Toward a systems approach to the human cytochrome P450 ensemble: interactions between CYP2D6 and CYP2E1 and their functional consequences

Abstract

Functional cross-talk among human drug-metabolizing cytochromes P450 through their association is a topic of emerging importance. Here we studied the interactions of human CYP2D6, a major metabolizer of psychoactive drugs, with one of the most prevalent human P450 enzymes, ethanol-inducible CYP2E1. Detection of P450-P450 interactions was accomplished through luminescence resonance energy transfer (LRET) between labeled proteins incorporated into human liver microsomes and the microsomes of insect cells containing NADPH-cytochrome P450 reductase. The potential of CYP2D6 to form oligomers in the microsomal membrane is among the highest observed with human cytochromes P450 studied up to date. We also observed the formation of heteromeric complexes of CYP2D6 with CYP2E1 and CYP3A4, and found a significant modulation of these interactions by MDMA, a widespread drug of abuse metabolized by CYP2D6. Our results demonstrate an ample alteration of the catalytic properties of CYP2D6 and CYP2E1 caused by their association. In particular, we demonstrated that pre-incubation of microsomes containing co-incorporated CYP2D6 and CYP2E1 with CYP2D6-specific substrates resulted in considerable time-dependent activation of CYP2D6, which presumably occurs via a slow substrate-induced reorganization of CYP2E1-CYP2D6 heterooligomers. Furthermore, we demonstrated that the formation of heteromeric complexes between CYP2E1 and CYP2D6 affects the stoichiometry of futile cycling and substrate oxidation by CYP2D6 by means of decreasing the electron leakage through the peroxide-generating pathways. Our results further emphasize the role of P450-P450 interactions in regulatory cross-talk in human drug-metabolizing ensemble and suggest a role of interactions of CYP2E1 with CYP2D6 in pharmacologically important instances of alcohol-drug interactions.

Alcohol-Induced Increase in the Content of CYP2E1 in Human Liver Microsomes Causes a Multifold Activation of CYP3A4 and Attenuates its Cooperativity

“…The finding that the activation of BQ metabolism and attenuation of its cooperativity may be reproduced in HLM-N samples via incorporation of the exogenous CYP2E1 into their membranes suggests that these effects, at least in a part, represent direct results of the alcohol-induced increase in CYP2E1 content. The striking effects of one P450 protein on the functional properties of another P450 enzyme observed in this study and evidenced in earlier reports from our group and by others (Backes et al, 1998;Kelley et al, 2006;Reed et al, 2010;Davydov et al, 2015;Davydov et al, 2017) may be best explained with a concept of "positional heterogeneity" in P450 oligomers (Davydov, 2011;Davydov et al, 2015;Davydov, 2016;Reed and Backes, 2017;Davydova et al, 2019). This concept is based on considering heteromeric complexes of multiple P450 species as a predominant state of the P450 enzymes in the microsomal membrane.…”

“…International, New Brunswick, NJ (Davydov et al, 2017). In the experiments with Cary Eclipse the excitation was performed with a monochromatic light centered at 405 nm with 5 nm bandwidth.…”

“…In order to probe the crosstalk between CYP2E1 and other P450 species and elucidate its role in alcohol-drug interactions we established a model of alcohol-induced increase in CYP2E1 content that implements enrichment of HLM samples with CYP2E1 through membrane incorporation of the purified protein (Davydov et al, 2015;Davydov et al, 2017;Davydova et al, 2019). We demonstrated that the adopted CYP2E1 becomes a fully-functional member of the drug-metabolizing ensemble and interacts with other P450 enzymes with the formation of heteromeric complexes (Davydova et al, 2019).…”

Non-additive effects of changing the cytochrome P450 ensemble: incorporation of CYP2E1 into human liver microsomes and its impact on CYP1A2

“…recent studies on a large selection of human liver microsomes (HLM) demonstrated the lack of a straight correlation between the composition of human cytochrome P450 ensemble and the profile of drug metabolism [4]. At the same time, there is an emerging recognition of the functional importance of physical interactions between multiple P450 species co-localized in the microsomal membrane [5–9]. Due to complex effects of these interactions any change in the content of a particular P450 enzyme may affect drug metabolism in a complex, hard-to-predict manner.…”

“…In our recent study we also explored the interactions between CYP2E1 with CYP2D6 and observed the formation of their heteromeric complexes in microsomal membrane. We demonstrated that these interactions inhibit the activity of CYP2E1, while exerting a time-dependent activating effect on CYP2D6 in the presence of its specific substrates, 3-[2-(N,N-diethyl-N-methyl ammonium)ethyl]-7-methoxy-4-methylcoumarin (AMMC) and bufuralol [9]. Of particular importance might be the effect of CYP2E1 on the stoichiometry of futile cycling and substrate oxidation by CYP2D6 revealed in a decrease in the electron leakage from CYP2D6 through the peroxide-generating pathways [9].…”