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Drug Metabolism and Disposition Fast Forward
First published on April 17, 2008; DOI: 10.1124/dmd.108.021261

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Received for publication March 3, 2008.
Revised April 14, 2008.
Accepted for publication April 16, 2008.

Functional expression and comparative characterisation of nine murine cytochrome P450s by fluorescent inhibition screening

Lesley A McLaughlin 1, Leslie J Dickmann 2, C Roland Wolf 1, Colin James Henderson 3*

1 Cancer Research UK Molecular Pharmacology Unit, University of Dundee 2 Biochemistry and Biophysics Group, Pharmacokinetics and Drug Metabolism, Amgen, Inc., 3 Cancer Research UK Molecular Pharmacology Unit

* Address correspondence to: E-mail: c.j.henderson{at}dundee.ac.uk

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 P450s has become essential, particularly with the recent advances in "humanised" 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 (POR) to generate functional monooxygenase systems in E. 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 provides 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.


Key words: CYP cloning, CYP expression, CYP inhibition, cytochrome P450, cytochrome P450 catalyzed oxidations, cytochrome P450 function, cytochrome P450 isoforms, enzyme inhibitors, enzyme kinetics, microsomes




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