Archives of Toxicology

, Volume 92, Issue 4, pp 1435–1451 | Cite as

Identification of approved drugs as potent inhibitors of pregnane X receptor activation with differential receptor interaction profiles

  • Oliver Burk
  • Maria Kuzikov
  • Thales Kronenberger
  • Judith Jeske
  • Oliver Keminer
  • Wolfgang E. Thasler
  • Matthias Schwab
  • Carsten Wrenger
  • Björn Windshügel
Molecular Toxicology

Abstract

Activation of pregnane X receptor (PXR) results in the induction of first-pass metabolism and drug efflux. Hereby, PXR may cause adverse drug reactions or therapeutic failure of drugs. PXR inhibition is thus an attractive option to minimise adverse effects or to improve therapeutic efficiencies; however, only a limited number of antagonists have been identified so far. We performed a cell-based high-throughput screen to identify PXR antagonists, using a library of approved and investigational drugs. Two approved drugs, pimecrolimus and pazopanib, emerged as novel potent antagonists of PXR activation, with IC50 values of 1.2 and 4.1 µM, respectively. We further characterised these with respect to receptor specificity, assembly of the PXR ligand-binding domain (LBD) and interactions with co-factors. In vitro and in silico assays were carried out to identify the site(s) of interaction with the PXR LBD. Primary human hepatocytes were used to investigate antagonism of the induction of endogenous PXR target genes. Pimecrolimus and pazopanib did not affect the transcriptional activity of other nuclear receptors. Both induced the release of co-repressor from PXR and likewise interfered with agonist-induced recruitment of co-activator. Cumulative evidence from cellular and in vitro assays, as well as molecular docking, suggested additional or exclusive binding outside the PXR ligand-binding pocket for both. The compounds differentially antagonised the induction of PXR-regulated genes by rifampicin in primary human hepatocytes. In conclusion, we here have identified two approved drugs as novel potent PXR inhibitors with differential receptor interaction profiles and gene selectivity in primary human hepatocytes.

Keywords

Pregnane X Receptor Antagonist High-throughput screening Pimecrolimus Pazopanib 

Notes

Acknowledgements

We appreciate the expert technical assistance of K. Abuazi-Rincones. The non-profit 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. Primary human hepatocytes were kindly prepared by M. Demmel (Hepacult GmbH, München, Germany). This work was supported by the São Paulo state funding agency FAPESP (Grants 2014/03644-9, 2014/27313-1) and CNPq-DAAD Science Without Borders programme (T.K.), by the Robert Bosch Foundation, Stuttgart, Germany (O.B., M.S.), the Horizon 2020-PHC-2015 Grant U-PGx 668353 (M.S.) and by the Interfaculty Center for Pharmacogenomics and Pharma Research of the University of Tübingen, Germany (O.B., J.J.).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

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.

Human and animal rights statement

This article does not contain any studies with animals performed by any of the authors.

Informed consent

Informed consent was obtained from all individual participants included in the study.

Supplementary material

204_2018_2165_MOESM1_ESM.docx (1.2 mb)
Supplementary material 1 (DOCX 1185 KB)

