Novel Cell-based Reporter Assay System Using Epitope-tagged Protein for the Identification of Agonistic Ligands of Constitutive Androstane Receptor (CAR)

doi:10.2133/dmpk.DMPK-12-RG-112

Drug Metabolism and Pharmacokinetics

Volume 28, Issue 4, 2013, Pages 290-298

https://doi.org/10.2133/dmpk.DMPK-12-RG-112 Get rights and content

Summary:

Constitutive androstane receptor (CAR) plays vital roles in multiple liver functions including xenobiotic and energy metabolisms, and also in hepatocarcinogenesis. CAR shows species-dependent ligand selectivity, leading to species differences in the actions of xenobiotics. Thus, to know whether a compound of interest activates human CAR or not is helpful for the chemical safety evaluation. However, it is very difficult to observe clear ligand response for CAR in cell-based assays, because of its high basal transcriptional activity in the absence of its ligands. In this study, we found that reporter assays using HepG2 cells human and mouse CAR fused with an epitope-tag at its C-terminus showed low basal activity and high response to the corresponding agonists but not to their indirect activator, phenobarbital. Using this system, 176 industrial chemicals were screened for their abilities to activate human and mouse CAR, and 6 compounds moderately or strongly activated human and/or mouse CAR. The CAR-mediated transcriptions induced by these compounds were blocked by clotrimazole or androstanol, the human and mouse CAR inverse agonists, respectively, suggesting that the newly identified CAR activators are agonistic ligands of CAR. Taken together, our reporter assay system may be a promising tool to assess chemicals’ agonistic activities toward CAR.

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Cited by (17)

