Functional Expression and Comparative Characterization of Nine Murine Cytochromes P450 by Fluorescent Inhibition Screening

Abstract

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 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.

CYP1B1 expression promotes the proangiogenic phenotype of endothelium through decreased intracellular oxidative stress and thrombospondin-2 expression

“…However, the substrate specificity of murine CYP2A4 was recently shown to be very different from that of CYP1B1. 58 There is not much known about the expression of CYP2A4 in the endothelium and its potential function in angiogenesis, especially in the absence of CYP1B1, and this requires future investigation. The lack of effects on capillary morphogenesis of retinal ECs in the presence of 17-ODYA suggest a minimal role for CYP4A and CYP2J, which catalyze the formation of 20-HETE and EET from arachidonic acid, 34 in these processes.…”

Modeling Human Cytochrome P450 2D6 Metabolism and Drug-Drug Interaction by a Novel Panel of Knockout and Humanized Mouse Lines

“…For example, the only functional CYP2D gene in humans is CYP2D6, compared with nine functional genes in the mouse (Nelson et al, 2004). Furthermore, recent studies revealed marked differences in the substrate specificities between murine Cyp2d22 or Cyp2d9 on the one hand and human CYP2D6 on the other (Smith et al, 1998;McLaughlin et al, 2008).…”

“…A limitation of this approach, however, is the presence of the murine Cyp2d genes in this model. Some of the mouse Cyp2d enzymes have the ability to metabolize CYP2D6 substrates at considerable rates (Bogaards et al, 2000;McLaughlin et al, 2008), so that WT mice do not generically represent the human "poor metabolizer" phenotype. Furthermore, because of the variability among random transgenic lines as a result of the integration at different sites of the genome, it is difficult to standardize this approach as required for a comparison between lines expressing different allelic variants.…”

Role of the Drug-Metabolizing Enzyme CYP during Mouse Liver Development

“…To date, 58 types of CYP molecular species have been identified in humans, and 108 types have been identified in mice. [1][2][3] As the CYP molecular species share high amino acid sequence homology in several substrate-recognition sites, in addition to the abovementioned functions of CYP, it may also play a major role in development and differentiation in the body. Because CYP begins to be expressed close to the time of hematopoiesis initiation during the fetal stage and CYP requires heme for its structure, CYP is considered to be involved in the development of the liver during the fetal stage.…”