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 (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-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. Twenty-three FabI inhibitors were analyzed and a mathematical model was used to estimate limits to the tR values of each inhibitor based on percent 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 10-100 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 °C to 37 °C .

Evaluating the Contribution of Transition-State Destabilization to Changes in the Residence Time of Triazole-Based InhA Inhibitors

“…28 Briefly, the reaction velocities were measured by monitoring the oxidation of NADH to NAD + at 340 nm. The enzyme reaction was initiated by adding 100 nM enzyme to C8-CoA (340 λM), NADH (250 μM), NAD + (200 μM), DMSO (2% v/v), inhibitor (0 – 20 μM) and 8% glycerol in 30 mM PIPES pH 6.8 buffer containing 150 mM NaCl and 1 mM EDTA.…”

“…28 Briefly, 2 mmol trans -2-octenoic acid was dissolved in 3 mL dry tetrahydrofuran with 6 mmol triethylamine under nitrogen. Following the addition of 6 mmol ethyl chloroformate, the reaction was stirred at room temperature for 3 h under nitrogen.…”

“…Progress curve kinetics studies were performed on a Cary 100 UV−vis spectrophotometer (Varian) at 20 or 25 °C as described previously but with minor modifications. 28 Briefly, the reaction velocities were measured by monitoring the oxidation of NADH to NAD + at 340 nm. The enzyme reaction was initiated by adding 100 nM enzyme to C8-CoA (340 μM), NADH (250 μM), NAD + (200 μM), DMSO (2% v/v), inhibitor (0−20 μM), and 8% glycerol in 30 mM PIPES buffer, pH 6.8, containing 150 mM NaCl and 1 mM EDTA.…”

“…In a separate experiment, the value of k off was also determined for selected inhibitors using a direct dissociation method for comparison with the values of k 6 obtained by global fitting of the progress curve data. 28 These studies revealed that the triazole-diphenyl ethers had overall association rate constants (k on,overall ) that were 1−3 orders of magnitude slower than the association rates of the alkyldiphenyl ethers. In addition, whereas values for k 5 , the rate constant for conversion of EI to EI*, varied by ∼100-fold across all compounds, K i app , the dissociation constant for the initial EI complex, varied by ∼4000-fold, indicating that variation in inhibitor structure had a more profound impact on the barrier leading to formation of EI than on the barrier between EI and EI*.…”

Rationalizing the Binding Kinetics for the Inhibition of the Burkholderia pseudomallei FabI1 Enoyl-ACP Reductase

“…20, 25, 28, 42–44 PT400 , PT401 , PT406 , PT407 , PT408 , PT409 , and PT444 were synthesized as described in the supplement.…”