Journal
JOURNAL OF BIOMOLECULAR SCREENING
Volume 10, Issue 1, Pages 56-66Publisher
SAGE PUBLICATIONS INC
DOI: 10.1177/1087057104269731
Keywords
cytochrome p450; CYP1A2; CYP2C9; CYP2D6; CYP3A4; CYP2C19; fluorescent substrate; drug metabolism; P450 assay; P450 inhibition; high-throughput screening (HTS); ultra-high-throughput screening (uHTS)
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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.
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