Diazepam Promotes Translocation of Human Constitutive Androstane Receptor (CAR) via Direct Interaction with the Ligand-Binding Domain
The constitutive androstane receptor (CAR) is the essential regulator of genes involved both in xenobiotic and endobiotic metabolism. Diazepam has been shown as a potent stimulator of CAR nuclear translocation and is assumed as an indirect CAR activator not interacting with the CAR cavity. In this s...
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MDPI AG
2020-11-01
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| Online Access: | https://www.mdpi.com/2073-4409/9/12/2532 |
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| author | Josef Skoda Jan Dusek Martin Drastik Alzbeta Stefela Klara Dohnalova Karel Chalupsky Tomas Smutny Stanislav Micuda Sabine Gerbal-Chaloin Petr Pavek |
| author_facet | Josef Skoda Jan Dusek Martin Drastik Alzbeta Stefela Klara Dohnalova Karel Chalupsky Tomas Smutny Stanislav Micuda Sabine Gerbal-Chaloin Petr Pavek |
| author_sort | Josef Skoda |
| collection | DOAJ |
| description | The constitutive androstane receptor (CAR) is the essential regulator of genes involved both in xenobiotic and endobiotic metabolism. Diazepam has been shown as a potent stimulator of CAR nuclear translocation and is assumed as an indirect CAR activator not interacting with the CAR cavity. In this study, we sought to determine if diazepam is a ligand directly interacting with the CAR ligand binding domain (LBD) and if it regulates its target genes in a therapeutically relevant concentration. We used different CAR constructs in translocation and luciferase reporter assays, recombinant CAR-LBD in a TR-FRET assay, and target genes induction studied in primary human hepatocytes (PHHs), HepaRG cells, and in CAR humanized mice. We also used in silico docking and CAR-LBD mutants to characterize the interaction of diazepam and its metabolites with the CAR cavity. Diazepam and its metabolites such as nordazepam, temazepam, and oxazepam are activators of CAR+Ala in translocation and two-hybrid assays and fit the CAR cavity in docking experiments. In gene reporter assays with CAR3 and in the TR-FRET assay, only diazepam significantly interacts with CAR-LBD. Diazepam also promotes up-regulation of CYP2B6 in PHHs and in HepaRG cells. However, in humanized CAR mice, diazepam significantly induces neither <i>CYP2B6</i> nor <i>Cyp2b10</i> genes nor does it regulate critical genes involved in glucose and lipids metabolism and liver proliferation. Thus, we demonstrate that diazepam interacts with human CAR-LBD as a weak ligand, but it does not significantly affect expression of tested CAR target genes in CAR humanized mice. |
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| last_indexed | 2024-03-10T14:37:00Z |
| publishDate | 2020-11-01 |
| publisher | MDPI AG |
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| series | Cells |
| spelling | doaj.art-7c2b6c2d073147058fe91e7104860ab22023-11-20T22:04:40ZengMDPI AGCells2073-44092020-11-01912253210.3390/cells9122532Diazepam Promotes Translocation of Human Constitutive Androstane Receptor (CAR) via Direct Interaction with the Ligand-Binding DomainJosef Skoda0Jan Dusek1Martin Drastik2Alzbeta Stefela3Klara Dohnalova4Karel Chalupsky5Tomas Smutny6Stanislav Micuda7Sabine Gerbal-Chaloin8Petr Pavek9Department of Pharmacology and Toxicology, Faculty of Pharmacy, Charles University, Heyrovskeho 1203, 500 05 Hradec Kralove, Czech RepublicDepartment of Pharmacology and Toxicology, Faculty of Pharmacy, Charles University, Heyrovskeho 1203, 500 05 Hradec Kralove, Czech RepublicDepartment of Physical Chemistry and Biophysics, Faculty of Pharmacy, Charles University, Heyrovskeho 1203, 500 05 Hradec Kralove, Czech RepublicDepartment of Pharmacology and Toxicology, Faculty of Pharmacy, Charles University, Heyrovskeho 1203, 500 05 Hradec Kralove, Czech Republic1<sup>st</sup> Medical