title: Transactivation and Coactivator Recruitment Assays for Measuring Farnesoid X Receptor Activity.
abstract: 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.
authors: National Center for Advancing Translational Sciences, National Institutes of Health, Building C, MSC: 3375, 9800 Medical Center Drive, Bethesda, MD, 20892, USA.
authors: Chia-Wen Amy
authors: CW
authors: Hsu
authors: National Center for Advancing Translational Sciences, National Institutes of Health, Building C, MSC: 3375, 9800 Medical Center Drive, Bethesda, MD, 20892, USA.
authors: Jinghua
authors: J
authors: Zhao
authors: National Center for Advancing Translational Sciences, National Institutes of Health, Building C, MSC: 3375, 9800 Medical Center Drive, Bethesda, MD, 20892, USA. mxia@mail.nih.gov.
authors: Menghang
authors: M
authors: Xia
mesh_terms: D002635:Chenodeoxycholic Acid / Q000494:pharmacology; D031541:Fluorescence Resonance Energy Transfer; D017930:Genes, Reporter; D057809:HEK293 Cells; D057166:High-Throughput Screening Assays; D006801:Humans; D011282:Pregnenediones / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D018160:Receptors, Cytoplasmic and Nuclear / Q000819:agonists / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D054852:Small Molecule Libraries / Q000633:toxicity*; D013329:Structure-Activity Relationship; D015533:Transcriptional Activation; D001618:beta-Lactamases / Q000235:genetics / Q000378:metabolism; D000099150:Receptor, Farnesoid X-Activated
chemical_list: D002635:Chenodeoxycholic Acid; D011282:Pregnenediones; D018160:Receptors, Cytoplasmic and Nuclear; D054852:Small Molecule Libraries; D001618:beta-Lactamases; D000099150:Receptor, Farnesoid X-Activated; C023617:pregna-4,17-diene-3,16-dione
keywords: Beta-lactamase; Coactivator recruitment; Drugs; Environmental chemicals; FRET; Farnesoid X receptor; Fluorescence resonance energy transfer; HTS; High-throughput screening; Nuclear receptor; Reporter gene