A high throughput screening assay to screen for CYP2E1 metabolism and inhibition using a fluorogenic Vivid® P450 substrate
A high throughput screening assay to screen for CYP2E1 metabolism and inhibition using a fluorogenic Vivid® P450 substrate
复制标题
DOI：
10.1089/154065802761001329
复制
发表时间：
2002-11-01
影响因子：
1.8
通讯作者：
Trubetskoy, OV
中科院分区：
文献类型：
--
作者：
Marks, BD;Smith, RW;Trubetskoy, OV
大规模筛选多个化合物文库和组合文库的药理活性是现代药物发现过程中的新方法之一。应用同工酶特异性高通量筛选(HTS)分析来表征潜在候选药物与主要的人类药物代谢细胞色素P450酶(P450)的相互作用，正成为这一过程的重要组成部分。基于荧光的HTS分析已成功地用于体外评估药物与药物的相互作用和对几种P450亚型的酶抑制作用，包括CYP3A4、CYP2D6、CYP2C9和CYP2C19。在这里，我们描述了一种基于荧光的HTS方法，用于检测药物代谢和人CYP2E1的抑制作用。CYP2E1在多种药物、多种溶剂和毒素的代谢中起着重要作用，因此被反复与多种病理因素联系在一起，包括癌症、肝肾毒性、糖尿病和酒精中毒。这种分析是基于一种药物与荧光Vivid(R)CYP2E1 Blue底物竞争CYP2E1代谢的能力，从而使快速筛选铅分子的抑制潜力成为可能。我们利用这一方法筛选了一组药物和化合物，以了解它们对CYP2E1代谢和抑制的影响。我们的结果证明了该方法在鉴定CYP2E1底物和抑制剂以及深入表征它们与CYP2E1同工酶的相互作用方面的有效性。我们还介绍了该分析的详细特征，包括其动态范围和Z‘-因子值，这表明这一稳健的分析非常适合于HTS格式的动力学和抑制研究。
Large-scale screening of multiple compound libraries and combinatorial libraries for pharmacological activity is one of the novel approaches of the modern drug discovery process. The application of isozyme-specific high-throughput screening (HTS) assays for characterizing the interactions of potential drug candidates with major human drug-metabolizing cytochrome P450 enzymes (P450s) is newly becoming an essential part of this process. Fluorescence-based HTS assays have been successfully employed for in vitro assessment of drug - drug interactions and enzyme inhibition with several P450 isoforms, including CYP3A4, CYP2D6, CYP2C9, and CYP2C19. Here we describe a fluorescence-based HTS assay for detecting drug metabolism and inhibition with human CYP2E1. CYP2E1 plays an important role in the metabolism of several drugs, many solvents, and toxins and therefore has been repeatedly linked to numerous pathologies, including cancer, liver and kidney toxicity, diabetes, and alcoholism. The assay is based on the ability of a drug to compete with the fluorogenic Vivid(R) CYP2E1 Blue Substrate for CYP2E1 metabolism and thus enables rapid screening of lead molecules for their inhibitory potential. We have used this assay to screen a panel of drugs and compounds for their effects on CYP2E1 metabolism and inhibition. Our results demonstrate the assay's usefulness in identifying CYP2E1 substrates and inhibitors and in enabling in-depth characterization of their interactions with the CYP2E1 isozyme. We also present detailed characteristics of the assay, including its dynamic range and Z'-factor values, which indicate that this robust assay is well suited for kinetic and inhibition studies in HTS formats.