Mechanism-based inactivation of cytochrome P450 2B1 by 2-ethynylnaphthalene: Identification of an active-site peptide

Abstract

The 7-ethoxycoumarin O-deethylase activity of rat liver cytochrome P450 2B1 reconstituted with NADPH-cytochrome P450 reductase and lipid was inactivated by 2-ethynylnaphthalene (2EN) in a time- and NADPH-dependent manner, and the loss of activity followed pseudo-first-order kinetics. The extrapolated KI and kinactivation were 0.08 microM and 0.83 min-1, respectively. The loss of 7-ethoxycoumarin O-deethylation activity displayed a number of characteristics consistent with mechanism-based inactivation, including irreversibility, saturability, protection by an alternate substrate, and the lack of an effect of exogenous nucleophiles on the inactivation. The inactivation was not accompanied by a concomitant loss of spectrally detectable cytochrome P450. HPLC analysis showed that [3H]2EN was irreversibly bound to the protein moiety of cytochrome P450 and the stoichiometry of inactivation was approximately 1.3 mol of 2EN bound per mole of cytochrome P450. Liquid chromatographic and GC-MS analyses of the organic extracts from these incubations showed that the major metabolite was 2-naphthylacetic acid, and a partition ratio of 4-5 mol of acid produced per mole of cytochrome P450 2B1 inactivated was determined. A radiolabeled peptide, approximately 6.5 kDa when analyzed by Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), was isolated by HPLC from a tryptic digest of the [3H]2EN-inactivated cytochrome P450 and NADPH-cytochrome P450 reductase. Sequence data were obtained after cyanogen bromide cleavage of this amino-terminally blocked peptide. These results in conjunction with the results from the cleavage of the intact [3H]2EN-inactivated cytochrome P450 by cyanogen bromide and separation of the peptides either by HPLC or by Tricine-SDS-PAGE followed by transfer of the peptides to a poly(vinylidene difluoride) membrane and sequencing of the labeled peptides from both experiments, led to the identification of a 2EN-modified active-site peptide with the sequence ISLLSLFFAGTETSSTTLRYGFLLM. This corresponds to positions 290-314 in cytochrome P450 2B1. Sequence alignments of cytochrome P450 2B1 with cytochrome P450 2B1 with cytochrome P450 101 predict that this region might correspond to helix I of the bacterial protein [Poulos, T.L. (1988) Pharm. Res. 5, 67-75] that contains a highly conserved threonine residue involved in oxygen binding.

Inhibition of CYP2B4 by the mechanism-based inhibitor 2-ethynylnaphthalene: Inhibitory potential of 2EN is dependent on the size of the substrate

“…2EN has been characterized as a selective mechanism based inhibitor for CYP2B4 (4)(5)(6). The goal of this study was to examine the ability of 2EN to inhibit CYP2B4-dependent monooxygenase activities.…”

“…Certain compounds containing the acetylenic functional group have been shown to act both as reversible inhibitors and mechanism-based inactivators that alkylate the protein moiety of cytochrome P450 in microsomes and reconstituted systems (2)(3)(4)(5)(6)(7)(8). Ethynes, such as 9-ethynylphenanthrene, are effective mechanism-based inhibitors of CYP2B-dependent reactions, whereas propynes like 1-(1-propynyl)-pyrene are more selective inhibitors of CYP1A enzymes (2;7).…”

“…Ethynes, such as 9-ethynylphenanthrene, are effective mechanism-based inhibitors of CYP2B-dependent reactions, whereas propynes like 1-(1-propynyl)-pyrene are more selective inhibitors of CYP1A enzymes (2;7). 2-Ethynylnaphthalene (2EN) has previously been shown to be a mechanism-based inactivator of P450, and the radiolabeled compound was found to irreversibly bind to the protein moiety of rat CYP2B1, rat CYP1A1 and CYP1A2, and rabbit CYP1A2 with a binding stoichiometry of approximately 0.5-1.3 nmol of adduct/nmol of P450 (4)(5)(6). 2EN has also been reported to be an effective mechanism-based inhibitor of the 7-ethoxycoumarin O-deethylase activity of CYP2B4 with 0.83 mol of adduct bound per mole of P450 (2;3).…”

Naphthalene metabolism in relation to target tissue anatomy, physiology, cytotoxicity and tumorigenic mechanism of action

“…Rates of formation of naphthalene 1R,2S-oxide in mouse, rat, and hamster airway explant incubations correlate well with immunolocalization of CYP2F2, but not with CYP2B4 that also is found in pulmonary Clara cells; and CYP2B4 inhibition does not block naphthalene metabolism by mouse lung microsomal enzymes (Buckpitt et al, 1995;Roberts et al, 1993). A subsequent immunolocalization study (complemented by peptide mass fingerprinting, and RT-PCR analysis of CYP2F mRNA expression) failed to detect CYP2F in rhesus macaque tissue of any kind studied other than nasal ethmoturbinates, where levels were 10-and 20-fold lower than in corresponding rat and mouse tissue, respectively (Baldwin et al, 2004).…”

Identification of the Residue in Human CYP3A4 That Is Covalently Modified by Bergamottin and the Reactive Intermediate That Contributes to the Grapefruit Juice Effect

“…The [ 14 C]BG-labeled apo-3A4 was separated from the reductase by SDS-PAGE and recovered by electroblotting to a nitrocellulose (NC) membrane or to a polyvinylidene difluoride (PVDF) membrane for digestion by lysyl endopeptidase (Lys C) or cyanogen bromide (CNBr), respectively (Roberts et al, 1993). All of the peptides that eluted from the PVDF membrane or the NC membrane were further separated by Tricine-SDS-PAGE and subjected to autoradiography (Roberts et al, 1993). These methods provided information about the stability of the BG-inactivated apoprotein to enzymatic digestion and the approximate size of BG-inactivated peptides.…”