Functional expression and comparative characterization of nine murine cytochromes P450 by fluorescent inhibition screening.
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 IC(50), 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.
McLaughlin
Lesley A
LA
Biomedical Research Centre, University of Dundee, Ninewells Hospital and Medical School, Dundee DD1 9SY, UK.
Dickmann
Leslie J
LJ
Wolf
C Roland
CR
Henderson
Colin J
CJ
D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018076:DNA, Complementary; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005456:Fluorescent Dyes / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D008024:Ligands; D051379:Mice; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid
D065607:Cytochrome P-450 Enzyme Inhibitors; D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D008024:Ligands; D003577:Cytochrome P-450 Enzyme System