Detail
Article
Online article
FT
Medvik - BMC
  • Something wrong with this record ?

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

J. Skoda, J. Dusek, M. Drastik, A. Stefela, K. Dohnalova, K. Chalupsky, T. Smutny, S. Micuda, S. Gerbal-Chaloin, P. Pavek

. 2020 ; 9 (12) : . [pub] 20201124

Language English Country Switzerland

Document type Journal Article, Research Support, Non-U.S. Gov't

Persistent link   https://www.medvik.cz/link/bmc21019798

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.

References provided by Crossref.org

000      
00000naa a2200000 a 4500
001      
bmc21019798
003      
CZ-PrNML
005      
20210830101409.0
007      
ta
008      
210728s2020 sz f 000 0|eng||
009      
AR
024    7_
$a 10.3390/cells9122532 $2 doi
035    __
$a (PubMed)33255185
040    __
$a ABA008 $b cze $d ABA008 $e AACR2
041    0_
$a eng
044    __
$a sz
100    1_
$a Skoda, Josef $u Department of Pharmacology and Toxicology, Faculty of Pharmacy, Charles University, Heyrovskeho 1203, 500 05 Hradec Kralove, Czech Republic
245    10
$a Diazepam Promotes Translocation of Human Constitutive Androstane Receptor (CAR) via Direct Interaction with the Ligand-Binding Domain / $c J. Skoda, J. Dusek, M. Drastik, A. Stefela, K. Dohnalova, K. Chalupsky, T. Smutny, S. Micuda, S. Gerbal-Chaloin, P. Pavek
520    9_
$a 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.
650    _2
$a dospělí $7 D000328
650    _2
$a zvířata $7 D000818
650    _2
$a buněčné linie $7 D002460
650    _2
$a proliferace buněk $x účinky léků $7 D049109
650    _2
$a diazepam $x farmakologie $7 D003975
650    _2
$a ženské pohlaví $7 D005260
650    _2
$a reportérové geny $x účinky léků $x genetika $7 D017930
650    _2
$a hepatocyty $x účinky léků $7 D022781
650    _2
$a lidé $7 D006801
650    _2
$a ligandy $7 D008024
650    _2
$a játra $x účinky léků $7 D008099
650    _2
$a mužské pohlaví $7 D008297
650    _2
$a myši $7 D051379
650    _2
$a lidé středního věku $7 D008875
650    _2
$a proteinové domény $x účinky léků $7 D000072417
650    _2
$a transport proteinů $x účinky léků $7 D021381
650    _2
$a receptory cytoplazmatické a nukleární $x metabolismus $7 D018160
655    _2
$a časopisecké články $7 D016428
655    _2
$a práce podpořená grantem $7 D013485
700    1_
$a Dusek, Jan $u Department of Pharmacology and Toxicology, Faculty of Pharmacy, Charles University, Heyrovskeho 1203, 500 05 Hradec Kralove, Czech Republic
700    1_
$a Drastik, Martin $u Department of Physical Chemistry and Biophysics, Faculty of Pharmacy, Charles University, Heyrovskeho 1203, 500 05 Hradec Kralove, Czech Republic
700    1_
$a Stefela, Alzbeta $u Department of Pharmacology and Toxicology, Faculty of Pharmacy, Charles University, Heyrovskeho 1203, 500 05 Hradec Kralove, Czech Republic
700    1_
$a Dohnalova, Klara $u 1st Medical Faculty, Charles University, Katerinská 32, 121 08 Prague, Czech Republic $u Czech Centre for Phenogenomics, Institute of Molecular Genetics of the Czech Academy of Sciences, Vídeňská 1083, 142 20 Prague, Czech Republic
700    1_
$a Chalupsky, Karel $u Czech Centre for Phenogenomics, Institute of Molecular Genetics of the Czech Academy of Sciences, Vídeňská 1083, 142 20 Prague, Czech Republic
700    1_
$a Smutny, Tomas $u Department of Pharmacology and Toxicology, Faculty of Pharmacy, Charles University, Heyrovskeho 1203, 500 05 Hradec Kralove, Czech Republic
700    1_
$a Micuda, Stanislav $u Department of Pharmacology, Medical Faculty in Hradec Kralove, Charles University, Simkova 870, 500 03 Hradec Kralove, Czech Republic
700    1_
$a Gerbal-Chaloin, Sabine $u IRMB, University of Montpellier, INSERM, 34295 Montpellier, France
700    1_
$a Pavek, Petr $u Department of Pharmacology and Toxicology, Faculty of Pharmacy, Charles University, Heyrovskeho 1203, 500 05 Hradec Kralove, Czech Republic
773    0_
$w MED00194911 $t Cells $x 2073-4409 $g Roč. 9, č. 12 (2020)
856    41
$u https://pubmed.ncbi.nlm.nih.gov/33255185 $y Pubmed
910    __
$a ABA008 $b sig $c sign $y p $z 0
990    __
$a 20210728 $b ABA008
991    __
$a 20210830101409 $b ABA008
999    __
$a ok $b bmc $g 1690582 $s 1140244
BAS    __
$a 3
BAS    __
$a PreBMC
BMC    __
$a 2020 $b 9 $c 12 $e 20201124 $i 2073-4409 $m Cells $n Cells $x MED00194911
LZP    __
$a Pubmed-20210728

Find record

Citation metrics

Archiving options