Identification of novel agonists by high-throughput screening and molecular modelling of human constitutive androstane receptor isoform 3

Keminer, Oliver; Windshügel, Björn; Eßmann, Frank; Lee, Serene M. L.; Schiergens, Tobias S.; Schwab, Matthias; Burk, Oliver

doi:10.1007/s00204-019-02495-6

Cited by 5 publications

(18 citation statements)

References 53 publications

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“…It is likely that these in silico methods will not be discriminating enough to quickly and reliably detect and distinguish between all CAR agonists and inverse agonists, but they are valuable as supportive tools either for pre-selection before or mechanistic binding evaluations after the functional assays (e.g. Küblbeck et al, 2011aKüblbeck et al, , 2011bLynch et al, 2013;Keminer et al, 2019) that will be described below.…”

Section: Structural Features That Contribute To High Constitutive Act...mentioning

confidence: 99%

“…The second approach relies on the use of natural (CAR3; Keminer et al, 2019;Skoda et al, 2020) or artificial CAR variants where the basal activity is reduced by addition of amino acids to the human (Chen et al, 2010;Imai et al, 2013;Kanno et al, 2010) or animal CAR LBDs (Omiecinski et al, 2011;Pinne et al, 2016). This approach carries the risk of affecting selectivity of the LBP (CAR2 or insertions near H12) or the CAR/RXR binding (CAR3 or insertion of single alanine instead of APYLT) that may affect the ligand-dependent changes in coactivator recruitment.…”

Section: Strategies In Identifying Car Modulatorsmentioning

confidence: 99%

“…Clotrimazole has repeatedly shown to be a CAR3 agonist (Chen et al, 2010;Omiecinski et al, 2011;Keminer et al, 2019;. Given the very high similarity of CAR1 and CAR3 LBPs and their similar activation profiles (Keminer et al, 2019), the above discrepancies are likely due to assay-related reasons.…”

Section: Strategies In Identifying Car Modulatorsmentioning

confidence: 99%

“…The dynamic range of the employed reporter assays, as defined by the maximal response of established positive controls such as TCPOBOP or CITCO relative to the DMSO vehicle, varies significantly. These values range from about two-fold (Yao et al, 2010;Wahlang et al, 2014;Hardesty et al, 2018), to ~5-fold (Mäkinen et al, 2003;Lynch et al, 2019) and to 21-fold (Küblbeck et al, 2011a) with CAR1, and between 6-to 26-fold with CAR3 (Skoda et al, 2020;Keminer et al, 2019). A low dynamic range may limit the sensitivity to detect true activators, and more importantly, observed lack of reporter activity in a suboptimal assay does not indicate discovery of an indirect CAR activator.…”

Section: Strategies In Identifying Car Modulatorsmentioning

confidence: 99%

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Searching for Constitutive Androstane Receptor Modulators

Honkakoski

2022

Drug Metabolism and Disposition

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AhR, aryl hydrocarbon receptor; CAR, constitutive androstane receptor; CITCO, 6-(4chlorophenyl)imidazo(2,1-b)(1,3)thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime; CYP, cytochrome P450; DMSO, dimethyl sulfoxide; EGF, epidermal growth factor; ERK, extracellular signal-regulated kinase; LBD, ligand-binding domain; LBP, ligand-binding pocket; MAPK, mitogen-activated protein kinase; NRF2, nuclear factor erythroid 2-related factor 2; PP2A, protein phosphatase 2A; PK11195, 1-(2-chlorophenyl)-N-methyl-N-(1methylpropyl)-3-isoquinoline carboxamide; PB, phenobarbital; PPAR, peroxisome proliferator-activated receptor; PXR, pregnane X receptor; RAR, retinoid acid receptor; RXR, retinoid X receptor; TCPOBOP, 1,4-bis-[2-(3,5-dichloropyridyloxy)]benzene.

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Section: Structural Features That Contribute To High Constitutive Act...mentioning

confidence: 99%

Section: Strategies In Identifying Car Modulatorsmentioning

confidence: 99%

Section: Strategies In Identifying Car Modulatorsmentioning

confidence: 99%

Section: Strategies In Identifying Car Modulatorsmentioning

confidence: 99%

See 2 more Smart Citations

Searching for Constitutive Androstane Receptor Modulators

Honkakoski

2022

Drug Metabolism and Disposition

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“…The discovery of a hCAR activator suitable for clinical studies or one already used in pharmacotherapy would be a milestone in the characterization of hCAR as a molecular target in pharmacotherapy. Among the variety of compounds characterized as hCAR activators using in vitro high-throughput screenings, only a few compounds are on the market [7,14,15]. For instance, the routinely used pharmaceuticals diazepam and phenytoin have been described as hCAR activators in vitro, but only one solitary drug-to-drug interaction possibly connected with hCAR activation has been reported [16].…”

Section: Introductionmentioning

confidence: 99%

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

Skoda

Dušek

Drastík

et al. 2020

Cells

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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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Filling the Blank Space: Branched 4-Nonylphenol Isomers Are Responsible for Robust Constitutive Androstane Receptor (CAR) Activation by Nonylphenol

Rashidian,

Dušek,

Drastik

et al. 2024

Environ. Sci. Technol.

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4-Nonylphenol (4-NP), a para-substituted phenolic compound with a straight or branched carbon chain, is a ubiquitous environmental pollutant and food contaminant. 4-NP, particularly the branched form, has been identified as an endocrine disruptor (ED) with potent activities on estrogen receptors. Constitutive Androstane Receptor (CAR) is another crucial nuclear receptor that regulates hepatic lipid, glucose, and steroid metabolism and is involved in the ED mechanism of action. An NP mixture has been described as an extremely potent activator of both human and rodent CAR. However, detailed mechanistic aspects of CAR activation by 4-NP are enigmatic, and it is not known if 4-NP can directly interact with the CAR ligand binding domain (LBD). Here, we examined interactions of individual branched (22NP, 33NP, and 353NP) and linear 4-NPs with CAR variants using molecular dynamics (MD) simulations, cellular experiments with various CAR expression constructs, recombinant CAR LBD in a TR-FRET assay, or a differentiated HepaRG hepatocyte cellular model. Our results demonstrate that branched 4-NPs display more stable poses to activate both wild-type CAR1 and CAR3 variant LBDs in MD simulations. Consistently, branched 4-NPs activated CAR3 and CAR1 LBD more efficiently than linear 4-NP. Furthermore, in HepaRG cells, we observed that all 4-NPs upregulated CYP2B6 mRNA, a relevant hallmark for CAR activation. This is the first study to provide detailed insights into the direct interaction between individual 4-NPs and human CAR-LBD, as well as its dominant variant CAR3. The work could contribute to the safer use of individual 4-NPs in many areas of industry.

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Identification of novel agonists by high-throughput screening and molecular modelling of human constitutive androstane receptor isoform 3

Cited by 5 publications

References 53 publications

Searching for Constitutive Androstane Receptor Modulators

Searching for Constitutive Androstane Receptor Modulators

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

Filling the Blank Space: Branched 4-Nonylphenol Isomers Are Responsible for Robust Constitutive Androstane Receptor (CAR) Activation by Nonylphenol

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