Abstract

Mifepristone (RU486), an 11β-substituted nor-steroid containing a 17α-1-propynyl group used clinically as an antiprogestin agent for medical abortions, was demonstrated to be a selective mechanism-based inactivator of human cytochrome P-450-3A4 (CYP-3A4). The loss of testosterone 6β-hydroxylation activity was time- and concentration-dependent as well as requiring metabolism of mifepristone in a purified CYP-3A4 reconstituted system. The inactivation exhibited pseudofirst-order kinetics. The values forK_I andk_inactivation were 4.7 μM and 0.089 min⁻¹, respectively. The reduced-CO spectrum of CYP-3A4 was decreased by 76%, whereas approximately 81% of the activity was lost following incubation with mifepristone in the reconstituted system in the presence of NADPH. However, the Soret peak of the inactivated CYP-3A4 was slightly increased. High-performance liquid chromatography analysis of the incubation mixture showed that the peak containing the heme dissociated from the inactivated CYP3A4 was almost identical with that seen for the −NADPH control. Covalent binding of [³H]mifepristone to apoCYP3A4 was demonstrated by SDS-PAGE and high-pressure liquid chromatography analyses of the reconstituted system containing CYP-3A4, NADPH-CYP reductase, cytochrome b₅ and lipids in the presence of NADPH. The stoichiometry was determined to be approximately 1 mol of mifepristone bound per 1 mol of CYP-3A4 inactivated. Therefore, the mechanism of inactivation of CYP-3A4 by mifepristone involves irreversible modification of the apoprotein at the enzyme active site instead of being the result of heme adduct formation or heme fragmentation. Mifepristone exhibits selectivity for CYP-3A4 as evidenced by the fact that it did not show mechanism-based inactivation of CYPs 1A, 2B, 2D6, and 2E1, although a competitive inhibition of CYP 2B1 and 2D6 was observed.