Mechanism-based inactivation of cytochrome P-450-3A4 by mifepristone (RU486).

Mifepristone (RU486), an 11beta-substituted nor-steroid containing a 17alpha-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 6beta-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 for KI and kinactivation were 4.7 microM 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.This study demonstrates mifepristone (RU-486) as a potent and selective mechanism-based inactivator of cytochrome P-450-3A4 (CYP-3A4) via irreversible modification of the apoprotein. The results of this clinical research indicate that loss of testosterone 6-beta-hydroxylation activity was time- and concentration-dependent, as well as requiring metabolism of mifepristone in purified CYP-3A4 reconstituted system. Inactivation using several different concentrations of mifepristone exhibited pseudo-first-order kinetics. Reduced-CO difference spectrum of CYP-3A4 decreased by 76%, whereas approximately 81% of CYP-3A4 activity was lost following incubation with mifepristone, indicating the occurrence of N-heme adduct formation detected by HPLC and UV-visible spectroscopy. The peak containing the heme dissociated from the mifepristone-inactivated CYP-3A4 was almost identical with that of the -NADPH control when the incubation mixtures were analyzed by HPLC. 3H-mifepristone proved to be covalently bound to the apoCYP-3A4 by HPLC and SDC-PAGE. The stoichiometry for the binding of the mifepristone was determined to be 1.02 +or- 0.15, approximately 1 mol of mifepristone bound per mole of inactivated CYP-3A4. In summary, mifepristone has been shown to be a potent mechanism-based inactivator of human CYP-3A4. The mechanism of the inactivation showed to involve irreversible modification of the apoprotein at the enzyme active site instead of heme adduct formation or heme fragmentation.