References

  1. 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.  https://doi.org/10.1186/1478-1336-2-1 CrossRefPubMedPubMedCentralGoogle Scholar
  2. 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.  https://doi.org/10.1007/s00204-014-1348-x CrossRefPubMedGoogle Scholar
  3. Boulin M, Guiu S, Chauffert B et al (2011) Screening of anticancer drugs for chemoembolization of hepatocellular carcinoma. Anticancer Drugs 22:741–748.  https://doi.org/10.1097/CAD.0b013e328346a0c5 CrossRefPubMedGoogle Scholar
  4. Bros M, Ross X-L, Pautz A et al (2003) The human fascin gene promoter is highly active in mature dendritic cells due to a stage-specific enhancer. J Immunol 171:1825–1834.  https://doi.org/10.4049/jimmunol.171.4.1825 CrossRefPubMedGoogle Scholar
  5. Burk O, Tegude H, Koch I et al (2002) Molecular mechanisms of polymorphic CYP3A7 expression in adult human liver and intestine. J Biol Chem 277:2428–24288.  https://doi.org/10.1074/jbc.M202345200 CrossRefGoogle Scholar
  6. 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.  https://doi.org/10.1124/mol.104.009019 CrossRefPubMedGoogle Scholar
  7. Chai SC, Cherian MT, Wang Y-M, Chen T (2016) Small-molecule modulators of PXR and CAR. Biochim Biophys Acta 1859:1141–1154.  https://doi.org/10.1016/j.bbagrm.2016.02.013 CrossRefPubMedPubMedCentralGoogle Scholar
  8. Chen T (2010) Overcoming drug resistance by regulating nuclear receptors. Adv Drug Deliv Rev 62:1257–1264.  https://doi.org/10.1016/j.addr.2010.07.008 CrossRefPubMedPubMedCentralGoogle Scholar
  9. Chen Y, Tang Y, Robbins GT, Nie D (2010) Camptothecin attenuates cytochrome P450 3A4 induction by blocking the activation of human pregnane X receptor. J Pharmacol Exp Ther 334:999–1008.  https://doi.org/10.1124/jpet.110.168294 CrossRefPubMedPubMedCentralGoogle Scholar
  10. 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 83:1112–1226.  https://doi.org/10.1016/j.bcp.2012.01.030 CrossRefPubMedPubMedCentralGoogle Scholar
  11. Chrencik JE, Orans J, Moore LB et al (2005) Structural disorder in the complex of human pregnane X receptor and the macrolide antibiotic rifampicin. Mol Endocrinol 19:1125–1134.  https://doi.org/10.1210/me.2004-0346 CrossRefPubMedGoogle Scholar
  12. Das B, Madhukumar A, Anguiano J et al (2008) Synthesis of novel ketoconazole derivatives as inhibitors of the human pregnane X receptor (PXR; NR1I2; also termed SXR, PAR). Bioorg Med Chem Lett 15:3974–3977.  https://doi.org/10.1016/j.bmcl.2008.06.018 CrossRefGoogle Scholar
  13. Deng Y, Sychterz C, Suttle AB et al (2013) Bioavailability, metabolism and disposition of oral pazopanib in patients with advanced cancer. Xenobiotica 43:443–453.  https://doi.org/10.3109/00498254.2012.734642 CrossRefPubMedGoogle Scholar
  14. Ekins S, Kholodovych V, Ai N et al (2008) Computational discovery of novel low micromolar human pregnane X receptor antagonists. Mol Pharmacol 74:662–672.  https://doi.org/10.1124/mol.108.049437 CrossRefPubMedGoogle Scholar
  15. Elentner A, Ortner D, Clausen B et al (2015) Skin response to a carcinogen involve sthe xenobiotic receptor pregnane X receptor. Exp Dermatol 24:835–840.  https://doi.org/10.1111/exd.12766 CrossRefPubMedGoogle Scholar
  16. Geick A, Eichelbaum M, Burk O (2001) Nuclear receptor response elements mediate induction of intestinal MDR1 by rifampin. J Biol Chem 276:14581–14587.  https://doi.org/10.1074/jbc.M010173200 CrossRefPubMedGoogle Scholar
  17. Handschin C, Meyer UA (2003) Induction of drug metabolism: the role of nuclear receptors. Pharmacol Rev 55:649–673.  https://doi.org/10.1124/pr.55.4.2.649 CrossRefPubMedGoogle Scholar
  18. Healan-Greenberg C, Waring J, Kempf D, Blomme EA, Tirona RG, Kim RB (2008) A human immunodeficiency virus protease inhibitor is a novel functional inhibitor of human pregnane X receptor. Drug Metab Dispos 36:500–507.  https://doi.org/10.1124/dmd.107.019547 CrossRefPubMedGoogle Scholar
  19. 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.  https://doi.org/10.1111/j.1476-5381.2011.01665.x CrossRefPubMedPubMedCentralGoogle Scholar
