Quantitative High-Throughput Luciferase Screening in Identifying CAR Modulators

  • Caitlin Lynch
  • Jinghua Zhao
  • Hongbing Wang
  • Menghang XiaEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1473)


The constitutive androstane receptor (CAR, NR1I3) is responsible for the transcription of multiple drug metabolizing enzymes and transporters. There are two possible methods of activation for CAR, direct ligand binding and a ligand-independent method, which makes this a unique nuclear receptor. Both of these mechanisms require translocation of CAR from the cytoplasm into the nucleus. Interestingly, CAR is constitutively active in immortalized cell lines due to the basal nuclear location of this receptor. This creates an important challenge in most in vitro assay models because immortalized cells cannot be used without inhibiting the high basal activity. In this book chapter, we go into detail of how to perform quantitative high-throughput screens to identify hCAR1 modulators through the employment of a double stable cell line. Using this line, we are able to identify activators, as well as deactivators, of the challenging nuclear receptor, CAR.

Key words

Constitutive Androstane Receptor (CAR) Cytochrome P450 2B6 (CYP2B6) Luciferase Quantitative high-throughput screening (qHTS) 


  1. 1.
    Honkakoski P, Zelko I, Sueyoshi T, Negishi M (1998) The nuclear orphan receptor CAR-retinoid X receptor heterodimer activates the phenobarbital-responsive enhancer module of the CYP2B gene. Mol Cell Biol 18(10):5652–5658CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Wang H, LeCluyse E (2003) Role of orphan nuclear receptors in the regulation of drug-metabolising enzymes. Clin Pharmacokinet 42(15):1331–1357. doi: 10.2165/00003088-200342150-00003 CrossRefPubMedGoogle Scholar
  3. 3.
    Lynch C, Pan Y, Li L, Heyward S, Moeller T, Swaan PW, Wang H (2014) Activation of the constitutive androstane receptor inhibits gluconeogenesis without affecting lipogenesis or fatty acid synthesis in human hepatocytes. Toxicol Appl Pharmacol 279(1):33–42, doi: Google Scholar
  4. 4.
    Dong B, Saha PK, Huang W, Chen W, Abu-Elheiga LA, Wakil SJ, Stevens RD, Ilkayeva O, Newgard CB, Chan L, Moore DD (2009) Activation of nuclear receptor CAR ameliorates diabetes and fatty liver disease. 106. doi:10.1073/pnas.0909731106Google Scholar
  5. 5.
    Chen T, Tompkins LM, Li L, Li H, Kim G, Zheng Y, Wang H (2010) 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 332(1):106–115CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Li H, Chen T, Cottrell J, Wang H (2009) 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. Drug Metab Dispos 37(5):1098–1106. doi: 10.1124/dmd.108.026005 Google Scholar
  7. 7.
    Zelko I, Sueyoshi T, Kawamoto T, Moore R, Negishi M (2001) The peptide near the C terminus regulates receptor CAR nuclear translocation induced by xenochemicals in mouse liver. Mol Cell Biol 21(8):2838–2846. doi: 10.1128/mcb.21.8.2838-2846.2001 CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Huang W, Zhang J, Wei P, Schrader WT, Moore DD (2004) Meclizine is an agonist ligand for mouse Constitutive Androstane Receptor (CAR) and an inverse agonist for human CAR. Mol Endocrinol 18(10):2402–2408. doi: 10.1210/me.2004-0046 CrossRefPubMedGoogle Scholar
  9. 9.
    Li L, Chen T, Stanton JD, Sueyoshi T, Negishi M, Wang H (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(2):443–453. doi:10.1124/mol.108.046656Google Scholar
  10. 10.
    Moore LB, Parks DJ, Jones SA, Bledsoe RK, Consler TG, Stimmel JB, Goodwin B, Liddle C, Blanchard SG, Willson TM, Collins JL, Kliewer SA (2000) Orphan nuclear receptors constitutive androstane receptor and pregnane X receptor share xenobiotic and steroid ligands. J Biol Chem 275(20):15122–15127. doi: 10.1074/jbc.M001215200 CrossRefPubMedGoogle Scholar
  11. 11.
    Lau AJ, Yang G, Rajaraman G, Baucom CC, Chang TKH (2011) Differential effect of meclizine on the activity of human pregnane X receptor and constitutive androstane receptor. J Pharmacol Exp Ther 336(3):816–826. doi: 10.1124/jpet.110.175927 CrossRefPubMedGoogle Scholar
  12. 12.
    Mäkinen J, Frank C, Jyrkkärinne J, Gynther J, Carlberg C, Honkakoski P (2002) Modulation of mouse and human phenobarbital-responsive enhancer module by nuclear receptors. Mol Pharmacol 62(2):366–378. doi: 10.1124/mol.62.2.366 CrossRefPubMedGoogle Scholar
  13. 13.
    Toell A, Kröncke K-D, Kleinert H, Carlberg C (2002) Orphan nuclear receptor binding site in the human inducible nitric oxide synthase promoter mediates responsiveness to steroid and xenobiotic ligands. J Cell Biochem 85(1):72–82. doi: 10.1002/jcb.10104 CrossRefPubMedGoogle Scholar
  14. 14.
    Freitas J, Miller N, Mengeling BJ, Xia M, Huang R, Houck K, Rietjens IMCM, Furlow JD, Murk AJ (2014) Identification of thyroid hormone receptor active compounds using a quantitative high-throughput screening platform. Curr Chem Genomics Transl Med 8:36–46. doi: 10.2174/2213988501408010036 CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Shukla SJ, Sakamuru S, Huang R, Moeller TA, Shinn P, VanLeer D, Auld DS, Austin CP, Xia M (2011) Identification of clinically used drugs that activate pregnane X receptors. Drug Metab Dispos 39(1):151–159. doi: 10.1124/dmd.110.035105 CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Lynch C, Zhao J, Huang R, Xiao J, Li L, Heyward S, Xia M, Wang H (2015) Quantitative High-Throughput Identification of Drugs as Modulators of Human Constitutive Androstane Receptor. Sci Rep 5. doi:10.1038/srep10405.

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Caitlin Lynch
    • 1
  • Jinghua Zhao
    • 1
  • Hongbing Wang
    • 2
  • Menghang Xia
    • 1
    Email author
  1. 1.National Center for Advancing Translational SciencesNational Institutes of HealthBethesdaUSA
  2. 2.Department of Pharmaceutical SciencesUniversity of Maryland School of PharmacyBaltimoreUSA

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