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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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Autores principales: Skoda, Josef, Dusek, Jan, Drastik, Martin, Stefela, Alzbeta, Dohnalova, Klara, Chalupsky, Karel, Smutny, Tomas, Micuda, Stanislav, Gerbal-Chaloin, Sabine, Pavek, Petr
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2020
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Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7761063/
https://www.ncbi.nlm.nih.gov/pubmed/33255185
http://dx.doi.org/10.3390/cells9122532
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author Skoda, Josef
Dusek, Jan
Drastik, Martin
Stefela, Alzbeta
Dohnalova, Klara
Chalupsky, Karel
Smutny, Tomas
Micuda, Stanislav
Gerbal-Chaloin, Sabine
Pavek, Petr
author_facet Skoda, Josef
Dusek, Jan
Drastik, Martin
Stefela, Alzbeta
Dohnalova, Klara
Chalupsky, Karel
Smutny, Tomas
Micuda, Stanislav
Gerbal-Chaloin, Sabine
Pavek, Petr
author_sort Skoda, Josef
collection PubMed
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 CYP2B6 nor Cyp2b10 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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spelling pubmed-77610632020-12-26 Diazepam Promotes Translocation of Human Constitutive Androstane Receptor (CAR) via Direct Interaction with the Ligand-Binding Domain Skoda, Josef Dusek, Jan Drastik, Martin Stefela, Alzbeta Dohnalova, Klara Chalupsky, Karel Smutny, Tomas Micuda, Stanislav Gerbal-Chaloin, Sabine Pavek, Petr Cells Article 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 CYP2B6 nor Cyp2b10 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. MDPI 2020-11-24 /pmc/articles/PMC7761063/ /pubmed/33255185 http://dx.doi.org/10.3390/cells9122532 Text en © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Skoda, Josef
Dusek, Jan
Drastik, Martin
Stefela, Alzbeta
Dohnalova, Klara
Chalupsky, Karel
Smutny, Tomas
Micuda, Stanislav
Gerbal-Chaloin, Sabine
Pavek, Petr
Diazepam Promotes Translocation of Human Constitutive Androstane Receptor (CAR) via Direct Interaction with the Ligand-Binding Domain
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 Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7761063/
https://www.ncbi.nlm.nih.gov/pubmed/33255185
http://dx.doi.org/10.3390/cells9122532
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