Development of a strategy to identify and evaluate direct and indirect activators of constitutive androstane receptor in rats
2022, Food and Chemical Toxicology
Citation Excerpt :
Insertion of a short loop of amino acid residues between helix 11 (H11) and AF2 was shown to force a realignment of the configuration of AF2 to deactivate the receptor in unliganded states (Chen et al., 2010; Cherian et al., 2018; Dussault et al., 2002; Kanno and Inouye, 2010). In addition, we have reported that hCAR tagged with V5 and 6×His tags or Myc and 6×His tags directly following the AF2 domain showed decreased basal activity and that ligands, but not indirect activators, activated the tagged hCAR (Imai et al., 2013). These results suggest that the configuration of AF2 is a key determinant of the basal activity of CAR.
Constitutive androstane receptor (CAR) is a nuclear receptor that plays a key role in drug metabolism and disposition and in the development of liver tumors in rodents. CAR is activated by ligands and indirect activators, which do not bind to the receptor but activate it through cellular signaling. In this study, we sought to identify direct and indirect activators of rat CAR (rCAR). Assessment of the influence of mutations on the transcriptional activity of rCAR identified a mutant termed rCAR-3A-G354Q that displays low constitutive activity and high ligand responsiveness. Reporter assays using the mutant were performed with compounds that increased the mRNA levels of Cyp2b1, a CAR target gene, in rat primary hepatocytes. Several compounds activated rCAR-3A-G354Q and were implicated as rCAR ligands. Since indirect CAR activators are considered to display little species differences, we then determined CYP2B6 mRNA levels in human hepatocyte-like HepaRG cells after treatment with compounds that increased Cyp2b1 mRNA levels in rat hepatocytes but did not activate rCAR-3A-G354Q. The results demonstrated six compounds as possible rCAR indirect activators. Taken together, the combined measurement of Cyp2b1 mRNA levels in rat primary hepatocytes and rCAR-3A-G354Q activation in reporter assays can be useful for evaluating rCAR activation by chemicals.
Computational prediction of cytochrome P450 inhibition and induction
2020, Drug Metabolism and Pharmacokinetics
Citation Excerpt :
CAR is responsible for CYP2B6 induction and regulates the expression of numerous other drug-metabolizing enzymes and transporters, including CYP2C, CYP3A, glutathione S-transferases, SULT, UGT1A1, organic anion-transporting polypeptide 1, multidrug resistance-associated proteins 2 and 3 (MRP2 and MRP3) [113–118]. A wide variety of compounds, including clinically used drugs, steroids, natural compounds, pesticides, industrial chemicals, and various synthetic compounds, are known as the activators of CAR [119–122]. CAR normally resides in the cytoplasmic compartment; translocation to the nucleus occurs after exposure to chemical activators or ligands such as 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichloro-ben-zyl)oxime.
Cytochrome P450 (CYP) enzymes play an important role in the phase I metabolism of many xenobiotics. Most drug–drug interactions (DDIs) associated with CYP are caused by either CYP inhibition or induction. The early detection of potential DDIs is highly desirable in the pharmaceutical industry because DDIs can cause serious adverse events, which can lead to poor patient health and drug development failures. Recently, many computational studies predicting CYP inhibition and induction have been reported. The current computational modeling approaches for CYP metabolism are classified as ligand- and structure-based; various techniques, such as quantitative structure–activity relationships, machine learning, docking, and molecular dynamic simulation, are involved in both the approaches. Recently, combining these two approaches have resulted in improvements in the prediction accuracy of DDIs.
In this review, we present important, recent developments in the computational prediction of the inhibition of four clinically crucial CYP isoforms (CYP1A2, 2C9, 2D6, and 3A4) and three nuclear receptors (aryl hydrocarbon receptor, constitutive androstane receptor, and pregnane X receptor) involved in the induction of CYP1A2, 2B6, and 3A4, respectively.
High-content analysis of constitutive androstane receptor (CAR) translocation identifies mosapride citrate as a CAR agonist that represses gluconeogenesis
2019, Biochemical Pharmacology
Citation Excerpt :
Despite these challenges, many efforts have been made to identify compounds that modulate hCAR activity because of its ability to mediate DDIs and control important physiological processes in the liver. A number of screening methods have been developed to identify hCAR modulators, employing approaches from computational modeling to cell-based luciferase reporter assays with varying success [12–16]. While computational and in situ approaches have had some success finding hCAR ligands, cell-based assays are the most widely used to identify chemical modulators of this receptor.
The constitutive androstane receptor (CAR) plays an important role in hepatic drug metabolism and detoxification but has recently been projected as a potential drug target for metabolic disorders due to its repression of lipogenesis and gluconeogenesis. Thus, identification of physiologically-relevant CAR modulators has garnered significant interest. Here, we adapted the previously characterized human CAR (hCAR) nuclear translocation assay in human primary hepatocytes (HPH) to a high-content format and screened an FDA-approved drug library containing 978 compounds. Comparison of hCAR nuclear translocation results with the Tox21 hCAR luciferase reporter assay database in 643 shared compounds revealed significant overlap between these two assays, with approximately half of hCAR agonists also mediating nuclear translocation. Further validation of these compounds in HPH and/or using published data from literature demonstrated that hCAR translocation exhibits a higher correlation with the induction of hCAR target genes, such as CYP2B6, than the luciferase assay. In addition, some CAR antagonists which repress CYP2B6 mRNA expression in HPH, such as sorafenib, rimonabant, and CINPA1, were found to translocate hCAR to the nucleus of HPH. Notably, both the translocation assay and the luciferase assay identified mosapride citrate (MOS), a gastroprokinetic agent that is known to reduce fasting blood glucose levels in humans, as a novel hCAR activator. Further studies with MOS in HPH uncovered that MOS can repress the expression of gluconeogenic genes and decrease glucose output from hepatocytes, providing a previously unidentified liver-specific mechanism by which MOS modulates blood glucose levels.
