Marks Bryan D, Smith Ronald W, Braun Heidi A, Goossens Tony A, Christenson Marie, Ozers Mary S, Lebakken Connie S, Trubetskoy Olga V
Pan Vera LLC, Madison, WI, USA.
Assay Drug Dev Technol. 2002 Nov;1(1 Pt 1):73-81. doi: 10.1089/154065802761001329.
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 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.
对多个化合物库和组合库进行大规模药理活性筛选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）分析来表征潜在药物候选物与主要人体药物代谢细胞色素P450酶（P450s）之间的相互作用，正新成为这一过程的重要组成部分。基于荧光的HTS分析已成功用于体外评估药物与药物之间的相互作用以及对几种P450同工型（包括CYP3A4、CYP2D6、CYP2C9和CYP2C19）的酶抑制作用。在此，我们描述一种基于荧光的HTS分析方法，用于检测人CYP2E1的药物代谢和抑制作用。CYP2E1在几种药物、许多溶剂和毒素的代谢中起重要作用，因此多次与包括癌症、肝肾毒性、糖尿病和酒精中毒在内的多种病理状况相关联。该分析基于药物与荧光性Vivid CYP2E1蓝色底物竞争CYP2E1代谢的能力，从而能够快速筛选先导分子的抑制潜力。我们已使用该分析方法筛选了一组药物和化合物对CYP2E1代谢和抑制的影响。我们的结果证明了该分析方法在鉴定CYP2E1底物和抑制剂以及深入表征它们与CYP2E1同工酶相互作用方面的有用性。我们还展示了该分析方法的详细特性，包括其动态范围和Z'因子值，这表明这种稳健的分析方法非常适合高通量筛选形式下的动力学和抑制研究。
