Center for Toxicogenomics and Human Health, NOVA Medical School/Faculdade de Ciências Médicas, Universidade NOVA de Lisboa, Lisbon, Portugal. Electronic address: http://www.fcm.unl.pt.
Center for Toxicogenomics and Human Health, NOVA Medical School/Faculdade de Ciências Médicas, Universidade NOVA de Lisboa, Lisbon, Portugal.
Biochem Pharmacol. 2018 Dec;158:134-140. doi: 10.1016/j.bcp.2018.10.006. Epub 2018 Oct 9.
Cytochrome P450s (CYPs) are key enzymes involved in drug and xenobiotic metabolism. A wide array of in vitro methodologies, including recombinant sources, are currently been used to assess CYP catalysis, to identify the metabolic profile of compounds, potential drug-drug interactions, protein-protein interactions in the CYP enzyme complex and the role of polymorphic enzymes. We report here on a bacterial whole-cells high-throughput method for the activity evaluation of human CYP1A2, 2A6, and 3A4, when sustained by NADPH cytochrome P450 oxidoreductase (CPR), in the absence or presence of cytochrome b (CYB5). This new assay consists of a microplate real-time fluorometric method, with direct measurement of metabolite formation, in a suspension of Escherichia coli BTC-CYP bacteria, a human CYP competent tester strain when incubated with specific fluorogenic substrates. Overall, the maximum turnover (k) velocities of the three human CYPs resulting from the whole-BTC cells assays were similar to those obtained when applying the corresponding standard reference membrane fractions assays. CYP activity screening with co-expression of CYB5 suggests an enhancing effect of CYB5 on the k of specific isoforms, when using the whole-BTC cells assay. Our results demonstrate that this new approach can offer an efficient high-throughput method for screening of CYP1A2, 2A6 and 3A4 activity and can be potentially applicable for other human CYPs. This can be of particular use for timely and efficient screening of chemical libraries or mutant libraries of CYP enzyme complex proteins, without the necessity for labor intensive isolation of subcellular fractions.
细胞色素 P450 酶（CYPs）是参与药物和外源性化合物代谢的关键酶。目前，广泛使用包括重组来源在内的各种体外方法学来评估 CYP 催化作用，以确定化合物的代谢谱、潜在的药物-药物相互作用、CYP 酶复合物中的蛋白质-蛋白质相互作用以及多态性酶的作用。我们在此报告了一种细菌全细胞高通量方法，用于在 NADPH 细胞色素 P450 氧化还原酶（CPR）存在或不存在细胞色素 b（CYB5）的情况下，评估人 CYP1A2、2A6 和 3A4 的活性。该新测定法由微板实时荧光法组成，在与特定荧光底物孵育时，对大肠杆菌 BTC-CYP 细菌悬浮液中代谢物形成进行直接测量，该细菌是一种具有人 CYP 能力的测试菌株。总体而言，来自全 BTC 细胞测定的三种人 CYP 的最大周转（k）速度与应用相应的标准参考膜部分测定时获得的速度相似。用 CYB5 共表达进行 CYP 活性筛选表明，当使用全 BTC 细胞测定时，CYB5 对特定同工型的 k 具有增强作用。我们的结果表明，这种新方法可以提供一种有效的高通量方法来筛选 CYP1A2、2A6 和 3A4 的活性，并且可能适用于其他人类 CYP。对于及时有效地筛选 CYP 酶复合物蛋白质的化学文库或突变体文库，这可能特别有用，而无需费力地分离亚细胞部分。
