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Surface display of human cytochrome P450 enzymes 3A4, 1A2, 2C9, 2C19 and 2D6 with cytochrome P450 reductase for drug metabolism studies
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1  Institute of Pharmaceutical and Medicinal Chemistry, PharmaCampus, Westfälische Wilhelms-Universität Münster, Corrensstraße 48, 48149 Münster

Published: 30 October 2019 by MDPI in 5th International Electronic Conference on Medicinal Chemistry session ECMC-5

Cytochrome P450 monooxygenases (CYPs) are responsible for the biotransformation of most known drugs and xenobiotics in human body [1]. As part of the phase-I-metabolism they catalyze a broad diversity of oxidation reactions in an extensive spectrum of substrates. The utilization of CYPs as biocatalysts is limited due to their low stability and their requirement of a membrane surrounding to fold into an active form [2]. Autodisplay of CYPs on the surface of E. coli has been shown an appropriate tool to overcome these limitations [3, 4].

In order to establish an in vitro system to study drug metabolism, the five most important CYPs, CYP 3A4, CYP 1A2, CYP 2C9, CYP 2C19 and CYP 2D6 were displayed on the surface of E. coli. The catalytic activity of CYP 3A4 was shown by testosterone as a substrate using a HPLC assay with external addition of the cytochrome P450 reductase (CPR) [5]. A co‑expression of CYP 1A2 and CPR was established with both enzymes being displayed on the surface of E. coli. Surface display was confirmed by a protease accessibility test and by flow cytometry. Surface displayed CYP 1A2 with co-expressed CPR was able to convert phenacetin to paracetamol, as well as 7‑ethoxyresorufin and 3-cyano-7-ethoxycoumarin to the fluorescent products resorufin [6] and 3-cyano-7-hydroxycoumarin. CYP 2C9, CYP 2C19 and CYP 2D6 were co‑expressed with CPR on the surface of E. coli as well. Combining cells with these five CYP enzymes in an active form on the bacterial cell surface is supposed to provide a suitable approach for the in vitro simulation of drug metabolism.

Keywords: cytochrome P450 monooxygenase; surface display; drug metabolism; autodisplay