Profiling of bisphenol A and eight its analogues on transcriptional activity via human nuclear receptors
2019, Toxicology
Several bisphenol A (BPA) analogues have been detected in environmental samples, foodstuffs, and/or human biological samples, and there is concern regarding their potential endocrine-disrupting effects. In this study, we characterized the agonistic and/or antagonistic activities of BPA and eight its analogues against human estrogen receptors (ERα/β), androgen receptor (AR), glucocorticoid receptor (GR), pregnane X receptor (PXR), and constitutive androstane receptor (CAR). All the test compounds, except for bisphenol P (BPP), showed both ERα and ERβ agonistic activities, with bisphenol AF (BPAF) being the most potent. On the other hand, BPAF and BPP showed ERα and ERβ antagonistic activities. Interestingly, their ER activities demonstrated a preference toward ERβ. All the test compounds, except for bisphenol S, showed AR antagonistic activities, with bisphenol E being the most potent. Weak GR antagonistic activities were also found in BPA and five its analogues. PXR agonistic activity was observed in the six compounds, with bisphenol Z being the most potent. Results of the CAR assay revealed that BPA and five its analogues acted as CAR inverse agonists. Taken together, these results suggested that BPA analogues demonstrate multiple effects via human nuclear receptors in a similar manner to BPA, and several analogues might have more potent endocrine-disrupting activity than does BPA.
Precise prediction of activators for the human constitutive androstane receptor using structure-based three-dimensional quantitative structure–activity relationship methods
2017, Drug Metabolism and Pharmacokinetics
Citation Excerpt :
Thus, consideration of the CAR activating potency of novel compounds is important in the early drug-discovery process. A wide variety of compounds, including clinically used drugs, steroids, natural compounds, pesticides, industrial chemicals, and various synthetic compounds, are known to be activators of CAR [10–14]. In vivo and in primary hepatic cultures, CAR normally resides in the cytoplasmic compartment; translocation to the nucleus occurs after exposure to chemical activators or ligands such as 6-(4-chlorophenyl) imidazo [2,1-b] [1,3] thiazole-5-carbaldehyde O-(3,4-dichloro-ben-zyl) oxime (CITCO).
The constitutive androstane receptor (CAR, NR1I3) regulates the expression of numerous drug-metabolizing enzymes and transporters. The upregulation of various enzymes, including CYP2B6, by CAR activators is a critical problem leading to clinically severe drug–drug interactions (DDIs). To date, however, few effective computational approaches for identifying CAR activators exist. In this study, we aimed to develop three-dimensional quantitative structure–activity relationship (3D-QSAR) models to predict the CAR activating potency of compounds emerging in the drug-discovery process. Models were constructed using comparative molecular field analysis (CoMFA) based on the molecular alignments of ligands binding to CAR, which were obtained from ensemble ligand-docking using 28 compounds as a training set. The CoMFA model, modified by adding a lipophilic parameter with calculated logD_7.4 (S+logD_7.4), demonstrated statistically good predictive ability (r² = 0.99, q² = 0.74). We also confirmed the excellent predictability of the 3D-QSAR model for CAR activation (r²_pred = 0.71) using seven compounds as a test set for external validation. Collectively, our results indicate that the 3D-QSAR model developed in this study provides precise prediction of CAR activating potency and, thus, should be useful for selecting drug candidates with minimized DDI risk related to enzyme-induction in the early drug-discovery stage.
Development of pharmacophore-based classification model for activators of constitutive androstane receptor
2017, Drug Metabolism and Pharmacokinetics
Citation Excerpt :
The overall modeling procedure is depicted in Fig. 1. A set of 548 compounds used in this study were collected from previous studies [17,18,22–55]. The compounds in the data set were classified in several ways as shown in Fig. 2.
Constitutive androstane receptor (CAR) is predominantly expressed in the liver and is important for regulating drug metabolism and transport. Despite its biological importance, there have been few attempts to develop in silico models to predict the activity of CAR modulated by chemical compounds. The number of in silico studies of CAR may be limited because of CAR's constitutive activity under normal conditions, which makes it difficult to elucidate the key structural features of the interaction between CAR and its ligands. In this study, to address these limitations, we introduced 3D pharmacophore-based descriptors with an integrated ligand and structure-based pharmacophore features, which represent the receptor-ligand interaction. Machine learning methods (support vector machine and artificial neural network) were applied to develop an in silico model with the descriptors containing significant information regarding the ligand binding positions. The best classification model built with a solvent accessibility volume-based filter and the support vector machine showed good predictabilities of 87%, and 85.4% for the training set and validation set, respectively. This demonstrates that our model can be used to accurately predict CAR activators and offers structural information regarding ligand/protein interactions.

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: This study was supported in part by a Grant-in-Aid from the Ministry of Education, Culture, Sports, Sciences and Technology of Japan [22390027, 24659061] and a grant from the Japan Chemical Industry Association (JCIA) Long-range Research Initiative (LRI).

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Regular ArticleNovel Cell-based Reporter Assay System Using Epitope-tagged Protein for the Identification of Agonistic Ligands of Constitutive Androstane Receptor (CAR)

Summary:

J. Biol. Chem.

Arch. Biochem. Biophys.

Drug Metab. Pharmacokinet.

J. Biol. Chem.

FEBS Lett.

J. Pharmacol. Toxicol. Methods

Chem. Biol. Interact.

Chem. Biol. Interact.

Biochem. Pharmacol.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

The nuclear orphan receptor CAR-retinoid X receptor heterodimer activates the phenobarbital-responsive enhancer module of the CYP2B gene

Mol. Cell. Biol.

CAR, driving into the future

Mol Endocrinol.

Regulation of drug-metabolizing enzymes by xenobiotic receptors: PXR and CAR

Adv. Drug Deliv. Rev.

Regular Article
Novel Cell-based Reporter Assay System Using Epitope-tagged Protein for the Identification of Agonistic Ligands of Constitutive Androstane Receptor (CAR)