Functional expression and comparative characterization of nine murine cytochromes P450 by fluorescent inhibition screening

Lesley A. McLaughlin, Leslie J. Dickmann, C. Roland Wolf, Colin J. Henderson

    Research output: Contribution to journalArticlepeer-review

    33 Citations (Scopus)

    Abstract

    The increasing number of transgenic or gene knockout mouse models generated for use in drug metabolism studies has meant that a greater understanding of the function and substrate specificities of murine cytochromes P450 (P450s) has become essential, particularly with the recent advances in "humanized" mouse models. In this study, we have heterologously expressed nine murine P450s-Cyp1a1, Cyp1a2, Cyp1b1, Cyp2a4, Cyp2b20, Cyp2c29, Cyp2d22, Cyp2e1, and Cyp3a11 - individually with human P450 oxidoreductase to generate functional monooxygenase systems in Escherichia coli. We have identified a suitable fluorogenic probe for each P450 and determined the apparent kinetic parameters. These probes have enabled the screening of a panel of 31 test compounds classified as "drugs," "natural compounds," "endogenous compounds," and "pesticides" by measurement of IC50, thus allowing the comparison of binding affinities. Human P450s CYP2C9, CYP2D6, and CYP3A4 were also included in the study to enable direct comparisons to be made with the mouse enzymes. Although there were general similarities between human and mouse P450s, perhaps the most significant finding in this study was the observation that, despite 77% amino acid identity, Cyp2d22 and CYP2D6 were remarkably dissimilar in a range of enzymatic properties, with potentially serious implications for pharmacokinetic studies using CYP2D substrates. The data presented in this study provide a solid foundation with which to assess the degree of similarity (or difference) between mouse and human P450s involved in xenobiotic metabolism and can be used as a basis for further studies.

    Original languageEnglish
    Pages (from-to)1322-1331
    Number of pages10
    JournalDrug Metabolism and Disposition
    Volume36
    Issue number7
    DOIs
    Publication statusPublished - Jul 2008

    Keywords

    • HUMAN LIVER-MICROSOMES
    • ESCHERICHIA-COLI
    • SUBSTRATE RECOGNITION
    • TRANSGENIC MICE
    • MOUSE
    • GENE
    • REDUCTASE
    • ACID
    • KNOCKOUT
    • SYSTEM

    Cite this