  20. Huang H, Wang H, Sinz M et al (2007) Inhibition of drug metabolism by blocking the activation of nuclear receptors by ketoconazole. Oncogene 26:258–268.  https://doi.org/10.1038/sj.onc.1209788 CrossRefPubMedGoogle Scholar
  21. 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 29:1454–1459PubMedGoogle Scholar
  22. Iversen PW, Eastwood BJ, Sittampalam GS, Cox KL (2006) A comparison of assay performance measures in screening assays: signal window, Z′ factor, and assay variability ratio. J Biomol Screen 11:247–252.  https://doi.org/10.1177/1087057105285610 CrossRefPubMedGoogle Scholar
  23. Jeske J, Windshugel B, Thasler WE, Schwab M, Burk O (2017) Human pregnane X receptor is activated by dibenzazepine carbamate-based inhibitors of constitutive androstane receptor. Arch Toxicol.  https://doi.org/10.1007/s00204-017-1948-3 PubMedGoogle Scholar
  24. Kandel BA, Thomas M, Winter S et al (2016) Genomewide comparison of the inducible transcriptomes of nuclear receptors CAR, PXR and PPARα in primary human hepatocytes. Biochim Biophys Acta 1859:1218–1227.  https://doi.org/10.1016/j.bbagrm.2016.03.007 CrossRefPubMedGoogle Scholar
  25. Krausova L, Stejskalova L, Wang H et al (2011) Metformin suppresses pregnane X receptor (PXR)-regulated transactivation of CYP3A4 gene. Biochem Pharmacol 1:1771–1780.  https://doi.org/10.1016/j.bcp.2011.08.023 CrossRefGoogle Scholar
  26. Lee SM, Schelcher C, Demmel M et al (2013) Isolation of human hepatocytes by a two-step collagenase perfusion procedure. J Vis Exp.  https://doi.org/10.3791/50615 Google Scholar
  27. 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.  https://doi.org/10.1124/mol.106.033415 CrossRefPubMedGoogle Scholar
  28. Lim Y-P, Ma C-Y, Liu C-L et al (2012) Sesamin: a naturally occurring lignan inhibits CYP3A4 by antagonizing the pregnane X receptor activation. Evid Based Complement Alternat Med 2012:242810.  https://doi.org/10.1155/2012/242810 PubMedPubMedCentralGoogle Scholar
  29. Lin W, Wang YM, Chai SC et al (2017) SPA70 is a potent antagonist of human pregnane X receptor. Nat Commun 8:741.  https://doi.org/10.1038/s41467-017-00780-5 CrossRefPubMedPubMedCentralGoogle Scholar
  30. Liu C-L, Lim Y-P, Hu M-L (2012) Fucoxanthin attenuates rifampin-induced cytochrome P450 3A4 (CYP3A4) and multiple drug resistance 1 (MDR1) gene expression through pregnane X receptor (PXR)-mediated pathways in human hepatoma HepG2 and colon adenocarcinoma LS174T cells. Mar Drugs 10:242–257.  https://doi.org/10.3390/md10010242 CrossRefPubMedPubMedCentralGoogle Scholar
  31. Moore TW, Mayne CG, Katzenellenbogen JA (2010) Minireview: not picking pockets: nuclear receptor alternate-site modulators (NRAMs). Mol Endocrinol 24:683–695.  https://doi.org/10.1210/me.2009-0362 CrossRefPubMedGoogle Scholar
  32. 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.  https://doi.org/10.1128/AAC.04140-14 CrossRefPubMedGoogle Scholar
  33. 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 pii]CrossRefPubMedGoogle Scholar
  34. Ratajewski M, Grzelak I, Wiśniewska K et al (2015) Screening of a chemical library reveals novel PXR-activating pharmacologic compounds. Toxicol Lett 232:193–202.  https://doi.org/10.1016/j.toxlet.2014.10.009 CrossRefPubMedGoogle Scholar
  35. Riedmaier S, Klein K, Hofmann U et al (2010) UDP-glucuronosyltransferase (UGT) polymorphisms affect atorvastatin lactonization in vitro and in vivo. Clin Pharmacol Ther 87:65–73.  https://doi.org/10.1038/clpt.2009.181 CrossRefPubMedGoogle Scholar
  36. Sinz M, Kim S, Zhu Z et al (2006) Evaluation of 170 xenobiotics as transactivators of human pregnane X receptor (hPXR) and correlation to known CYP3A4 drug interactions. Curr Drug Metab 7:375–388CrossRefPubMedGoogle Scholar
  37. Synold TW, Dussault I, Forman BM (2001) The orphan nuclear receptor SXR coordinately regulates drug metabolism and efflux. Nat Med 7:584–590.  https://doi.org/10.1038/87912 CrossRefPubMedGoogle Scholar
  38. Tabb MM, Kholodovych V, Grün F et al (2004) Highly chlorinated PCBs inhibit the human xenobiotic response mediated by the steroid and xenobiotic receptor (SXR). Environ Health Perspect 112:163–169CrossRefPubMedPubMedCentralGoogle Scholar
