Characterization of cytochrome P450 2D6.1 (CYP2D6.1), CYP2D6.2, and CYP2D6.17 activities toward model CYP2D6 substrates dextromethorphan, bufuralol, and debrisoquine
Characterization of cytochrome P450 2D6.1 (CYP2D6.1), CYP2D6.2, and CYP2D6.17 activities toward model CYP2D6 substrates dextromethorphan, bufuralol, and debrisoquine
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DOI：
10.1124/dmd.30.5.595
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发表时间：
2002-05-01
影响因子：
3.9
通讯作者：
Gaedigk, A
中科院分区：
文献类型：
--
作者：
Marcucci, KA;Pearce, RE;Gaedigk, A
已经描述了超过50种编码完全功能、活性降低或无功能蛋白质的细胞色素P450 2D 6（CYP 2D 6）的等位基因变体。与高加索人相比，在黑人人群中的研究表明，CYP 2D 6活性有降低的趋势，部分原因是存在与活性降低相关的变异等位基因，即CYP 2D 6 *17等位基因。为了研究这种变体蛋白的动力学特征，制备编码CYP2D6.1、CYP2D6.2和CYP2D6.17基因产物的表达构建体，并转染到哺乳动物COS-7和昆虫（粉纹夜蛾）细胞中进行表达。含有表达蛋白的微粒体组分用于确定模型底物美沙芬、丁呋洛尔和异喹喹的动力学参数Km、V-max和固有清除率（Cl-int）。相对于COS-7细胞中表达的野生型CYP2D6.1蛋白，CYP2D6.17表现出高2倍的K-m和使用美沙芬作为底物的V-max降低50%。相比之下，CYP2D6.17未观察到丁呋洛尔K-m的明显变化，而V-max降低了50%。当在杆状病毒表达系统中表达时，与CYP 2D6.1相比，CYP2D6.17表现出以美沙芬为底物时K-m增加6倍，但V-max无变化，以丁呋洛尔为底物时K-m增加2倍，V-max降低50%，以异喹喹为底物时K-m增加3倍，V-max无变化。这些数据表明，CYP2D6.17对所有三种常用的CYP 2D 6底物的代谢活性均降低，尽管对底物亲和力和转换的特定影响表现出一定的底物依赖性。
Over 50 allelic variants of cytochrome P450 2D6 (CYP2D6) encoding fully functional, reduced-activity, or nonfunctional proteins have been described. Compared with Caucasians, studies in black populations demonstrate a tendency toward slower CYP2D6 activity, attributed in part to the presence of a variant allele associated with reduced activity, the CYP2D6*17 allele. To investigate the kinetic characteristics of this variant protein, expression constructs coding for CYP2D6.1, CYP2D6.2, and CYP2D6.17 gene products were prepared and transfected into mammalian COS-7 and insect (Trichoplusia ni) cells for expression. Microsomal fractions containing the expressed proteins were used to determine the kinetic parameters K-m, V-max, and intrinsic clearance (Cl-int) for the model substrates dextromethorphan, bufuralol, and debrisoquine. Relative to the wild-type CYP2D6.1 protein expressed in COS-7 cells, CYP2D6.17 exhibited a 2-fold higher K-m and a 50% reduction in V-max using dextromethorphan as the substrate. In contrast, no appreciable change in bufuralol K-m was observed with CYP2D6.17 whereas V-max was decreased by 50%. When expressed in the baculovirus expression system, CYP2D6.17 exhibited a 6-fold increase in K-m but no change in V-max with dextromethorphan as the substrate, a 2-fold higher K-m and 50% reduction in V-max with bufuralol, and a 3-fold increase in K-m and no change in V-max with debrisoquine relative to CYP2D6.1. These data indicate that CYP2D6.17 exhibits reduced metabolic activity toward all three commonly used CYP2D6 substrates, although specific effects on substrate affinity and turnover demonstrate some substrate dependence.