title: Mechanism-based inactivation of cytochrome P450 2A6 and 2A13 by Rhinacanthus nasutus constituents.
abstract: 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 KI and kinact 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 KI and kinact 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 KI values of 0.69 and 0.42 µM, respectively and apparent kinact 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.
authors.affiliation: Department of Biochemistry, Faculty of Science, Mahidol University.
authors.forename: Phisit
authors.initials: P
authors.lastname: Pouyfung
authors.forename: Aruna
authors.initials: A
authors.lastname: Prasopthum
authors.forename: Songklod
authors.initials: S
authors.lastname: Sarapusit
authors.forename: Ekaruth
authors.initials: E
authors.lastname: Srisook
authors.forename: Pornpimol
authors.initials: P
authors.lastname: Rongnoparut
mesh_terms: D027901:Acanthaceae / Q000737:chemistry*; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000037:antagonists & inhibitors* / Q000737:chemistry; D003374:Coumarins / Q000737:chemistry; D065637:Cytochrome P-450 CYP2A6; D006801:Humans; D006900:Hydroxylation; D007700:Kinetics; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry; D009285:Naphthoquinones / Q000737:chemistry*; D010936:Plant Extracts / Q000737:chemistry; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry
chemical_list: D003374:Coumarins; D009285:Naphthoquinones; D010936:Plant Extracts; D011994:Recombinant Proteins; C575763:rhinacanthin-A; C575762:rhinacanthin-B; C515073:rhinacanthin-C; C030123:coumarin; D001189:Aryl Hydrocarbon Hydroxylases; C414206:CYP2A13 protein, human; C585710:CYP2A6 protein, human; D065637:Cytochrome P-450 CYP2A6; D009251:NADPH-Ferrihemoprotein Reductase