  39. Takeshita A, Taguchi M, Koibuchi N, Ozawa Y (2002) Putative role of the orphan nuclear receptor SXR (steroid and xenobiotic receptor) in the mechanism of CYP3A4 inhibition by xenobiotics. J Biol Chem 277:32453–32458.  https://doi.org/10.1074/jbc.M111245200 CrossRefPubMedGoogle Scholar
  40. 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.  https://doi.org/10.1023/A:1026392429112 CrossRefPubMedGoogle Scholar
  41. Tice CM, Zheng Y-J (2016) Non-canonical modulators of nuclear receptors. Bioorg Med Chem Lett 26:2157–2164.  https://doi.org/10.1016/j.bmcl.2016.07.067 Google Scholar
  42. Tolson AH, Wang H (2010) Regulation of drug-metabolizing enzymes by xenobiotic receptors: PXR and CAR. Adv Drug Deliv Rev 62:1238–1249.  https://doi.org/10.1016/j.addr.2010.08.006 CrossRefPubMedPubMedCentralGoogle Scholar
  43. van Geel RM, Beijnen JH, Schellens JH (2012) Concise drug review: pazopanib and axitinib. Oncologist 17:1081–1089.  https://doi.org/10.1634/theoncologist.2012-0055 CrossRefPubMedPubMedCentralGoogle Scholar
  44. Venkatesh M, Wang H, Cayer J et al (2011) In vivo and in vitro characterization of a first-in-class novel azole analog that targets pregnane X receptor activation. Mol Pharmacol 80:124–135.  https://doi.org/10.1124/mol.111.071787 CrossRefPubMedPubMedCentralGoogle Scholar
  45. 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.  https://doi.org/10.1074/jbc.M212482200 CrossRefPubMedGoogle Scholar
  46. Wang H, Huang H, Li H et al (2007) Activated pregnenolone X-receptor is a target for ketoconazole and its analogs. Clin Cancer Res 13:2488–2495.  https://doi.org/10.1158/1078-0432.CCR-06-1592 CrossRefPubMedGoogle Scholar
  47. Wang H, Li H, Moore LB et al (2008) The phytoestrogen coumestrol is a naturally occurring antagonist of the human pregnane X receptor. Mol Endocrinol 22:838–857.  https://doi.org/10.1210/me.2007-0218 CrossRefPubMedPubMedCentralGoogle Scholar
  48. Wang H, Venkatesh M, Li H et al (2011) Pregnane X receptor activation induces FGF19-dependent tumor aggressiveness in humans and mice. J Clin Invest 121:3220–3232.  https://doi.org/10.1172/JCI41514 CrossRefPubMedPubMedCentralGoogle Scholar
  49. 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.  https://doi.org/10.1517/17425255.2012.685237 CrossRefPubMedPubMedCentralGoogle Scholar
  50. Zhang J, Chung TDY, Oldenburg KR (1999) A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J Biomol Screen 4:67–73.  https://doi.org/10.1177/108705719900400206 CrossRefPubMedGoogle Scholar
  51. Zhou C, Poulton E-J, Grün F et al (2007) The dietary isothiocyanate sulforaphane is an antagonist of the human steroid and xenobiotic nuclear receptor. Mol Pharmacol 71:220–229.  https://doi.org/10.1124/mol.106.029264 CrossRefPubMedGoogle Scholar
  52. Zhu Z, Kim S, Chen T et al (2004) Correlation of high-throughput pregnane X receptor (PXR) transactivation and binding assays. J Biomol Screen 9:533–540.  https://doi.org/10.1177/1087057104264902 CrossRefPubMedGoogle Scholar
  53. Zhuo W, Hu L, Lv J et al (2014) Role of pregnane X receptor in chemotherapeutic treatment. Cancer Chemother Pharmacol 74:217–227.  https://doi.org/10.1007/s00280-014-2494-9 CrossRefPubMedPubMedCentralGoogle Scholar
  54. Zollinger M, Waldmeier F, Hartmann S et al (2006) Pimecrolimus: absorption, distribution, metabolism, and excretion in healthy volunteers after a single oral dose and supplementary investigations in vitro. Drug Metab Dispos 34:765–774.  https://doi.org/10.1124/dmd.105.007732 CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Dr. Margarete Fischer-Bosch-Institute of Clinical PharmacologyStuttgartGermany
  2. 2.University of TübingenTübingenGermany
  3. 3.Fraunhofer Institute for Molecular Biology and Applied Ecology IMEHamburgGermany
  4. 4.Department of Parasitology, Institute of Biomedical SciencesUniversity of São PauloSão PauloBrazil
  5. 5.Department of General, Visceral, Transplantation, and Vascular SurgeryUniversity of MunichMunichGermany
  6. 6.Department of Clinical PharmacologyUniversity Hospital TübingenTübingenGermany
  7. 7.Department of Pharmacy and BiochemistryUniversity of TübingenTübingenGermany

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