Mechanism-Based Inactivation of Cytochrome P450 3A4 by 17α-Ethynylestradiol: Evidence for Heme Destruction and Covalent Binding to Protein

Abstract

17␣-Ethynylestradiol (EE), a major constituent of many oral contraceptives, inactivated the testosterone 6␤-hydroxylation activity of purified P450 3A4 reconstituted with phospholipid and NADPH-cytochrome P450 reductase in a mechanismbased manner. The inactivation of P450 3A4 followed pseudo first order kinetics and was dependent on NADPH. The values for the K I and k inact were 18 M and 0.04 min Ϫ1 , respectively, and the t 1/2 was 16 min. Incubation of 50 M EE with P450 3A4 at 37°C for 30 min resulted in a 67% loss of testosterone 6␤-hydroxylation activity accompanied by a 35% loss of the spectral absorbance of the native protein at 415 nm and a 70% loss of the spectrally detectable P450-CO complex. The inactivation of P450 3A4 by EE was irreversible. Testosterone, an alternate substrate, was able to protect P450 3A4 from EEdependent inactivation. The partition ratio was ϳ50. (HPLC) analysis demonstrated that the inactivation resulting from EE metabolism led to the destruction of approximately half the heme with the concomitant generation of modified heme and EE-labeled heme fragments and produced covalently radiolabeled P450 3A4 apoprotein. Electrospray mass spectrometry demonstrated that the fraction corresponding to the major radiolabeled product of EE metabolism has a mass (M Ϫ H) Ϫ of 479 Da. HPLC and gas chromatography-mass spectometry analyses revealed that EE metabolism by P450 3A4 generated one major metabolite, 2-hydroxyethynylestradiol, and at least three additional metabolites. In conclusion, our results demonstrate that EE is an effective mechanism-based inactivator of P450 3A4 and that the mechanism of inactivation involves not only heme destruction, but also the irreversible modification of the apoprotein at the active site.

Identification of 17-α-Ethynylestradiol-Modified Active Site Peptides and Glutathione Conjugates Formed during Metabolism and Inactivation of P450s 2B1 and 2B6

“…The metabolism of 17EE by human P450 enzymes such as P450s 3A4 and 2B6 results in the production of reactive intermediates that are able to alkylate the P450 heme or modify the apoprotein (16)(17)(18). This observation not only raises a potential concern about drug interactions with other therapeutics that are also metabolized by these enzymes but the ensuing reactive 17EE intermediates may also lead to toxic or carcinogenic effects (3).…”

“…The resultant intermediate then undergoes a rearrangement involving ring expansion to an aldehyde that could potentially be further oxidized to the corresponding carboxylic acid (36). A metabolite corresponding to this 17-formyl-D-homosteroid has been observed in incubation mixtures from P450 2B and 3A enzymes that were inactivated by 17EE (17,18). Alternatively such a reactive intermediate could also react with either the heme or the apoprotein.…”

“…Previously, 17EE was shown to be a mechanism-based inactivator for P450s 3A4, 2B1, and 2B6 (16)(17)(18). Interestingly, the activity of P450s 2B2 and 2B4, which share a greater than 70% sequence identity with P450 2B1, was only minimally affected by incubations with 17EE in the presence of NADPH (17).…”

CYP3A4 overexpression enhances apoptosis induced by anticancer agent imidazoacridinone C-1311, but does not change the metabolism of C-1311 in CHO cells

“…C-1311-mediated inhibition of CYP3A4 might modulate the metabolism of other therapeutics that are sensitive to the action of this enzyme. Many reports have demonstrated the modulation of CYP3A4 activity by drugs such as carbamazepine [39,40] or 17α-ethynylestradiol [41] . The latter strongly inhibited CYP3A4 while also being metabolized by it.…”

Detection of Covalent Adducts to Cytochrome P450 3A4 Using Liquid Chromatography Mass Spectrometry

“…Numerous studies with radiolabeled compounds have been reported in the literature definitively showing that metabolic activation leads to covalent labeling of P450s. These studies have also demonstrated specific labeling of individual isoforms (7)(8)(9)(10)(11)(12)(13)(14)(15)(16). More recently, liquid chromatography coupled with electrospray mass spectrometry (LC-ESMS) has been employed to detect covalent adducts using unlabeled compounds.…”