zhang
authors: Institute of Chemical Biology and Drug Discovery, Department of Chemistry, Stony Brook University, Stony Brook, NY 11790, USA.
authors: Nina
authors: N
authors: Liu
authors: Great Neck South High School, Great Neck, NY 11020, USA.
authors: Christina
authors: C
authors: Lai
authors: Great Neck South High School, Great Neck, NY 11020, USA.
authors: James
authors: J
authors: Truglio
authors: Institute of Chemical Biology and Drug Discovery, Department of Chemistry, Stony Brook University, Stony Brook, NY 11790, USA. Electronic address: peter.tonge@stonybrook.edu.
authors: Peter J
authors: PJ
authors: Tonge
mesh_terms: D000213:Acyl Carrier Protein; D001671:Biochemistry / Q000379:methods*; D003198:Computer Simulation; D050753:Enoyl-(Acyl-Carrier-Protein) Reductase (NADH) / Q000037:antagonists & inhibitors* / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005240:Feasibility Studies; D057166:High-Throughput Screening Assays; D007700:Kinetics; D009243:NAD / Q000378:metabolism*; D010761:Phosphorus Radioisotopes; D015203:Reproducibility of Results; D013696:Temperature; D013997:Time Factors
chemical_list: D000213:Acyl Carrier Protein; D004791:Enzyme Inhibitors; D010761:Phosphorus Radioisotopes; D009243:NAD; D050753:Enoyl-(Acyl-Carrier-Protein) Reductase (NADH)
keywords: Enoyl ACP reductase; High-throughput screening; Methodology; Penefsky column; Residence time (t(R)); Steady-state kinetics