使用7-甲氧基-4-（氨基甲基）-香豆素对细胞色素P450 2D6的新型高通量分析方法的评估。,European Journal of Pharmaceutical Sciences

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使用7-甲氧基-4-（氨基甲基）-香豆素对细胞色素P450 2D6的新型高通量分析方法的评估。
European Journal of Pharmaceutical Sciences ( IF 4.3 ) Pub Date : 2000-12-05 , DOI: 10.1016/s0928-0987(00)00150-0
J Venhorst ₁ , R C Onderwater , J H Meerman , N P Vermeulen , J N Commandeur

Affiliation

我们最近报道了人类细胞色素P450 2D6（CYP2D6），7-甲氧基-4-（氨基甲基）-香豆素（MAMC）的新型和选择性底物的设计，合成和表征。在这里，我们描述了一种高通量的酶标仪检测方法，该方法利用MAMC作为荧光探针来测定CYP2D6在异源表达系统和人肝微粒体中的抑制和活性。高通量筛选（HTS）分析可用于终点和实时配置，并且易于使用，快速且灵敏。另外，还已经成功地进行了通过流动注射分析的终点测量。通过对CYP2D6的几种低亲和力和高亲和力配体（n = 6）进行抑制实验来验证HTS分析的有效性，并将结果与通过右美沙芬的标准O-脱甲基化分析获得的结果进行比较。结果表明，所有测试的化合物在MAMC和右美沙芬测定中均显示竞争性抑制，并且K（i）值显示两种测定之间的很好相关性（R（2）= 0.984）。为进一步证明HTS分析的有用性，已确定了CYP2D6的5个3,4-亚甲基二氧基苯丙胺的N取代类似物系列的IC（50）值。获得的结果表明，当前的HTS测定法代表了对先前测定法的显着改进，MAMC的更新量更高，对CYP2D6的选择性更高。和K（i）值揭示了这两种测定法之间的很好的相关性（R（2）= 0.984）。为进一步证明HTS分析的有用性，已确定了CYP2D6的5个3,4-亚甲基二氧基苯丙胺的N取代类似物系列的IC（50）值。获得的结果表明，当前的HTS测定法代表了对先前测定法的显着改进，MAMC的更新量更高，对CYP2D6的选择性更高。和K（i）值揭示了这两种测定之间的很好的相关性（R（2）= 0.984）。为进一步证明HTS分析的有用性，已确定了CYP2D6的5个3,4-亚甲基二氧基苯丙胺的N取代类似物系列的IC（50）值。获得的结果表明，当前的HTS测定法代表了对先前测定法的显着改进，MAMC的更新量更高，对CYP2D6的选择性更高。

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Evaluation of a novel high-throughput assay for cytochrome P450 2D6 using 7-methoxy-4-(aminomethyl)-coumarin.

We recently reported on the design, synthesis and characterisation of a novel and selective substrate of human cytochrome P450 2D6 (CYP2D6), 7-methoxy-4-(aminomethyl)-coumarin (MAMC). Here, we describe a high-throughput microplate reader assay, which makes use of MAMC as a fluorescent probe for determining the inhibition and activity of CYP2D6 in heterologously expressed systems and human liver microsomes. The high-throughput screening (HTS) assay can be used both in an end-point and real-time configuration, and is easy to use, rapid and sensitive. In addition, end-point measurements by means of flow injection analysis have also successfully been performed. The HTS-assay was validated by performing inhibition experiments for several low- and high-affinity ligands (n=6) of CYP2D6, and comparing the findings to those obtained with the standard O-demethylation assay of dextromethorphan. The results indicate that all compounds tested display competitive inhibition in both the MAMC and dextromethorphan assay, and the K(i) values reveal a very good correlation (R(2)=0.984) between the two assays. To further demonstrate the usefulness of the HTS-assay, IC(50) values of a series of five N-substituted analogs of 3, 4-methylenedioxyamphetamine for CYP2D6 have been determined. The results obtained demonstrate that the current HTS-assay represents a significant improvement over previous assays, with a higher turnover of MAMC and a higher selectivity for CYP2D6.

更新日期：2019-11-01

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