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High-Throughput Screening Assays for the Assessment of CYP2C9*1, CYP2C9*2, and CYP2C9*3 Metabolism Using Fluorogenic Vivid® Substrates

Invitrogen Drug Discovery, Madison, Wisconsin

David V. Thompson

Invitrogen Drug Discovery, Madison, Wisconsin

Tony A. Goossens

Invitrogen Drug Discovery, Madison, Wisconsin

Olga V. Trubetskoy

Invitrogen Drug Discovery, Madison, Wisconsin

CYP2C9 is a genetically polymorphic human cytochrome P450 isozyme involved in the oxidative metabolism of many drugs, including nonsteroidal anti-inflammatory compounds. Individuals genotyped heterozygous or homozygous for CYP2C9 allelic variants have demonstrated altered metabolism of some drugs primarily metabolized by CYP2C9. The ability to expand screening of CYP2C9 allelic variants to a larger set of drugs and pharmaceutical agents would contribute to a better understanding of the significance of CYP2C9 polymorphisms in the population and to predictions of possible outcomes. The authors report the development of an in vitro fluorescence-based assay employing recombinant CYP2C9 variants (CYP2C9*1, CYP2C9*2, and CYP2C9*3) and fluorogenic Vivid® CYP2C9 substrates to explore the effects of CYP2C9 polymorphisms on drug metabolism, using drugs primarily metabolized by CYP2C9. Several chemically diverse fluorogenic substrates (Vivid® CYP2C9 blue, green, and red substrates) were used as prototypic probes to obtain in vitro CYP2C9 metabolic rates and kinetic parameters, such as apparent K_m, V_max, and V_max/K_m ratios for each allelic variant. In addition, a diverse panel of drugs was screened as assay modifiers with CYP2C9*1, CYP2C9*2, CYP2C9*3, and the fluorogenic Vivid® CYP2C9 substrates. The inhibitory potential of this large group of chemically diverse drugs and compounds has been assessed on the basis of their ability to compete with Vivid® CYP2C9 substrates in fluorescent reporter assays, thus providing a sensitive and quick assessment of polymorphism-dependent changes in CYP2C9 metabolism.

Key Words: Cytochrome P450 • CYP2C9 • polymorphism • fluorescent substrate • drug metabolism • high-throughput screening (HTS)

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