Mechanism-based inactivation of cytochrome P450 2A6 and 2A13 by Rhinacanthus nasutus constituents

Human cytochrome P450 CYP2A6 and CYP2A13 catalyze nicotine metabolisms and mediate activation of tobacco-specific carcinogens including 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and 4-(methylnitrosamino)- 1-(3-pyridyl)-1-butanol (NNAL). In this study, we found rhinacanthins A, B, and C isolated from Rhinacanthus nasutus potentially inhibited coumarin 7-hydroxylation mediated by reconstituted purified recombinant CYP2A6 and CYP2A13. Rhinacanthins A-C are mechanism-based inactivators of CYP2A6 and CYP2A13 as they cause concentration, time and NADPH-dependent inhibition. Among the three rhinacanthins, rhinacanthin-B possessed highest inhibitory potency against CYP2A13 with apparent K_I and k_inact of 0.16 μM and 0.1 min^-1, respectively, while values of 0.44 μM and 0.12 min^-1 were found against CYP2A6. Rhinacanthin-C had least inhibition potency, with apparent K_I and k_inact of 0.97 μM and 0.07 min^-1 for CYP2A6, respectively, and values of 1.68 μM and 0.05 min^-1 for CYP2A13. Rhinacanthin-A inhibited CYP2A6 and CYP2A13 with apparent K_I values of 0.69 and 0.42 μM, respectively and apparent k_inact of 0.18 and 0.06 min^-1, respectively. The inhibition of both enzymes by rhinacanthins A-C could not be prevented by addition of trapping agents or reversed by dialysis or potassium ferricyanide. These findings demonstrated that rhinacanthins A-C, which are 1,4-naphthoquinone derivatives, irreversibly inhibited CYP2A6 and CYP2A13 in a mechanism-based inhibition mode.