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.

Inhibition of human cytochromes P450 2A6 and 2A13 by flavonoids, acetylenic thiophenes and sesquiterpene lactones from Pluchea indica and Vernonia cinerea

“…Inhibitory effect of plant extracts, fractions, subfractions and constituents was determined by inhibition assay on CYP2A6- and CYP2A13-mediated coumarin 7-hydroxylation. The recombinant human CYP2A6, CYP2A13 and rat NADPH-dependent cytochrome P450 oxidoreductase (CYPOR) proteins were expressed, purified, according to the methods previously described 20 . Enzymatic reconstitution assay, containing CYP2A, CYPOR as a redox partner enzyme and coumarin probe substrate (2 µM, close to K m value), was performed at room temperature as described 20 .…”

“…The recombinant human CYP2A6, CYP2A13 and rat NADPH-dependent cytochrome P450 oxidoreductase (CYPOR) proteins were expressed, purified, according to the methods previously described 20 . Enzymatic reconstitution assay, containing CYP2A, CYPOR as a redox partner enzyme and coumarin probe substrate (2 µM, close to K m value), was performed at room temperature as described 20 . Rate of 7-hydroxycoumarin product formation was measured on spectrofluorometer (Shimadzu, Kyoto, Japan) at λ ex = 355 nm, λ em = 460 nm.…”

“…Rate of 7-hydroxycoumarin product formation was measured on spectrofluorometer (Shimadzu, Kyoto, Japan) at λ ex = 355 nm, λ em = 460 nm. Inhibition effects were determined by incubation with extracts, fractions or purified compounds with coumarin substrate in the reaction mixture and calculated as per cent relative inhibition compared with vehicle control reaction 20 and IC 50 values were calculated using GraphPad Prism 5 (GraphPad Software Inc., La Jolla, CA).…”

“…MBI requires NADPH and time to generate reactive intermediate as inhibitor and ultimately inactivates the enzyme. MBI was evaluated by pre-incubation with each test inhibitor in the reaction at different time periods (0, 10, 20 and 30 min) in the presence of NADPH for time-dependent assay, as described 20 . The apparent inactivation constants, K I and K inact , were obtained from double reciprocal plots of k obs versus inhibitor concentrations by linear regression analysis on GraphPad Prism 5.…”

“…Methoxsalen was used as a control mechanism-based inhibitor for time- and NADPH-dependent inhibition assays. Effects of trapping agents (2 mM GSH, 5 mM semicarbazide and 2000 U catalase), 70 µM potassium ferricyanide and dialysis were performed as previously described with compounds that exhibited MBI pattern 20 .…”