ABSTRACT:The metabolism of phencyclidine (PCP) has been studied previously in cytochrome P450 (P450)-containing microsomal systems. However, the reactive intermediate(s) that covalently binds to the P450 and leads to inactivation or leaves the active site to modify other proteins has not been identified. In this study two electrophilic intermediates of PCP were identified by mass spectrometry and by trapping with reduced glutathione (GSH) or N-acetyl cysteine (NAC). The tentative structures of these electrophilic intermediates were determined using mass spectrometry. P450s 2B1 and 2B4 formed a metabolite that exhibited an m/z of 240 corresponding to the mass of the 2,3-dihydropyridinium species of PCP or its conjugate base, the 1,2-dihydropyridine. Chemical reduction of the incubation mixture using NaBH

ABSTRACT:Phencyclidine (PCP) was analyzed for its ability to inactivate human cytochrome P450 (P450) 2B6. PCP inactivated the 7-ethoxy-4-(trifluoromethyl)coumarin O-deethylation activity of P450 2B6 in a concentration-, time-, and NADPH-dependent manner and exhibited pseudo-first order kinetics. The K I was 10 M, k inact was 0.01 min

The mechanism of inactivation of cytochrome P450 2B1 (CYP2B1) by 4-tert-butylphenylacetylene (BPA) has been characterized previously to be caused by the covalent binding of a reactive intermediate to the apoprotein rather than heme destruction (J Pharmacol Exp Ther 331:392-403, 2009). The identification of a BPA-glutathione conjugate and the increase in the mass of the BPA-adducted apoprotein have indicated that the mass of adduct is 174 Da, equivalent to the mass of BPA plus one oxygen atom. To identify the adducted residue, BPA-inactivated CYP2B1 was digested with trypsin, and the digest was then analyzed by using capillary liquid chromatography with a LTQ linear ion trap mass spectrometer as the detector. A mass shift of 174 Da was used for a SEQUEST database search. The tandem mass spectrometry fragmentation of the modified peptide and the identity of modified residue were determined. The results revealed a mass increase of 174 Da for the peptide sequence 296 FFAGTSSTTLR 308 in the I-helix of CYP2B1 and that the site of adduction formation is Thr302. Homology modeling and ligand docking studies showed that BPA binds in close proximity to both the heme iron and Thr302 with the distances being 2.96 and 3.42 Å, respectively. The identification of Thr302 in the CYP2B1 active site as the site of covalent modification leading to inactivation by BPA supports previous hypotheses that this conserved Thr residue may play a crucial role for various functions in P450s.

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ABSTRACT:The mechanism behind the observed inactivation of human P450 2B6 by phencyclidine (PCP) has been evaluated over the past 2 decades. The scope of the current investigation was to contribute to the fundamental knowledge of PCP oxidation and perhaps the mechanism behind P450 inactivation. To study the chemistry of PCP oxidation, we subjected PCP to the Fenton reagent. Under Fenton chemistry conditions, oxidation on all three PCP rings was observed by liquid chromatography/tandem mass spectrometry (LC-MS/MS). When PCP was incubated with the Fenton system in the presence of glutathione (GSH), three GSH-PCP conjugates were identified. Subsequent LC-MS/MS analysis of these conjugates revealed two species that had GSH attached to the cyclohexane ring of PCP and a third conjugate in which GSH was adducted to the piperidine ring. When PCP was incubated across a panel of P450 enzymes, several enzymes, including P450s 2D6 and 3A4, were able to catalyze the formation of the PCP iminium ion, whereas P450s 2B6 and 2C19 were exclusively able to hydroxylate secondary carbons on the cyclohexane ring of PCP. Subsequent mechanistic experiments revealed that only P450s 2B6 and 2C19 demonstrated loss of catalytic activity after preincubation with 10 M PCP. Finally, investigation of P450 2B6 inactivation using structural analogs of PCP revealed that blocking the para-carbon atom on the cyclohexane ring of PCP from oxidation protected the P450 2B6 from inactivation, which suggests that a reactive intermediate generated during the hydroxylation of the cyclohexane ring may be linked to the mechanism of inactivation of P450 2B6 by PCP.The biotransformation of phencyclidine, PCP (Fig. 1), by liver proteins was shown to result in the formation of reactive metabolites leading to P450 inactivation and the formation of covalent adducts with hepatic macromolecules more than 20 years ago (Castagnoli et al., 1997). Originally, covalent binding and enzyme inactivation was thought to occur via alkylation of protein-based nucleophiles by an iminium ion metabolite, M4 (Fig. 1), that arose from the P450-dependent two-electron ␣-carbon oxidation of PCP (Sayre et al., 1997). This hypothesis was supported by the formation of PCP iminium-cyanide adducts and the observation that the presence of cyanide in microsomal incubations protected against the formation of covalent adducts with proteins (Ward et al., 1982b;Hoag et al., 1984). However, subsequent studies demonstrated that the P450 inactivation by the PCP iminium ion (PCP-Im) required the presence of NADPH (Hoag et al., 1984;Osawa and Coon, 1989;Sayre et al., 1991), which inferred that metabolism of PCP beyond the iminium species was required for P450 inactivation.Later, the PCP mechanism-based inactivation of P450 2B6, using purified, recombinant human P450 enzyme, was investigated in great detail (Jushchyshyn et al., 2003). Through a series of experiments, it was shown that P450 2B6 was inactivated via covalent modification of the P450 2B6 apoprotein. Moreover, inclusion of cyanide in the pu...