Transactivation and Coactivator Recruitment Assays for Measuring Farnesoid X Receptor Activity

  • Chia-Wen (Amy) Hsu
  • Jinghua Zhao
  • Menghang XiaEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1473)


The farnesoid X receptor (FXR) is a nuclear receptor responsible for homeostasis of bile acids, lipids, and glucose. Compounds that alter endogenous FXR signaling can be used as therapeutic candidates or identified as potentially hazardous compounds depending on exposure doses and health states. Therefore, there is an increasing need for high-throughput screening assays of FXR activity to profile large numbers of environmental chemicals and drugs. This chapter describes a workflow of FXR modulator identification and characterization. To identify compounds that modulate FXR transactivation at the cellular level, we first screen compounds from the Tox21 10 K compound library in an FXR-driven beta-lactamase reporter gene assay multiplexed with a cell viability assay in the same well of the 1536-well plates. The selected compounds are then tested biochemically for their ability to modulate FXR-coactivator binding interactions using a time-resolved fluorescence resonance energy transfer (TR-FRET) coactivator assay. The assay results from the workflow can be used to prioritize compounds for more extensive investigations.

Key words

Farnesoid X receptor Nuclear receptor Drugs Environmental chemicals High-throughput screening HTS Reporter gene Beta-lactamase Coactivator recruitment Fluorescence resonance energy transfer FRET 


  1. 1.
    Kuipers F, Bloks VW, Groen AK (2014) Beyond intestinal soap–bile acids in metabolic control. Nat Rev Endocrinol 10(8):488–498. doi: 10.1038/nrendo.2014.60 CrossRefPubMedGoogle Scholar
  2. 2.
    Merk D, Steinhilber D, Schubert-Zsilavecz M (2012) Medicinal chemistry of farnesoid X receptor ligands: from agonists and antagonists to modulators. Future Med Chem 4(8):1015–1036. doi: 10.4155/fmc.12.47 CrossRefPubMedGoogle Scholar
  3. 3.
    Lamers C, Schubert-Zsilavecz M, Merk D (2014) Medicinal chemistry and pharmacological effects of Farnesoid X Receptor (FXR) antagonists. Curr Top Med Chem 14(19):2188–2205CrossRefPubMedGoogle Scholar
  4. 4.
    Zheng ZH, Lv GP, Si SY, Dong YS, Zhao BH, Zhang H, He JG (2007) A cell-based high-throughput screening assay for Farnesoid X receptor agonists. Biomed Environ Sci 20(6):465–469Google Scholar
  5. 5.
    Wu X, Glickman JF, Bowen BR, Sills MA (2003) Comparison of assay technologies for a nuclear receptor assay screen reveals differences in the sets of identified functional antagonists. J Biomol Screen 8(4):381–392. doi: 10.1177/1087057103256466 CrossRefPubMedGoogle Scholar
  6. 6.
    Hsu CW, Zhao J, Huang R, Hsieh JH, Hamm J, Chang X, Houck K, Xia M (2014) Quantitative high-throughput profiling of environmental chemicals and drugs that modulate farnesoid X receptor. Sci Rep 4:6437. doi: 10.1038/srep06437 CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Han KC, Kim JH, Kim KH, Kim EE, Seo JH, Yang EG (2010) Identification of farnesoid X receptor modulators by a fluorescence polarization-based interaction assay. Anal Biochem 398(2):185–190. doi: 10.1016/j.ab.2009.11.008 CrossRefPubMedGoogle Scholar
  8. 8.
    Yu DD, Lin W, Chen T, Forman BM (2013) Development of time resolved fluorescence resonance energy transfer-based assay for FXR antagonist discovery. Bioorg Med Chem 21(14):4266–4278. doi: 10.1016/j.bmc.2013.04.069 CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Glickman JF, Wu X, Mercuri R, Illy C, Bowen BR, He Y, Sills M (2002) A comparison of ALPHAScreen, TR-FRET, and TRF as assay methods for FXR nuclear receptors. J Biomol Screen 7(1):3–10. doi: 10.1089/108705702753520288 CrossRefPubMedGoogle Scholar
  10. 10.
    Fujino T, Sato Y, Une M, Kanayasu-Toyoda T, Yamaguchi T, Shudo K, Inoue K, Nishimaki-Mogami T (2003) In vitro farnesoid X receptor ligand sensor assay using surface plasmon resonance and based on ligand-induced coactivator association. J Steroid Biochem Mol Biol 87(4-5):247–252CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Chia-Wen (Amy) Hsu
    • 1
  • Jinghua Zhao
    • 1
  • Menghang Xia
    • 1
    Email author
  1. 1.National Center for Advancing Translational SciencesNational Institutes of HealthBethesdaUSA

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