PT  - JOURNAL ARTICLE
AU  - Dermot F. McGinnity
AU  - Sarah J. Griffin
AU  - Graeme C. Moody
AU  - Mike Voice
AU  - Stephen Hanlon
AU  - Thomas Friedberg
AU  - Robert J. Riley
TI  - Rapid Characterization of the Major Drug-Metabolizing Human Hepatic Cytochrome P-450 Enzymes Expressed in <em>Escherichia coli</em>
DP  - 1999 Sep 01
TA  - Drug Metabolism and Disposition
PG  - 1017--1023
VI  - 27
IP  - 9
4099  - http://dmd.aspetjournals.org/content/27/9/1017.short
4100  - http://dmd.aspetjournals.org/content/27/9/1017.full
SO  - Drug Metab Dispos1999 Sep 01; 27
AB  - The major drug-metabolizing human hepatic cytochrome P-450s (CYPs; CYP1A2, 2C9, 2C19, 2D6, and 3A4) coexpressed functionally inEscherichia coli with human NADPH-P-450 reductase have been validated as surrogates to their counterparts in human liver microsomes (HLM) using automated technology. The dealkylation of ethoxyresorufin, dextromethorphan, and erythromycin were all shown to be specific reactions for CYP1A2, CYP2D6, and CYP3A4 that allowed direct comparison with kinetic data for HLM. For CYP2C9 and CYP2C19, the kinetics for the discrete oxidations of naproxen and diazepam were compared to data obtained using established, commercial CYP preparations. Turnover numbers of CYPs expressed in E. coli toward these substrates were generally equal to or even greater than those of the major commercial suppliers [CYP1A2 (ethoxyresorufin), E. coli 0.6 ± 0.2 min−1 versus B lymphoblasts 0.4 ± 0.1 min−1; CYP2C9 (naproxen), 6.7 ± 0.9 versus 4.9 min−1; CYP2C19 (diazepam), 3.7 ± 0.3 versus 0.2 ± 0.1 min−1; CYP2D6 (dextromethorphan), 4.7 ± 0.1 versus 4.4 ± 0.1 min−1; CYP3A4 (erythromycin), 3 ± 1.2 versus 1.6 min−1]. The apparentKm values for the specific reactions were also similar (Km ranges for expressed CYPs and HLM were: ethoxyresorufin 0.5–1.0 μM, dextromethorphan 1.3–5.9 μM, and erythromycin 18–57 μM), indicating little if any effect ofN-terminal modification on the E. coli-expressed CYPs. The data generated for all the probe substrates by HLM and recombinant CYPs also agreed well with literature values. In summary, E. coli-expressed CYPs appear faithful surrogates for the native (HLM) enzyme, and these data suggest that such recombinant enzymes may be suitable for predictive human metabolism studies. The American Society for Pharmacology and Experimental Therapeutics