Trubetskoy O V, Finel M, Kurkela M, Fitzgerald M, Peters N R, Hoffman F M, Trubetskoy V S
Quintessence Biosciences, University of Wisconsin, Madison, WI., School of Pharmacy, University of Wisconsin, Madison, WI 53719, USA.
Assay Drug Dev Technol. 2007 Jun;5(3):343-54. doi: 10.1089/adt.2006.050.
Development of high throughput screening (HTS) assays for evaluation of a compound's toxicity and potential for drug-drug interactions is a critical step towards production of better drug candidates and cost reduction in the drug development process. HTS assays for drug metabolism mediated by cytochrome P450s are now routinely used in compound library characterization and for computer modeling studies. However, development and application of HTS assays involving UDP-glucuronosyltransferases (UGTs) are lagging behind. Here we describe the development of a fluorescence-based HTS assay for UGT1A1 using recombinant enzyme and fluorescent substrate in the presence of an aqueous solution of PreserveX-QML (QBI Life Sciences, Madison, WI) polymeric micelles, acting as a stabilizer and a blocker of nonspecific interactions. The data include assay characteristics in 384-well plate format obtained with robotic liquid handling equipment and structures of hits (assay modifiers) obtained from the screening of a small molecule library at the University of Wisconsin HTS screening facility. The application of the assay for predicting UGT-related drug-drug interactions and building pharmacophore models, as well as the effects of polymeric micelles on the assay performance and compound promiscuity, is discussed.
开发用于评估化合物毒性和药物相互作用潜力的高通量筛选（HTS）分析方法，是在药物开发过程中生产出更好的候选药物并降低成本的关键一步。由细胞色素P450介导的药物代谢的HTS分析方法现在常用于化合物库表征和计算机建模研究。然而，涉及尿苷二磷酸葡萄糖醛酸基转移酶（UGT）的HTS分析方法的开发和应用却滞后了。在此，我们描述了一种基于荧光的UGT1A1的HTS分析方法的开发，该方法使用重组酶和荧光底物，并存在PreserveX-QML（QBI生命科学公司，威斯康星州麦迪逊）聚合物胶束的水溶液，其作为稳定剂和非特异性相互作用的阻断剂。数据包括使用机器人液体处理设备在384孔板格式中获得的分析特性，以及在威斯康星大学HTS筛选设施对小分子文库进行筛选获得的命中物（分析修饰剂）的结构。讨论了该分析方法在预测UGT相关药物相互作用和构建药效团模型方面的应用，以及聚合物胶束对分析性能和化合物混杂性的影响。
