Pharmaceutical Research

, Volume 30, Issue 2, pp 489–501 | Cite as

Identification of Novel Activators of Constitutive Androstane Receptor from FDA-Approved Drugs by Integrated Computational and Biological Approaches

  • Caitlin Lynch
  • Yongmei Pan
  • Linhao Li
  • Stephen S. Ferguson
  • Menghang Xia
  • Peter W. Swaan
  • Hongbing WangEmail author
Research Paper



The constitutive androstane receptor (CAR, NR1I3) is a xenobiotic sensor governing the transcription of numerous hepatic genes associated with drug metabolism and clearance. Recent evidence suggests that CAR also modulates energy homeostasis and cancer development. Thus, identification of novel human (h) CAR activators is of both clinical importance and scientific interest.


Docking and ligand-based structure-activity models were used for virtual screening of a database containing over 2000 FDA-approved drugs. Identified lead compounds were evaluated in cell-based reporter assays to determine hCAR activation. Potential activators were further tested in human primary hepatocytes (HPHs) for the expression of the prototypical hCAR target gene CYP2B6.


Nineteen lead compounds with optimal modeling parameters were selected for biological evaluation. Seven of the 19 leads exhibited moderate to potent activation of hCAR. Five out of the seven compounds translocated hCAR from the cytoplasm to the nucleus of HPHs in a concentration-dependent manner. These compounds also induce the expression of CYP2B6 in HPHs with rank-order of efficacies closely resembling that of hCAR activation.


These results indicate that our strategically integrated approaches are effective in the identification of novel hCAR modulators, which may function as valuable research tools or potential therapeutic molecules.


CAR CYP2B6 hepatocytes induction pharmacophore 



adenovirus expressing enhanced yellow fluorescent protein-tagged human CAR






constitutive androstane receptor


6-(4-chlorophenyl) imidazo[2,1-b][1,3]-thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl)oxime


cytochrome P450


dimethyl sulfoxide


glyceraldehyde-3-phosphate dehydrogenase


human primary hepatocytes


ligand-binding domain










pregnane X receptor


reverse transcription-polymerase chain reaction






Acknowledgments and Disclosures

The authors thank Dr. James Polli (The University of Maryland School of Pharmacy) for kindly offering multiple compounds and Dr. Alex MacKerell (The University of Maryland School of Pharmacy) for making the Discovery Studio available for this study. We also thank Dr. Sean Ekins (Collaborations in Chemistry, Jenkintown, PA) for offering initial CDD database and Dr. Taiji Oashi, a previous lab member from Dr. MacKerell’s lab for database optimization. The authors appreciatively acknowledge The University of Maryland Medical Center and Life Technologies (Durham, NC) for providing the human hepatocytes used in this study. The research is supported in part by the National Institutes of Health Grants DK061652 (H.W) and DK061425 (P.S).

Supplementary material

11095_2012_895_MOESM1_ESM.docx (20 kb)
ESM 1 (DOCX 19 kb)
11095_2012_895_Fig7_ESM.jpg (116 kb)
Figure S1

Chemical structures of training compounds. Seventeen known human CAR ligands extracted from literature were used as training compounds to generate the common features of pharmacophore model in this study. The chemical structures of these compounds were drawn using ChemDraw Ultra 10. (JPEG 115 kb)

11095_2012_895_MOESM2_ESM.tif (5.2 mb)
High Resolution Image (TIFF 5344 kb)
11095_2012_895_Fig8_ESM.jpg (152 kb)
Figure S2

Chemical structures of tested lead compounds. Nineteen lead compounds with optimal pharmacophore parameters from virtual screening of the FDA-approved drug data base were selected for biological assessment of CAR activation. The chemical structures of these compounds were drawn using ChemDraw Ultra 10. (JPEG 151 kb)

11095_2012_895_MOESM3_ESM.tif (6.4 mb)
High Resolution Image (TIFF 6602 kb)


