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

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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 (kinactivation) of 0.09 min−1and an apparent KI of 5.1 μM. 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 mechanism-based 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.

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