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Journal of Biomolecular Screening
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Highly Miniaturized Formats for In Vitro Drug Metabolism Assays Using Vivid® Fluorescent Substrates and Recombinant Human Cytochrome P450 Enzymes

Olga V. Trubetskoy

Quintessence Biosciences, Madison, WI, olgat{at}quintbio.com

Jasmin R. Gibson

Invitrogen Drug Discovery Solutions, Madison, WI

Bryan D. Marks

Invitrogen Drug Discovery Solutions, Madison, WI

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® 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® 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. (Journal of Biomolecular Screening 2005:56-66)

Key Words: cytochrome P450 • CYP1A2 • CYP2C9 • CYP2D6 • CYP3A4 • CYP2C19 • fluorescent substrate • drug metabolism • P450 assay • P450 inhibition • high-throughput screening (HTS) • ultra-high-throughput screening (uHTS)

Journal of Biomolecular Screening, Vol. 10, No. 1, 56-66 (2005)
DOI: 10.1177/1087057104269731
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[Abstract] [PDF]