ARYL ACETYLENES AS MECHANISM-BASED INHIBITORS OF CYTOCHROME P450-DEPENDENT MONOOXYGENASE ENZYMES

Aryl acetylenes have been investigated as inhibitors of cytochrome P45 0 (P450)-dependent alkoxyresorufin dealkylation activities in liver mi crosomes prepared from rats exposed to beta-naphthoflavone, isosafrole , or phenobarbital. Many of the acetylenes investigated produce pseudo -first-order time-dependent and NADPH-dependent losses of the dealkyla tion activities characteristic of mechanism-based irreversible inactiv ation (suicide inhibition). Replacing the terminal hydrogen of aryl ac etylenes with a methyl group to convert ethynes into propynes enhances the inhibition of P450 1A enzymes; in some instances, this modificati on converts a reversible inhibitor of P450s into a suicide inhibitor. In contrast, ethynes are more effective suicide inhibitors of P450 2B- dependent dealkylations than the corresponding propynes. Aryl acetylen es with an ethynyl group on the 2 position of naphthalene or on the 9 position of phenanthrene and arylalkyl acetylenes with alkyl chains co ntaining 2, 3, or 4 methylene groups are selective inhibitors of P450 2B1/2B2 in liver microsomes from rats. Aryl acetylenes also act as sui cide inhibitors of P450 1A2 in human liver microsomes, of purified P45 0 1A2 from rabbit or rat liver in reconstituted systems, and of purifi ed recombinant human P450 1A2 and 1A1 in reconstituted systems. 4-(1-P ropynyl)biphenyl (4PBi) inactivated P450 1A2-dependent ethoxyresourfin deethylation (EROD) activity in human liver microsomes in an NADPH-de pendent process (kappa(inactivation), 0.23 min(-1); K-I, 2.3 mu M). 4P Bi also inactivated purified recombinant human P450 1A2 (kappa(inactiv ation),0.24 min(-1);K-l,4.3 mu M). In agreement with previous reports [Yun, C.-H., Hammons, G. J., Jones, G., Martin, M. V., Hopkins, N. E., Alworth, W. L., and Guengerich, F. P. (1992) Biochemistry 31, 10556-1 0563], 2-ethynylnaphthalene (2EN) was not a suicide inhibitor of the P 450 1A2 activity in human liver microsomes but did inactivate purified human P450 1A2. Neither 4PBi nor 2EN affected diagnostic activities o f human microsomal P450 2E1, 2C9/10, 3A4, or 2C19. In the systems exam ined, the losses of P450-dependent activity produced by these aryl ace tylenes were not accompanied by corresponding decreases in the measure d P450 absorption spectra. Thus P450 inactivation by these aryl acetyl enes does not involve labeling and destruction of the heme. Incubation of 4PBi with microsomal P450 1A1 or 1A2 from rat liver under conditio ns that lead to P450-dependent enzyme inactivations generates a 2-biph enylylpropionic acid product. This suggests that the suicide inhibitio n of P450s by propynylaryl acetylenes proceeds via a methylaryl ketene formed by a 1,2-methyl rearrangement, analogous to the mechanism of s uicide inhibition by ethynyl acetylenes that proceed via ketene interm ediates formed by 1,a-hydrogen shifts [Ortiz de Montellano, P. R., and Kunze, K. L. (1981) Arch. Biochem. Biophys. 209, 710-712].