Faculty, Charles University, Katerinská 32, 121 08 Prague, Czech RepublicCzech Centre for Phenogenomics, Institute of Molecular Genetics of the Czech Academy of Sciences, Vídeňská 1083, 142 20 Prague, Czech RepublicDepartment of Pharmacology and Toxicology, Faculty of Pharmacy, Charles University, Heyrovskeho 1203, 500 05 Hradec Kralove, Czech RepublicDepartment of Pharmacology, Medical Faculty in Hradec Kralove, Charles University, Simkova 870, 500 03 Hradec Kralove, Czech RepublicIRMB, University of Montpellier, INSERM, 34295 Montpellier, FranceDepartment of Pharmacology and Toxicology, Faculty of Pharmacy, Charles University, Heyrovskeho 1203, 500 05 Hradec Kralove, Czech RepublicThe constitutive androstane receptor (CAR) is the essential regulator of genes involved both in xenobiotic and endobiotic metabolism. Diazepam has been shown as a potent stimulator of CAR nuclear translocation and is assumed as an indirect CAR activator not interacting with the CAR cavity. In this study, we sought to determine if diazepam is a ligand directly interacting with the CAR ligand binding domain (LBD) and if it regulates its target genes in a therapeutically relevant concentration. We used different CAR constructs in translocation and luciferase reporter assays, recombinant CAR-LBD in a TR-FRET assay, and target genes induction studied in primary human hepatocytes (PHHs), HepaRG cells, and in CAR humanized mice. We also used in silico docking and CAR-LBD mutants to characterize the interaction of diazepam and its metabolites with the CAR cavity. Diazepam and its metabolites such as nordazepam, temazepam, and oxazepam are activators of CAR+Ala in translocation and two-hybrid assays and fit the CAR cavity in docking experiments. In gene reporter assays with CAR3 and in the TR-FRET assay, only diazepam significantly interacts with CAR-LBD. Diazepam also promotes up-regulation of CYP2B6 in PHHs and in HepaRG cells. However, in humanized CAR mice, diazepam significantly induces neither <i>CYP2B6</i> nor <i>Cyp2b10</i> genes nor does it regulate critical genes involved in glucose and lipids metabolism and liver proliferation. Thus, we demonstrate that diazepam interacts with human CAR-LBD as a weak ligand, but it does not significantly affect expression of tested CAR target genes in CAR humanized mice.https://www.mdpi.com/2073-4409/9/12/2532diazepamgene regulationCARNR1I3cytochrome P450drug interaction |
| spellingShingle | Josef Skoda Jan Dusek Martin Drastik Alzbeta Stefela Klara Dohnalova Karel Chalupsky Tomas Smutny Stanislav Micuda Sabine Gerbal-Chaloin Petr Pavek Diazepam Promotes Translocation of Human Constitutive Androstane Receptor (CAR) via Direct Interaction with the Ligand-Binding Domain Cells diazepam gene regulation CAR NR1I3 cytochrome P450 drug interaction |
| title | Diazepam Promotes Translocation of Human Constitutive Androstane Receptor (CAR) via Direct Interaction with the Ligand-Binding Domain |
| title_full | Diazepam Promotes Translocation of Human Constitutive Androstane Receptor (CAR) via Direct Interaction with the Ligand-Binding Domain |
| title_fullStr | Diazepam Promotes Translocation of Human Constitutive Androstane Receptor (CAR) via Direct Interaction with the Ligand-Binding Domain |
| title_full_unstemmed | Diazepam Promotes Translocation of Human Constitutive Androstane Receptor (CAR) via Direct Interaction with the Ligand-Binding Domain |
| title_short | Diazepam Promotes Translocation of Human Constitutive Androstane Receptor (CAR) via Direct Interaction with the Ligand-Binding Domain |
| title_sort | diazepam promotes translocation of human constitutive androstane receptor car via direct interaction with the ligand binding domain |
| topic | diazepam gene regulation CAR NR1I3 cytochrome P450 drug interaction |
| url | https://www.mdpi.com/2073-4409/9/12/2532 |
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