Abstract
Unintentional activation of xenosensing nuclear receptors pregnane X receptor (PXR) and/or constitutive androstane receptor (CAR) by clinical drug use is known to produce severe side effects in patients, which may be overcome by co-administering antagonists. However, especially antagonizing CAR is hampered by the lack of specific inhibitors, which do not activate PXR. Recently, compounds based on a dibenzazepine carbamate scaffold were identified as potent CAR inhibitors. However, their potential to activate PXR was not thoroughly investigated, even if the lead compound was named “CAR inhibitor not PXR activator 1” (CINPA1). Thus, we performed a comprehensive analysis of the interaction of CINPA1 and four analogs with PXR. Cellular assays were used to investigate intra- and intermolecular interactions and transactivation activity of PXR as a function of the compounds. Modulation of PXR target gene expression was analyzed in primary human hepatocytes. Ligand binding to PXR was investigated by molecular docking and limited proteolytic digestion. We show here that CINPA1 induced the assembly of the PXR ligand-binding domain, released co-repressors from and recruited co-activators to the receptor. CINPA1 and its analogs induced the PXR-dependent activation of a CYP3A4 reporter gene and CINPA1 induced the expression of endogenous cytochrome P450 genes in primary hepatocytes, while not consistently inhibiting CAR-mediated induction. Molecular docking revealed favorable binding of CINPA1 and analogs to the PXR ligand-binding pocket, which was confirmed in vitro. Altogether, our data provide consistent evidence that compounds with a dibenzazepine carbamate scaffold, such as CINPA1 and its four analogs, bind to and activate PXR.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Arnold KA, Eichelbaum M, Burk O (2004) Alternative splicing affects the function and tissue-specific expression of the human constitutive androstane receptor. Nucl Recept 2:1
Bitter A, Rümmele P, Klein K et al (2015) Pregnane X receptor activation and silencing promote steatosis of human hepatic cells by distinct lipogenic mechanisms. Arch Toxicol 89:2089–2103
Burk O, Tegude H, Koch I et al (2002) Molecular mechanisms of polymorphic CYP3A7 expression in adult human liver and intestine. J Biol Chem 277:24280–24288
Burk O, Arnold KA, Nussler AK et al (2005) Antimalarial artemisinin drugs induce cytochrome P450 and MDR1 expression by activation of xenosensors pregnane X receptor and constitutive androstane receptor. Mol Pharmacol 67:1954–1965
Chai X, Zeng S, Xie W (2013) Nuclear receptors PXR and CAR: implications for drug metabolism regulation, pharmacogenomics and beyond. Expert Opin Drug Metab Toxicol 9:253–266
Chen T (2010) Overcoming drug resistance by regulating nuclear receptors. Adv Drug Deliv Rev 62:1257–1264
Chen Y, Tang Y, Guo C et al (2012) Nuclear receptors in the multidrug resistance through the regulation of drug-metabolizing enzymes and drug transporters. Biochem Pharmacol 83:1112–1126
Cherian MT, Lin W, Wu J, Chen T (2015) CINPA1 is an inhibitor of constitutive androstane receptor that does not activate pregnane X receptor. Mol Pharmacol 87:878–889
Cherian MT, Yang L, Chai SC et al (2016) Identification and characterization of CINPA1 metabolites facilitates structure-activity studies of the constitutive androstane receptor. Drug Metab Dispos 44:1759–1770
Emami Riedmaier A, Burk O, van Eijck BA et al (2015) Variability in hepatic expression of organic anion transporter 7/SLC22A9, a novel pravastatin uptake transporter: impact of genetic and regulatory factors. Pharmacogenomics J 16:341–351
Fujimura T, Sakuma H, Konishi S et al (2005) FK614, a novel peroxisome proliferator-activated receptor gamma modulator, induces differential transactivation through a unique ligand-specific interaction with transcriptional coactivators. J Pharmacol Sci 99:342–352
Gao J, Xie W (2012) Targeting xenobiotic receptors PXR and CAR for metabolic diseases. Trends Pharmacol Sci 33:552–558
Geick A, Eichelbaum M, Burk O (2001) Nuclear receptor response elements mediate induction of intestinal MDR1 by rifampin. J Biol Chem 276:14581–14587
Hoffart E, Ghebreghiorghis L, Nussler AK et al (2012) Effects of atorvastatin metabolites on induction of drug-metabolizing enzymes and membrane transporters through human pregnane X receptor. Br J Pharmacol 165:1595–1608
Hukkanen J, Hakkola J, Rysä J (2014) Pregnane X receptor (PXR) - a contributor to the diabetes epidemic? Drug Metabol Drug Interact 29:3–15
Hustert E, Zibat A, Presecan-Siedel E et al (2001) Natural protein variants of pregnane X receptor with altered transactivation activity toward CYP3A4. Drug Metab Dispos 29:1454–1459
Ihunnah CA, Jiang M, Xie W (2011) Nuclear receptor PXR, transcriptional circuits and metabolic relevance. Biochim Biophys Acta 1812:956–963
Jones SA, Moore LB, Shenk JL et al (2000) The pregnane X receptor: a promiscuous xenobiotic receptor that has diverged during evolution. Mol Endocrinol 14:27–39
