1-Aminobenzotriazole Coincubated with (S)-Warfarin Results in Potent Inactivation of CYP2C9

1-Aminobenzotriazole (ABT) is a nonselective, mechanism-based inactivator of cytochrome P450 (P450) and a useful tool compound to discern P450- from non-P450-mediated metabolism. ABT effectively inactivates major human P450 isoforms, with the notable exception of CYP2C9. Here we propose that ABT preferentially binds to the warfarin-binding pocket in the CYP2C9 active-site cavity; thus, ABT bioactivation and subsequent inactivation is not favored. Therefore, coincubation with ( S )-warfarin would result in displacement of ABT from the warfarin-binding pocket and subsequent binding to the active site, converting ABT into a potent inactivator of CYP2C9. To test this hypothesis, in vitro studies were conducted using various coincubation combinations of ABT and ( S )-warfarin or diclofenac to modulate the effectiveness of CYP2C9 inactivation by ABT. Coincubation of ABT with ( S )-warfarin (diclofenac probe substrate) resulted in potent inactivation, whereas weak inactivation was observed following coincubation of ABT with diclofenac [( S )-warfarin probe substrate]. The kinetic parameters of time-dependent inhibition of ABT for CYP2C9 in the absence and presence of ( S )-warfarin (20 μ M) were 0.0826 and 0.273 min −1 for k inact and 3.49 and 0.157 mM for K I , respectively. In addition, a 73.4-fold shift was observed in the in vitro potency ( k inact / K I ratio), with an increase from 23.7 ml/min/mmol (ABT alone) to 1740 ml/min/mmol [ABT with ( S )-warfarin (20 μ M)]. These findings were supported by in silico structural modeling, which showed ABT preferentially binding to the warfarin-binding pocket and the displacement of ABT to the active site in the presence of ( S )-warfarin.