Functional characterization of 40 CYP2B6 allelic variants by assessing efavirenz 8-hydroxylation
Functional characterization of 40 CYP2B6 allelic variants by assessing efavirenz 8-hydroxylation
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通过评估依非韦伦 8-羟基化来表征 40 个 CYP2B6 等位基因变体
DOI：
10.1016/j.bcp.2018.09.010
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发表时间：
2018
影响因子：
5.8
通讯作者：
Hirasawa Noriy
中科院分区：
文献类型：
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作者：
Watanabe Takashi;Saito Takahiro;Rico Evelyn Marie Gutierrez;Hishinuma Eiji;Kumondai Masaki;Maekawa Masamitsu;Oda Akifumi;Saigusa Daisuke;Saito Sakae;Yasuda Jun;Nagasaki Masao;Minegishi Naoko;Yamamoto Masayuki;Yamaguchi Hiroaki;Mano Nariyasu;Hirasawa Noriy
细胞色素P450 2B 6（CYP 2B 6）内的遗传变异导致临床重要药物（包括环磷酰胺、安非他酮、美沙酮和依法韦仑（EFZ））代谢的个体间变异。在这项研究中，我们进行了40个CYP 2B 6等位基因变异蛋白的体外分析，包括在1070个日本人中发现的7个新的变异。在293 FT细胞中异源表达野生型和39种变体蛋白，以估计EFZ 8-羟基化和7-乙氧基-4-三氟甲基香豆素（7-ETC）O-脱乙基化活性的动力学参数（Km，Vmax和CLint）。CYP 2B 6变体全酶的浓度通过使用一氧化碳（CO）还原差光谱法测量，野生型和28种变体在450 nm处显示峰。测定了野生型和24种变异蛋白的动力学参数。由于在使用的最高底物浓度下未检测到酶活性，因此无法测定其余15种变体的值。与野生型相比，6种变异体显示EFZ 8-羟基化CLint值显著降低，而这些值在另外6种变异体中显著增加，包括CYP2B6.6。虽然CYP 2B 6变体的7-ETCO-脱乙基CLint值与CYP2B6.1的没有显著差异，但7-ETCO-脱乙基的CLint值与EFZ 8-羟基化高度相关。此外，进行了三维结构建模分析，以阐明CYP 2B 6变体动力学变化的机制。我们的研究结果可以提供由这些变异等位基因编码的CYP 2B 6蛋白的特异性代谢活性的证据。
Genetic variations within cytochrome P450 2B6 (CYP2B6) contribute to inter-individual variation in the metabolism of clinically important drugs, including cyclophosphamide, bupropion, methadone and efavirenz (EFZ). In this study, we performed anin vitroanalysis of 40 CYP2B6 allelic variant proteins including seven novel variants identified in 1070 Japanese individuals. Wild-type and 39 variant proteins were heterologously expressed in 293FT cells to estimate the kinetic parameters (Km,Vmax, andCLint) of EFZ 8-hydroxylation and 7-ethoxy-4-trifluoromethylcoumarin (7-ETC)O-deethylation activities. The concentrations of CYP2B6 variant holo-enzymes were measured by using carbon monoxide (CO)-reduced difference spectroscopy, and the wild-type and 28 variants showed a peak at 450 nm. The kinetic parameters were measured for the wild-type and 24 variant proteins. The values for the remaining 15 variants could not be determined because the enzymatic activity was not detected at the highest substrate concentration used. Compared to wild-type, six variants showed significantly decreased EFZ 8-hydroxylationCLintvalues, while these values were significantly increased in another six variants, including CYP2B6.6. Although 7-ETCO-deethylationCLintvalues of CYP2B6 variants did not differ significantly from that of CYP2B6.1, theCLintratios obtained for 7-ETCO-deethylation were highly correlated with EFZ 8-hydroxylation. Furthermore, three-dimensional structural modeling analysis was performed to elucidate the mechanism of changes in the kinetics of CYP2B6 variants. Our findings could provide evidence of the specific metabolic activities of the CYP2B6 proteins encoded by these variant alleles.