Kakizaki S, Takizawa D, Tojima H et al (2009) Xenobiotic-sensing nuclear receptors CAR and PXR as drug targets in cholestatic liver disease. Curr Drug Targets 10:1156–1163
Küblbeck J, Jyrkkärinne J, Molnár F et al (2011) New in vitro tools to study human constitutive androstane receptor (CAR) biology: discovery and comparison of human CAR inverse agonists. Mol Pharm 8:2424–2433
Lee SM, Schelcher C, Demmel M et al (2013) Isolation of human hepatocytes by a two-step collagenase perfusion procedure. J Vis Exp. doi:10.3791/50615
Lemaire G, Benod C, Nahoum V et al (2007) Discovery of a highly active ligand of human pregnane x receptor: a case study from pharmacophore modeling and virtual screening to “in vivo” biological activity. Mol Pharmacol 72:572–581
Li L, Chen T, Stanton JD et al (2008) The peripheral benzodiazepine receptor ligand 1-(2-chlorophenyl-methylpropyl)-3-isoquinoline-carboxamide is a novel antagonist of human constitutive androstane receptor. Mol Pharmacol 74:443–453
Lim YP, Cheng CH, Chen WC et al (2015) Allyl isothiocyanate (AITC) inhibits pregnane X receptor (PXR) and constitutive androstane receptor (CAR) activation and protects against acetaminophen- and amiodarone-induced cytotoxicity. Arch Toxicol 89:57–72
Lin JH (2006) CYP induction-mediated drug interactions: in vitro assessment and clinical implications. Pharm Res 23:1089–1116
Lin W, Wu J, Dong H et al (2008) Cyclin-dependent kinase 2 negatively regulates human pregnane X receptor-mediated CYP3A4 gene expression in HepG2 liver carcinoma cells. J Biol Chem 283:30650–30657
Lin W, Yang L, Chai SC et al (2016) Development of CINPA1 analogs as novel and potent inverse agonists of constitutive androstane receptor. Eur J Med Chem 108:505–528
Maglich JM, Parks DJ, Moore LB et al (2003) Identification of a novel human constitutive androstane receptor (CAR) agonist and its use in the identification of CAR target genes. J Biol Chem 278:17277–17283
Mathäs M, Burk O, Qiu H et al (2012) Evolutionary history and functional characterization of the amphibian xenosensor CAR. Mol Endocrinol 26:14–26
Moore LB, Parks DJ, Jones SA et al (2000) Orphan nuclear receptors constitutive androstane receptor and pregnane X receptor share xenobiotic and steroid ligands. J Biol Chem 275:15122–15127
Piedade R, Traub S, Bitter A et al (2015) Carboxymefloquine, the major metabolite of the antimalarial drug mefloquine, induces drug-metabolizing enzyme and transporter expression by activation of pregnane X receptor. Antimicrob Agents Chemother 59:96–104
Pissios P, Tzameli I, Kushner P, Moore DD (2000) Dynamic stabilization of nuclear receptor ligand binding domains by hormone or corepressor binding. Mol Cell 6:245–253
Thasler WE, Weiss TS, Schillhorn K et al (2003) Charitable state-controlled foundation human tissue and cell research: ethic and legal aspects in the supply of surgically removed human tissue for research in the academic and commercial sector in Germany. Cell Tissue Bank 4:49–56
Tolson AH, Wang H (2010) Regulation of drug-metabolizing enzymes by xenobiotic receptors: PXR and CAR. Adv Drug Deliv Rev 62:1238–1249
Wallace BD, Redinbo MR (2013) Xenobiotic-sensing nuclear receptors involved in drug metabolism: a structural perspective. Drug Metab Rev 45:79–100
Wang H, Faucette S, Sueyoshi T et al (2003) A novel distal enhancer module regulated by pregnane X receptor/constitutive androstane receptor is essential for the maximal induction of CYP2B6 gene expression. J Biol Chem 278:14146–14152
Wang YM, Ong SS, Chai SC, Chen T (2012) Role of CAR and PXR in xenobiotic sensing and metabolism. Expert Opin Drug Metab Toxicol 8:803–817
Yan J, Chen B, Lu J et al (2015) Deciphering the roles of the constitutive androstane receptor in energy metabolism. Acta Pharmacol Sin 36:62–70
Acknowledgements
We appreciate the expert technical assistance of K. Abuazi-Rincones. The nonprofit foundation Human Tissue and Cell Research (Regensburg, Germany), which holds human tissue on trust, making it broadly available for research on an ethical and legal basis, kindly provided liver tissue for the preparation of human primary hepatocytes. This work was supported by the Robert Bosch Foundation, Stuttgart, Germany, and by the Interfaculty Center for Pharmacogenomics and Pharma Research of the University of Tübingen, Germany.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Ethical standards
All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.
This article does not contain any studies with animals performed by any of the authors.
Informed consent was obtained from all individual participants included in the study.
Conflict of interest
The authors declare that they have no conflict of interest.
Additional information
J. Jeske and B. Windshügel contributed equally to this work.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Jeske, J., Windshügel, B., Thasler, W.E. et al. Human pregnane X receptor is activated by dibenzazepine carbamate-based inhibitors of constitutive androstane receptor. Arch Toxicol 91, 2375–2390 (2017). https://doi.org/10.1007/s00204-017-1948-3
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00204-017-1948-3