  1. 1.
    Honkakoski P, Sueyoshi T, Negishi M. Drug-activated nuclear receptors CAR and PXR. Ann Med. 2003;35:172–82.PubMedCrossRefGoogle Scholar
  2. 2.
    Qatananiand M, Moore DD. CAR, the continuously advancing receptor, in drug metabolism and disease. Curr Drug Metab. 2005;6:329–39.CrossRefGoogle Scholar
  3. 3.
    Yap KY, Chui WK, Chan A. Drug interactions between chemotherapeutic regimens and antiepileptics. Clin Ther. 2008;30:1385–407.PubMedCrossRefGoogle Scholar
  4. 4.
    Maglich JM, Watson J, McMillen PJ, Goodwin B, Willson TM, Moore JT. The nuclear receptor CAR is a regulator of thyroid hormone metabolism during caloric restriction. J Biol Chem. 2004;279:19832–8.PubMedCrossRefGoogle Scholar
  5. 5.
    Yamamoto Y, Moore R, Goldsworthy TL, Negishi M, Maronpot RR. The orphan nuclear receptor constitutive active/androstane receptor is essential for liver tumor promotion by phenobarbital in mice. Cancer Res. 2004;64:7197–200.PubMedCrossRefGoogle Scholar
  6. 6.
    Gao J, He J, Zhai Y, Wada T, Xie W. The constitutive androstane receptor is an anti-obesity nuclear receptor that improves insulin sensitivity. J Biol Chem. 2009;284:25984–92.PubMedCrossRefGoogle Scholar
  7. 7.
    Kodama S, Koike C, Negishi M, Yamamoto Y. Nuclear Receptors CAR and PXR Cross Talk with FOXO1 To Regulate Genes That Encode Drug-Metabolizing and Gluconeogenic Enzymes. 2004, 24:7931–7940.Google Scholar
  8. 8.
    Huang W, Zhang J, Washington M, Liu J, Parant JM, Lozano G, et al. Xenobiotic stress induces hepatomegaly and liver tumors via the nuclear receptor constitutive androstane receptor. Mol Endocrinol. 2005;19:1646–53.PubMedCrossRefGoogle Scholar
  9. 9.
    Phillips JM, Burgoon LD, Goodman JI. Phenobarbital elicits unique, early changes in the expression of hepatic genes that affect critical pathways in tumor-prone B6C3F1 mice. Toxicol Sci. 2009;109:193–205.PubMedCrossRefGoogle Scholar
  10. 10.
    Kliewer SA. The nuclear pregnane X receptor regulates xenobiotic detoxification. J Nutr. 2003;133:2444S–7S.PubMedGoogle Scholar
  11. 11.
    Kawamoto T, Sueyoshi T, Zelko I, Moore R, Washburn K, Negishi M. Phenobarbital-responsive nuclear translocation of the receptor CAR in induction of the CYP2B gene. Mol Cell Biol. 1999;19:6318–22.PubMedGoogle Scholar
  12. 12.
    Omiecinski CJ, Coslo DM, Chen T, Laurenzana EM, Peffer RC. Multi-species Analyses of Direct Activators of the Constitutive Androstane Receptor. Toxicol Sci. 2011.Google Scholar
  13. 13.
    Auerbach SS, Stoner MA, Su S, Omiecinski CJ. Retinoid X receptor-alpha-dependent transactivation by a naturally occurring structural variant of human constitutive androstane receptor (NR1I3). Mol Pharmacol. 2005;68:1239–53.PubMedCrossRefGoogle Scholar
  14. 14.
    Chen T, Tompkins LM, Li L, Li H, Kim G, Zheng Y, et al. A single amino acid controls the functional switch of human constitutive androstane receptor (CAR) 1 to the xenobiotic-sensitive splicing variant CAR3. J Pharmacol Exp Ther. 2010;332:106–15.PubMedCrossRefGoogle Scholar
  15. 15.
    Jyrkkarinne J, Windshugel B, Ronkko T, Tervo AJ, Kublbeck J, Lahtela-Kakkonen M, et al. Insights into ligand-elicited activation of human constitutive androstane receptor based on novel agonists and three-dimensional quantitative structure-activity relationship. J Med Chem. 2008;51:7181–92.PubMedCrossRefGoogle Scholar
