Inhibition of cytochrome P450 2B6 activity by dihydromethysticin: Structural and mechanistic insights

(+)-Dihydromethysticin (DHM) is a major kavalactone isolated from kava plants. Previous studies have identified (+)-DHM as a CYP2B6 inhibitor, with intriguing structural specificity. It is the most potent CYP2B6 inhibitor identified to date, and CYP2B6 is the most sensitive of any CYP isoform to inhibition by DHM. This investigation evaluated the stereochemistry of DHM inhibition of CYP2B6 and the role of methylenedioxyphenyl group bioactivation in CYP2B6 inhibition, using expressed CYP2B6 and the probe substrates 7-ethoxy-4-trifluoromethyIcoumarin and S-ketamine. The unnatural enantiomer (-)-DHM and racemic (±)-DHM exhibited similar inhibitory activities. Both DHM enantiomers were noncompetitive inhibitors of CYP2B6, with Ki values of 0.2 μM. DHM analogs lacking a methylenedioxy group were devoid of inhibitory effects in both CYP2B6 metabolism assays. Difluoro substitution of the methylene hydrogens on DHM abolished DHM inhibitory activity, whereas dideuterio substitution had no effect on CYP2B6 inhibition. Both DHM enantiomers and the dideuterio analog, but not the difluorinated analog or methysticin, generated a difference spectrum consistent with a metabolite-inhibitor complex. Results suggest CYP2B6-catalyzed methylenedioxyphenyl bioactivation of DHM to a metabolite-inhibitor complex with subsequent enzyme inhibition. DHM may have potential clinical implications or application as a selective CYP2B6 index inhibitor probe. SIGNIFICANCE STATEMENT: Enantiomers of the kavalactone dihydromethysticin are among the most potent CYP2B6 inhibitors identified to date, undergo metabolite-inhibitor complex formation, and exhibit substrate-dependent competitive and noncompetitive inhibition, which may have potential clinical implications or application. Because there is presently no inhibitor of CYP2B6 recommended for in vitro studies and no strong index inhibitor available for CYP2B6 for clinical studies, due in part to specificity considerations, dihydromethysticin may be a candidate for this purpose.