Human cytochrome P450 expression in bacteria: Whole-cell high-throughput activity assay for CYP1A2, 2A6 and 3A4.
Biochem Pharmacol
; 158: 134-140, 2018 12.
Article
en En
| MEDLINE
| ID: mdl-30308189
ABSTRACT
Cytochrome P450s (CYPs) are key enzymes involved in drug and xenobiotic metabolism. A wide array of in vitro methodologies, including recombinant sources, are currently been used to assess CYP catalysis, to identify the metabolic profile of compounds, potential drug-drug interactions, protein-protein interactions in the CYP enzyme complex and the role of polymorphic enzymes. We report here on a bacterial whole-cells high-throughput method for the activity evaluation of human CYP1A2, 2A6, and 3A4, when sustained by NADPH cytochrome P450 oxidoreductase (CPR), in the absence or presence of cytochrome b5 (CYB5). This new assay consists of a microplate real-time fluorometric method, with direct measurement of metabolite formation, in a suspension of Escherichia coli BTC-CYP bacteria, a human CYP competent tester strain when incubated with specific fluorogenic substrates. Overall, the maximum turnover (kcat) velocities of the three human CYPs resulting from the whole-BTC cells assays were similar to those obtained when applying the corresponding standard reference membrane fractions assays. CYP activity screening with co-expression of CYB5 suggests an enhancing effect of CYB5 on the kcat of specific isoforms, when using the whole-BTC cells assay. Our results demonstrate that this new approach can offer an efficient high-throughput method for screening of CYP1A2, 2A6 and 3A4 activity and can be potentially applicable for other human CYPs. This can be of particular use for timely and efficient screening of chemical libraries or mutant libraries of CYP enzyme complex proteins, without the necessity for labor intensive isolation of subcellular fractions.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Citocromo P-450 CYP1A2
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Escherichia coli
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Citocromo P-450 CYP3A
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Citocromo P-450 CYP2A6
Límite:
Humans
Idioma:
En
Revista:
Biochem Pharmacol
Año:
2018
Tipo del documento:
Article
Pais de publicación:
ENGLAND
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ESCOCIA
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GB
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GREAT BRITAIN
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INGLATERRA
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REINO UNIDO
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SCOTLAND
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UK
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UNITED KINGDOM