Diazepam Promotes Translocation of Human Constitutive Androstane Receptor (CAR) via Direct Interaction with the Ligand-Binding Domain

被引:9
|
作者
Skoda, Josef [1 ]
Dusek, Jan [1 ]
Drastik, Martin [2 ]
Stefela, Alzbeta [1 ]
Dohnalova, Klara [3 ,4 ]
Chalupsky, Karel [4 ]
Smutny, Tomas [1 ]
Micuda, Stanislav [5 ]
Gerbal-Chaloin, Sabine [6 ]
Pavek, Petr [1 ]
机构
[1] Charles Univ Prague, Fac Pharm, Dept Pharmacol & Toxicol, Heyrovskeho 1203, Hradec Kralove 50005, Czech Republic
[2] Charles Univ Prague, Fac Pharm, Dept Phys Chem & Biophys, Heyrovskeho 1203, Hradec Kralove 50005, Czech Republic
[3] Charles Univ Prague, Med Fac 1, Katerinska 32, Prague 12108, Czech Republic
[4] Czech Acad Sci, Inst Mol Genet, Czech Ctr Phenogen, Videnska 1083, Prague 14220, Czech Republic
[5] Charles Univ Prague, Med Fac Hradec Kralove, Dept Pharmacol, Simkova 870, Hradec Kralove 50003, Czech Republic
[6] Univ Montpellier, INSERM, IRMB, F-34295 Montpellier, France
关键词
diazepam; gene regulation; CAR; cytochrome P450; drug interaction; PREGNANE-X-RECEPTOR; NUCLEAR TRANSLOCATION; HUMAN HEPATOCYTES; ACTIVATORS; MOUSE; CYP2B; LIVER; BENZODIAZEPINES; METABOLISM; INDUCTION;
D O I
10.3390/cells9122532
中图分类号
Q2 [细胞生物学];
学科分类号
071009 ; 090102 ;
摘要
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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页数:18
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