  16. 16.
    Pan Y, Li L, Kim G, Ekins S, Wang H, Swaan PW. Identification and Validation of Novel Human Pregnane X Receptor Activators among Prescribed Drugs via Ligand-Based Virtual Screening. 2011, 39:337–344.Google Scholar
  17. 17.
    Ekins S, Chang C, Mani S, Krasowski MD, Reschly EJ, Iyer M, et al. Human pregnane X receptor antagonists and agonists define molecular requirements for different binding sites. Mol Pharmacol. 2007;72:592–603.PubMedCrossRefGoogle Scholar
  18. 18.
    Xu RX, Lambert MH, Wisely BB, Warren EN, Weinert EE, Waitt GM, et al. A structural basis for constitutive activity in the human CAR/RXRalpha heterodimer. Mol Cell. 2004;16:919–28.PubMedCrossRefGoogle Scholar
  19. 19.
    Li L, Chen T, Stanton JD, Sueyoshi T, Negishi M, Wang H. The Peripheral Benzodiazepine Receptor Ligand 1-(2-Chlorophenyl-methylpropyl)-3-isoquinoline-carboxamide Is a Novel Antagonist of Human Constitutive Androstane Receptor. 2008, 74:443–453.Google Scholar
  20. 20.
    Maglich JM, Parks DJ, Moore LB, Collins JL, Goodwin B, Billin AN, et al. Identification of a novel human constitutive androstane receptor (CAR) agonist and its use in the identification of CAR target genes. J Biol Chem. 2003;278:17277–83.PubMedCrossRefGoogle Scholar
  21. 21.
    Kirchmair J, Laggner C, Wolber G, Langer T. Comparative analysis of protein-bound ligand conformations with respect to catalyst’s conformational space subsampling algorithms. J Chem Inf Model. 2005;45:422–30.PubMedCrossRefGoogle Scholar
  22. 22.
    Hohman M, Gregory K, Chibale K, Smith PJ, Ekins S, Bunin B. Novel web-based tools combining chemistry informatics, biology and social networks for drug discovery. Drug Discov Today. 2009;14:261–70.PubMedCrossRefGoogle Scholar
  23. 23.
    Ekinsand S, Williams AJ. Finding promiscuous old drugs for new uses. Pharm Res. 2011;28:1785–91.CrossRefGoogle Scholar
  24. 24.
    Ekins S, Williams AJ, Krasowski MD, Freundlich JS. In silico repositioning of approved drugs for rare and neglected diseases. Drug Discov Today. 2011;16:298–310.PubMedCrossRefGoogle Scholar
  25. 25.
    Chambers CC, Hawkins GD, Cramer CJ, Truhlar DG. Model for aqueous solvation based on class IV atomic charges and first solvation shell effects. J Phys Chem. 1996;100:16385–98.CrossRefGoogle Scholar
  26. 26.
    Li JB, Zhu TH, Cramer CJ, Truhlar DG. New class IV charge model for extracting accurate partial charges from wave functions. J Phys Chem A. 1998;102:1820–31.CrossRefGoogle Scholar
  27. 27.
    Kaminskiand G, Jorgensen WL. Performance of the AMBER94, MMFF94, and OPLS-AA force fields for modeling organic liquids. J Phys Chem. 1996;100:18010–3.CrossRefGoogle Scholar
  28. 28.
    Chang C, Bahadduri PM, Polli JE, Swaan PW, Ekins S. Rapid identification of P-glycoprotein substrates and inhibitors. Drug Metab Dispos. 2006;34:1976–84.PubMedCrossRefGoogle Scholar
  29. 29.
    Ruppert J, Welch W, Jain AN. Automatic identification and representation of protein binding sites for molecular docking. Protein Sci. 1997;6:524–33.PubMedCrossRefGoogle Scholar
  30. 30.
