Mechanism-Based Inactivation of Human Cytochrome P450 3A4 by Two Piperazine-Containing Compounds

Abstract

Human cytochrome P450 3A4 (CYP3A4) is responsible for the metabolism of more than half of pharmaceutic drugs, and inactivation of CYP3A4 can lead to adverse drug-drug interactions. The substi-(EMTPP) have been previously identified as mechanism-based inactivators (MBI) of CYP2D6. The present study shows that both SCH 66712 and EMTPP are also MBIs of CYP3A4. Inhibition of CYP3A4 by SCH 66712 and EMTPP was determined to be concentration, time, and NADPH dependent. In addition, inactivation of CYP3A4 by SCH 66712 was shown to be unaffected by the presence of electrophile scavengers. SCH 66712 displays type I binding to CYP3A4 with a spectral binding constant (K s ) of 42.9 6 2.9 mM. The partition ratios for SCH 66712 and EMTPP were 11 and 94, respectively. Whole protein mass spectrum analysis revealed 1:1 binding stoichiometry of SCH 66712 and EMTPP to CYP3A4 and a mass increase consistent with adduction by the inactivators without addition of oxygen. Heme adduction was not apparent. Multiple monooxygenation products with each inactivator were observed; no other products were apparent. These are the first MBIs to be shown to be potent inactivators of both CYP2D6 and CYP3A4.

CYP2D6 Allelic Variants *34, *17-2, *17-3, and *53 and a Thr309Ala Mutant Display Altered Kinetics and NADPH Coupling in Metabolism of Bufuralol and Dextromethorphan and Altered Susceptibility to Inactivation by SCH 66712

“…In whole-protein mass analysis of *53 and the Thr309Ala mutant after incubation with SCH 66712 and NADPH, evidence for adducted whole protein was not observed (data not shown). We have previously observed in mass spectral analysis whole-protein adduction of CYP3A4 and CYP2D6 by SCH 66712 (Bolles et al, 2014), and we have postulated that Thr309 of CYP2D6 might be the site of inactivation by SCH 66712.…”

“…The dissociation constant, K s , was determined using the following quadratic velocity equation: SCH 66712 Metabolite Analysis. Metabolites of SCH 66712 formed by all purified CYP2D6 enzymes were determined by LC-ESI-MS as previously described (Nagy et al, 2011;Bolles et al, 2014). Briefly, purified 2D6 enzymes (1 mM), lipids (30 mM), and reductase (2 mM) were reconstituted for 10 minutes at room temperature, followed by the addition of potassium phosphate (100 mM, pH 7.4), SCH 66712 (100 mM), and ultrapure water in a volume of 200 ml.…”

“…The *53 variant and the Thr309Ala mutant produced more of the first eluting peak, which corresponds to the SCH 66712 metabolite with mono-oxygenation on the phenyl ring [Supplemental Fig. 9, peak a, and (Nagy et al, 2011;Bolles et al, 2014)]. No other metabolites, such as dehydrogenation or dealkylation, were .…”