title: 1-Aminobenzotriazole coincubated with (S)-warfarin results in potent inactivation of CYP2C9.
abstract: 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 μM) were 0.0826 and 0.273 min(-1) for kinact and 3.49 and 0.157 mM for KI, respectively. In addition, a 73.4-fold shift was observed in the in vitro potency (kinact/KI ratio), with an increase from 23.7 ml/min/mmol (ABT alone) to 1740 ml/min/mmol [ABT with (S)-warfarin (20 μM)]. 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.
authors.affiliation: Department of Drug Metabolism and Pharmacokinetics (J.K.S., S.M., S.W., C.E.C.A.H., S.C.K., J.S.H.) and Department of Safety Assessment (K.A.F.), Genentech, Inc., South San Francisco, CA.
authors.forename: Jasleen K
authors.initials: JK
authors.lastname: Sodhi
authors.forename: Kevin A
authors.initials: KA
authors.lastname: Ford
authors.forename: Sophie
authors.initials: S
authors.lastname: Mukadam
authors.forename: Susan
authors.initials: S
authors.lastname: Wong
authors.forename: Cornelis E C A
authors.initials: CE
authors.lastname: Hop
authors.forename: S Cyrus
authors.initials: SC
authors.lastname: Khojasteh
authors.forename: Jason S
authors.initials: JS
authors.lastname: Halladay
mesh_terms: D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D065729:Cytochrome P-450 CYP2C9; D004008:Diclofenac / Q000008:administration & dosage / Q000737:chemistry / Q000494:pharmacology; D005260:Female; D006801:Humans; D066298:In Vitro Techniques; D008297:Male; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology; D062105:Molecular Docking Simulation; D015394:Molecular Structure; D011485:Protein Binding; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry; D013997:Time Factors; D014230:Triazoles / Q000008:administration & dosage / Q000737:chemistry / Q000494:pharmacology*; D014859:Warfarin / Q000008:administration & dosage / Q000737:chemistry / Q000494:pharmacology*
chemical_list: D014230:Triazoles; D004008:Diclofenac; C033020:1-aminobenzotriazole; D014859:Warfarin; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9