Davydov Dmitri R, Davydova Nadezhda Y, Rodgers John T, Rushmore Thomas H, Jones Jeffrey P
Department of Chemistry, Washington State University, Pullman, WA 99164, U.S.A.
Department of Pharmaceutical Sciences, University of the Sciences, Philadelphia, PA 19104, U.S.A.
Biochem J. 2017 Oct 10;474(20):3523-3542. doi: 10.1042/BCJ20170543.
Functional cross-talk among human drug-metabolizing cytochrome P450 through their association is a topic of emerging importance. Here, we studied the interactions of human CYP2D6, a major metabolizer of psychoactive drugs, with one of the most prevalent human P450 enzymes, ethanol-inducible CYP2E1. Detection of P450-P450 interactions was accomplished through luminescence resonance energy transfer between labeled proteins incorporated into human liver microsomes and the microsomes of insect cells containing NADPH-cytochrome P450 reductase. The potential of CYP2D6 to form oligomers in the microsomal membrane is among the highest observed with human cytochrome P450 studied up to date. We also observed the formation of heteromeric complexes of CYP2D6 with CYP2E1 and CYP3A4, and found a significant modulation of these interactions by 3,4-methylenedioxymethylamphetamine, a widespread drug of abuse metabolized by CYP2D6. Our results demonstrate an ample alteration of the catalytic properties of CYP2D6 and CYP2E1 caused by their association. In particular, we demonstrated that preincubation of microsomes containing co-incorporated CYP2D6 and CYP2E1 with CYP2D6-specific substrates resulted in considerable time-dependent activation of CYP2D6, which presumably occurs via a slow substrate-induced reorganization of CYP2E1-CYP2D6 hetero-oligomers. Furthermore, we demonstrated that the formation of heteromeric complexes between CYP2E1 and CYP2D6 affects the stoichiometry of futile cycling and substrate oxidation by CYP2D6 by means of decreasing the electron leakage through the peroxide-generating pathways. Our results further emphasize the role of P450-P450 interactions in regulatory cross-talk in human drug-metabolizing ensemble and suggest a role of interactions of CYP2E1 with CYP2D6 in pharmacologically important instances of alcohol-drug interactions.
人类药物代谢细胞色素P450通过其相互作用产生的功能串扰是一个日益重要的新兴话题。在此，我们研究了精神活性药物的主要代谢酶——人类CYP2D6与最普遍的人类P450酶之一、乙醇诱导型CYP2E1之间的相互作用。通过掺入人肝微粒体中的标记蛋白与含有NADPH - 细胞色素P450还原酶的昆虫细胞微粒体之间的发光共振能量转移来检测P450 - P450相互作用。在微粒体膜中，CYP2D6形成寡聚体的潜力是迄今为止所研究的人类细胞色素P450中观察到的最高水平之一。我们还观察到CYP2D6与CYP2E1和CYP3A4形成异源复合物，并发现3,4 - 亚甲基二氧甲基苯丙胺（一种由CYP2D6代谢的广泛滥用药物）对这些相互作用有显著调节作用。我们的结果表明，CYP2D6和CYP2E1的相互作用导致了它们催化特性的充分改变。特别是，我们证明了将共掺入CYP2D6和CYP2E1的微粒体与CYP2D6特异性底物预孵育会导致CYP2D6出现显著的时间依赖性激活，这可能是通过底物诱导的CYP2E1 - CYP2D6异源寡聚体缓慢重组而发生的。此外，我们证明了CYP2E1和CYP2D6之间异源复合物 的形成通过减少过氧化物生成途径的电子泄漏来影响CYP2D6无效循环和底物氧化的化学计量。我们的结果进一步强调了P450 - P450相互作用在人类药物代谢体系调节串扰中的作用，并表明CYP2E1与CYP2D6的相互作用在酒精 - 药物相互作用的药理学重要实例中发挥作用。
