Highly miniaturized formats for in vitro drug metabolism assays using Vivid® fluorescent substrates and recombinant human cytochrome P450 enzymes
Highly miniaturized formats for in vitro drug metabolism assays using Vivid® fluorescent substrates and recombinant human cytochrome P450 enzymes
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DOI：
10.1177/1087057104269731
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发表时间：
2005-02-01
影响因子：
--
通讯作者：
Marks, BD
中科院分区：
文献类型：
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作者：
Trubetskoy, OV;Gibson, JR;Marks, BD
开发了高度小型化的P450筛选试验，用于在1536孔板上方便地分析P450药物与主要人类细胞色素P450酶（CYP3A4, 2D6, 2C9, 2C19和1A2）和Vivid(R)荧光底物的相互作用。分析的详细表征包括重组P450酶的稳定性、均匀性和可重复性，以及它们与Vivid(R)荧光底物反应的动力学参数，重点关注每种成分的特定特征，以便在小体积1536孔板分析格式中进行筛选。筛选试验用于评估单个细胞色素P450抑制谱与一组选定的试验修饰剂，包括同工酶特异性底物和抑制剂。在96孔和1536孔板格式中获得的修饰剂的IC50值与在常规底物中获得的值相似且可比较。对1536孔分析统计数据（如信号与背景比和Z因子）的全面检查表明，这些分析是一种强大、成功和可靠的工具，可以以超高通量筛选形式筛选细胞色素P450的代谢和抑制。
Highly miniaturized P450 screening assays designed to enable facile analysis of P450 drug interactions in a 1536-well plate format with the principal human cytochrome P450 enzymes (CYP3A4, 2D6, 2C9, 2C19, and 1A2) and Vivid(R) fluorogenic substrates were developed. The detailed characterization of the assays included stability, homogeneity, and reproducibility of the recombinant P450 enzymes and the kinetic parameters of their reactions with Vivid(R) fluorogenic substrates, with a focus on the specific characteristics of each component that enable screening in a low-volume 1536-well plate assay format. The screening assays were applied for the assessment of individual cytochrome P450 inhibition profiles with a panel of selected assay modifiers, including isozyme-specific substrates and inhibitors. IC50 values obtained for the modifiers in 96- and 1536-well plate formats were similar and comparable with values obtained in assays with conventional substrates. An overall examination of the 1536-well assay statistics, such as signal-to-background ratio and Z' factor, demonstrated that these assays are a robust, successful, and reliable tool to screen for cytochrome P450 metabolism and inhibition in an ultra-high-throughput screening format.