Human cytochrome P450 expression in bacteria: Whole-cell high-throughput activity assay for CYP1A2, 2A6 and 3A4

Francisco Esteves, Diana Campelo, Philippe Urban, Sophie Bozonnet, Thomas Lautier, José Rueff, Gilles Truan, M Kranendonk

Research output: Contribution to journalArticle

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.

Original languageEnglish
Pages (from-to)134-140
Number of pages7
JournalBiochemical pharmacology
Volume158
Early online date8 Oct 2018
DOIs
Publication statusPublished - Dec 2018

Fingerprint

Cytochrome P-450 CYP1A2
Cytochromes
Cytochrome P-450 Enzyme System
Assays
Bacteria
Throughput
Cytochromes b5
Screening
Enzymes
Drug interactions
High-Throughput Screening Assays
Small Molecule Libraries
NADPH-Ferrihemoprotein Reductase
Proteins
Subcellular Fractions
Metabolome
Xenobiotics
Metabolites
Catalysis
Drug Interactions

Keywords

  • Cytochrome b5 (CYB5)
  • Microsomal cytochrome P450 monooxygenases (CYP)
  • NADPH cytochrome P450 oxidoreductase (CPR)
  • Real-time fluorometric measurement
  • Whole-cell high-throughput biocatalysis

Cite this

@article{e451490d7d934ec0876f10a0d0e25086,
title = "Human cytochrome P450 expression in bacteria: Whole-cell high-throughput activity assay for CYP1A2, 2A6 and 3A4",
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.",
keywords = "Cytochrome b5 (CYB5), Microsomal cytochrome P450 monooxygenases (CYP), NADPH cytochrome P450 oxidoreductase (CPR), Real-time fluorometric measurement, Whole-cell high-throughput biocatalysis",
author = "Francisco Esteves and Diana Campelo and Philippe Urban and Sophie Bozonnet and Thomas Lautier and Jos{\'e} Rueff and Gilles Truan and M Kranendonk",
note = "Copyright {\circledC} 2018 Elsevier Inc. All rights reserved.",
year = "2018",
month = "12",
doi = "10.1016/j.bcp.2018.10.006",
language = "English",
volume = "158",
pages = "134--140",
journal = "Biochemical pharmacology",
issn = "0006-2952",
publisher = "Elsevier Inc.",

}

Human cytochrome P450 expression in bacteria : Whole-cell high-throughput activity assay for CYP1A2, 2A6 and 3A4. / Esteves, Francisco; Campelo, Diana; Urban, Philippe; Bozonnet, Sophie; Lautier, Thomas; Rueff, José; Truan, Gilles; Kranendonk, M.

In: Biochemical pharmacology, Vol. 158, 12.2018, p. 134-140.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Human cytochrome P450 expression in bacteria

T2 - Whole-cell high-throughput activity assay for CYP1A2, 2A6 and 3A4

AU - Esteves, Francisco

AU - Campelo, Diana

AU - Urban, Philippe

AU - Bozonnet, Sophie

AU - Lautier, Thomas

AU - Rueff, José

AU - Truan, Gilles

AU - Kranendonk, M

N1 - Copyright © 2018 Elsevier Inc. All rights reserved.

PY - 2018/12

Y1 - 2018/12

N2 - 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.

AB - 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.

KW - Cytochrome b5 (CYB5)

KW - Microsomal cytochrome P450 monooxygenases (CYP)

KW - NADPH cytochrome P450 oxidoreductase (CPR)

KW - Real-time fluorometric measurement

KW - Whole-cell high-throughput biocatalysis

U2 - 10.1016/j.bcp.2018.10.006

DO - 10.1016/j.bcp.2018.10.006

M3 - Article

VL - 158

SP - 134

EP - 140

JO - Biochemical pharmacology

JF - Biochemical pharmacology

SN - 0006-2952

ER -