Mechanism-Based Inactivation of Cytochrome P450 2B6 by a Novel Terminal Acetylene Inhibitor

Abstract

This article is available online at http://dmd.aspetjournals.org ABSTRACT:N-(3,5-Dichloro-4-pyridyl)-3-(cyclopentyloxy)-4-methoxybenzamide (DCMB) is a known marker substrate for cytochrome P450 2B6. Based on the chemical template of DCMB, a novel terminal acetylene compound, N-(3,5-dichloro-4-pyridyl)-4-methoxy-3-(prop-2-ynyloxy)benzamide (TA) was synthesized and evaluated as a mechanism-based inactivator of P450 2B6. The pseudo first-order inactivation of expressed P450 2B6 by TA was both substrate and time-dependent. The kinetics of inhibition resulted in a maximal rate constant (k inactivation ) of 0.09 min ؊1 and an apparent K I of 5.1 M. Incubation of expressed P450 2B6 with TA and NADPH resulted in a 68% loss in enzyme activity and a concurrent 62% loss in the formation of a reduced carbon monoxide complex, suggesting that heme destruction is the primary mode of enzyme inactivation.Enzyme inactivation of P450 2B6 was not reduced by the presence of 10 mM glutathione and was protected by incubation of excess DCMB with TA. The production of the carboxylic acid metabolite, N-(3,5-Dichloro-4-pyridyl)-3-(2-carboxyethoxy)-4-methoxybenzamide (TA-COOH), during the incubation of TA with 2B6 suggests that inactivation proceeds through a ketene intermediate. For 2B6 inactivation, the partition ratio was approximately 1.5 nmol TA-COOH formed/ nmol P450 inactivated. Finally, TA was evaluated for mechanismbased inactivation of P450 3A4, 2C9, 2C19, 2D6, and 2E1 using human liver microsomes. In addition to 2B6, P450 2C forms were also found to be sensitive to TA-mediated inactivation, suggesting that subtle changes in the O-alkyl chain of the parent may be critical for the selectivity of enzyme inactivation.The cytochrome P450 enzyme family is responsible for the metabolism of many structurally diverse xenobiotics. To determine the role of specific human P450 1 forms in drug metabolism, an array of in vitro metabolism techniques including the use of P450 form-specific substrates, antibodies, and chemical inhibitors have been developed (Wrighton et al., 1993;Rodrigues, 1999). For chemical inhibition, both the mechanism and selectivity of enzyme inhibition are important characteristics for consideration. Mechanism-based enzyme inhibition, by definition, requires metabolism of a substrate to a reactive intermediate that can bind to the enzyme irreversibly, resulting in the loss of enzyme activity (Kent et al., 2001). This process of inactivation has been exploited in the design of P450 form-selective inactivators by the incorporation of dihalomethyl (Halpert et al., 1989) or acetylenic moieties (Ortiz de Montellano and Reich, 1984) at the preferred site of oxidation. Furthermore, the inactivation process can occur by heme alkylation and destruction and/or apoprotein modification (Halpert et al., 1985;Lin et al., 2002). Investigations of mechanism-based inactivation can therefore lead to detailed information on the interaction of the compound and the enzyme, i.e., the nature of the reactive intermediate formed, the efficiency ...

The Biochemistry of Drug Metabolism — An Introduction. Part 2. Redox Reactions and Their Enzymes

“…This is an electrophilic and highly reactive molecule which may or may not be the heme-alkylating species, but it is stable enough to leave the enzyme and cause major cellular damage by binding covalently to nucleophilic sites in proteins and even nucleic acids (see Part 5). [140]. Among positional isomers, a CC bond in a propynyl group (RÀCCÀCH 3 ) appears as reactive as a terminal ethynyl group in beeing activated to an inhibitory species.…”

Developmental Expression of Human Hepatic CYP2C9 and CYP2C19

“…The reaction product, 4-hydroxymephenytoin, was detected and quantified by high-performance liquid chromatography/ mass spectrometry as previously described (Fan et al, 2003). Sample areas were read from a linear regression of known standards (range, 4.68 -150.00 pmol) using 1/ϫ weighting.…”

“…The conversion of diclofenac (20-M final assay concentration) to 4-hydroxydiclofenac was used to measure CYP2C9 metabolic activity in individual microsomal preparations. The product, 4-hydroxydiclofenac, was separated and quantified by high-performance liquid chromatography using a previously described method (Fan et al, 2003). Sample areas were read from a linear regression of known standard amounts (range, 40 -5000 pmol) using no weighting.…”

Automated Assessment of Time-Dependent Inhibition of Human Cytochrome P450 Enzymes Using Liquid Chromatography-Tandem Mass Spectrometry Analysis

“…TDI is an unusual occurrence with most enzymes, but it is observed at a higher frequency in P450-catalyzed reactions, perhaps due to the reactivity of the oxygenated species formed during the course of the oxygenation reactions (Hollenberg, 2002). There are examples of irreversible or quasi-irreversible P450 inhibition across many classes of therapeutic drugs, recreational drugs, and herbal medicines (Zhou et al, 2005), and all of the major drug-metabolizing P450s have been implicated (Kunze and Trager, 1993;Lopez-Garcia et al, 1994;Newton et al, 1995;Koenigs and Trager, 1998; Chun et al., 2001a,b;Ha-Duong et al, 2001;Palamanda et al, 2001;Bertelsen et al, 2003;Fan et al, 2003;Lu et al, 2003;Polasek et al, 2004;Zhou et al, 2004).In a drug discovery setting, three different analytical endpoints are typically used for the study of P450 inhibition in vitro: liquid scintillation counting of radioactivity liberated during site-specific metabolism (Moody et al, 1999), selective analysis of fluorescent metabolites (Crespi and Stresser, 2000), and mass spectrometry (Yin et al,Article, publication date, and citation information can be found at …”