    LeCluyse EL, Alexandre E, Hamilton GA, Viollon-Abadie C, Coon DJ, Jolley S, et al. Isolation and culture of primary human hepatocytes. Methods Mol Biol. 2005;290:207–29.PubMedGoogle Scholar
  31. 31.
    Wang H, Faucette S, Sueyoshi T, Moore R, Ferguson S, Negishi M, et al. 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. 2003;278:14146–52.PubMedCrossRefGoogle Scholar
  32. 32.
    Chu V, Einolf HJ, Evers R, Kumar G, Moore D, Ripp S, et al. In vitro and in vivo induction of cytochrome p450: a survey of the current practices and recommendations: a pharmaceutical research and manufacturers of america perspective. Drug Metab Dispos. 2009;37:1339–54.PubMedCrossRefGoogle Scholar
  33. 33.
    FDA. Drug Interaction Studies-Study Design, Data Analysis, and Applications for Dosing and Labeling. FDA Guidance (2006).Google Scholar
  34. 34.
    Liand H, Wang H. Activation of xenobiotic receptors: driving into the nucleus. 2010, 6:409–426.Google Scholar
  35. 35.
    Li H, Chen T, Cottrell J, Wang H. Nuclear Translocation of Adenoviral-Enhanced Yellow Fluorescent Protein-Tagged-Human Constitutive Androstane Receptor (hCAR): A Novel Tool for Screening hCAR Activators in Human Primary Hepatocytes. 2009, 37:1098–1106.Google Scholar
  36. 36.
    Watkins RE, Wisely GB, Moore LB, Collins JL, Lambert MH, Williams SP, et al. The human nuclear xenobiotic receptor PXR: structural determinants of directed promiscuity. Science. 2001;292:2329–33.PubMedCrossRefGoogle Scholar
  37. 37.
    Dong B, Saha PK, Huang W, Chen W, Abu-Elheiga LA, Wakil SJ, et al. Activation of nuclear receptor CAR ameliorates diabetes and fatty liver disease. 2009, 106:18831–18836.Google Scholar
  38. 38.
    Kublbeck J, Laitinen T, Jyrkkarinne J, Rousu T, Tolonen A, Abel T, et al. Use of comprehensive screening methods to detect selective human CAR activators. Biochemical pharmacology. 2011.Google Scholar
  39. 39.
    DeKeyser JG, Stagliano MC, Auerbach SS, Prabhu KS, Jones AD, Omiecinski CJ. Di(2-ethylhexyl) phthalate is a highly potent agonist for the human constitutive androstane receptor splice variant CAR2. Mol Pharmacol. 2009;75:1005–13.PubMedCrossRefGoogle Scholar
  40. 40.
    Sinz M, Kim S, Zhu Z, Chen T, Anthony M, Dickinson K, et al. Evaluation of 170 xenobiotics as transactivators of human pregnane X receptor (hPXR) and correlation to known CYP3A4 drug interactions. Curr Drug Metab. 2006;7:375–88.PubMedCrossRefGoogle Scholar
  41. 41.
    Faucette SR, Sueyoshi T, Smith CM, Negishi M, Lecluyse EL, Wang H. Differential regulation of hepatic CYP2B6 and CYP3A4 genes by constitutive androstane receptor but not pregnane X receptor. J Pharmacol Exp Ther. 2006;317:1200–9.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2012

Authors and Affiliations

  • Caitlin Lynch
    • 1
  • Yongmei Pan
    • 1
  • Linhao Li
    • 1
  • Stephen S. Ferguson
    • 2
  • Menghang Xia
    • 3
  • Peter W. Swaan
    • 1
  • Hongbing Wang
    • 1
    Email author
  1. 1.Department of Pharmaceutical SciencesUniversity of Maryland School of PharmacyBaltimoreUSA
  2. 2.Life Technologies CorporationsDurhamUSA
  3. 3.NIH Chemical Genomics CenterNational Institutes of HealthBethesdaUSA

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