Aryl acetylenes as mechanism-based inhibitors of cytochrome P450-dependent monooxygenase enzymes
Aryl acetylenes as mechanism-based inhibitors of cytochrome P450-dependent monooxygenase enzymes
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DOI：
10.1021/tx960064g
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发表时间：
1997-01-01
影响因子：
4.1
通讯作者：
Guengerich, FP
中科院分区：
文献类型：
--
作者：
Foroozesh, M;Primrose, G;Guengerich, FP
在从暴露于β-萘啶酮、异黄樟油素或苯巴比妥的大鼠制备的肝微粒体中，研究了芳基乙炔作为细胞色素P450（P450）依赖性烷氧基试卤灵脱烷基化活性的抑制剂。许多研究的乙炔类产生伪一阶时间依赖性和NADPH依赖性的脱烷基化活性损失，其特征是基于机制的不可逆失活（自杀抑制）。用甲基取代芳基乙炔的末端氢以将乙炔转化为丙炔增强了对P450 1A酶的抑制;在某些情况下，这种修饰将P450的可逆抑制剂转化为自杀抑制剂。相比之下，乙炔是比相应的丙炔更有效的P450 2B依赖性脱烷基化的自杀抑制剂。在萘的2位或菲的9位上具有乙炔基的芳基乙炔和具有含有2、3或4个亚甲基的烷基链的芳基烷基乙炔是大鼠肝微粒体中P450 2B 1/2B 2的选择性抑制剂。芳基乙炔类化合物还可作为人肝微粒体中P450 1A 2、重组系统中兔或大鼠肝脏纯化P450 1A 2以及重组系统中纯化重组人P450 1A 2和1A 1的自杀抑制剂。在NADPH依赖性过程中，4-（1-丙炔基）联苯（4PBi）灭活人肝微粒体中P450 1A 2依赖性乙氧基资源精脱乙基（EROD）活性（kappa（灭活），0.23 min（-1）; K-I，2.3 μ M）。4PBi也使纯化的重组人P450 1A 2失活（kappa（失活），0.24 min（-1）; K-1，4.3 μ M）。与以前的报告一致[Yun，C.- H、Hammons，G. J.，琼斯，G.，马丁，M。五、霍普金斯，N. E、Alworth，W. L.，和Guengerich，F. P.（1992）Biochemistry 31，10556-10563]，2-乙炔基萘（2 EN）不是人肝微粒体中P450 1A 2活性的自杀性抑制剂，但对纯化的人P450 1A 2具有自杀性抑制作用。4PBi和2 EN均不影响人微粒体P450 2 E1、2C 9/10、3A 4或2C 19的诊断活性。在系统中检查，这些芳基乙炔产生的P450依赖的活性的损失并不伴随着相应的减少，在测量的P450吸收光谱。因此，P450失活这些芳基乙炔不涉及标记和血红素的破坏。在导致P450依赖性酶失活的条件下，将4PBi与来自大鼠肝脏的微粒体P450 1A 1或1A 2孵育，产生2-联苯基丙酸产物。这表明丙炔基芳基乙炔类对P450的自杀抑制通过由1，2-甲基重排形成的甲基芳基烯酮进行，类似于乙炔基乙炔类的自杀抑制机制，其通过由1，α-氢位移形成的烯酮中间体进行[Ortiz de蒙泰拉诺，P.R.，和Kunze，K. L.（1981）Arch. Biochem. Biophys. 209，710-712]。
Aryl acetylenes have been investigated as inhibitors of cytochrome P450 (P450)-dependent alkoxyresorufin dealkylation activities in liver microsomes 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 dealkylation activities characteristic of mechanism-based irreversible inactivation (suicide inhibition). Replacing the terminal hydrogen of aryl acetylenes with a methyl group to convert ethynes into propynes enhances the inhibition of P450 1A enzymes; in some instances, this modification 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 acetylenes with an ethynyl group on the 2 position of naphthalene or on the 9 position of phenanthrene and arylalkyl acetylenes with alkyl chains containing 2, 3, or 4 methylene groups are selective inhibitors of P450 2B1/2B2 in liver microsomes from rats. Aryl acetylenes also act as suicide inhibitors of P450 1A2 in human liver microsomes, of purified P450 1A2 from rabbit or rat liver in reconstituted systems, and of purified recombinant human P450 1A2 and 1A1 in reconstituted systems. 4-(1-Propynyl)biphenyl (4PBi) inactivated P450 1A2-dependent ethoxyresourfin deethylation (EROD) activity in human liver microsomes in an NADPH-dependent process (kappa(inactivation), 0.23 min(-1); K-I, 2.3 mu M). 4PBi also inactivated purified recombinant human P450 1A2 (kappa(inactivation),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, 10556-10563], 2-ethynylnaphthalene (2EN) was not a suicide inhibitor of the P450 1A2 activity in human liver microsomes but did inactivate purified human P450 1A2. Neither 4PBi nor 2EN affected diagnostic activities of human microsomal P450 2E1, 2C9/10, 3A4, or 2C19. In the systems examined, the losses of P450-dependent activity produced by these aryl acetylenes were not accompanied by corresponding decreases in the measured P450 absorption spectra. Thus P450 inactivation by these aryl acetylenes does not involve labeling and destruction of the heme. Incubation of 4PBi with microsomal P450 1A1 or 1A2 from rat liver under conditions that lead to P450-dependent enzyme inactivations generates a 2-biphenylylpropionic acid product. This suggests that the suicide inhibition of P450s by propynylaryl acetylenes proceeds via a methylaryl ketene formed by a 1,2-methyl rearrangement, analogous to the mechanism of suicide inhibition by ethynyl acetylenes that proceed via ketene intermediates formed by 1,a-hydrogen shifts [Ortiz de Montellano, P. R., and Kunze, K. L. (1981) Arch. Biochem. Biophys. 209, 710-712].