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

Weixuan Yu, Carla Neckles, Andrew Chang, Gopal Reddy Bommineni, Lauren Spagnuolo, Zhuo Zhang, Nina Liu, Christina Lai, James Truglio, Peter J. Tonge

Research output: Contribution to journalArticlepeer-review

7 Scopus citations


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-32P]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.

Original languageEnglish (US)
Pages (from-to)40-49
Number of pages10
JournalAnalytical Biochemistry
StatePublished - Apr 1 2015
Externally publishedYes


  • Enoyl ACP reductase
  • High-throughput screening
  • Methodology
  • Penefsky column
  • Residence time (t)
  • Steady-state kinetics

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology


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