Related Experiment Video
Updated: Mar 16, 2026
Reverse Yeast Two-hybrid System to Identify Mammalian Nuclear Receptor Residues that Interact with Ligands and/or Antagonists
Published on: November 15, 2013
Transactivation and Coactivator Recruitment Assays for Measuring Farnesoid X Receptor Activity
Chia-Wen Amy Hsu1, Jinghua Zhao1, Menghang Xia2
1National Center for Advancing Translational Sciences, National Institutes of Health, Building C, MSC: 3375, 9800 Medical Center Drive, Bethesda, MD, 20892, USA.
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.
Insights
A new workflow efficiently screens compounds for farnesoid X receptor (FXR) activity. This method identifies potential drug candidates and environmental hazards by assessing FXR modulation and cell viability.
Area of Science:
- Biochemistry
- Toxicology
- Pharmacology
Background:
- The farnesoid X receptor (FXR) is crucial for maintaining bile acid, lipid, and glucose homeostasis.
- Modulation of FXR signaling has therapeutic implications and can indicate potential environmental hazards.
- High-throughput screening assays are needed to evaluate numerous chemicals and drugs for FXR activity.
Purpose of the Study:
- To establish a workflow for identifying and characterizing compounds that modulate farnesoid X receptor (FXR) activity.
- To enable profiling of large compound libraries for FXR modulators.
Main Methods:
- Compounds were screened using an FXR-driven beta-lactamase reporter gene assay multiplexed with a cell viability assay in 1536-well plates.
- Selected compounds were further analyzed biochemically using a time-resolved fluorescence resonance energy transfer (TR-FRET) coactivator assay to assess FXR-coactivator binding interactions.
Main Results:
- The described workflow successfully identifies compounds modulating FXR transactivation at the cellular level.
- The combined cellular and biochemical assays provide a comprehensive profile of FXR modulators.
Conclusions:
- This workflow offers a robust method for the initial identification and characterization of FXR modulators.
- The assay results aid in prioritizing compounds for further in-depth investigation and safety assessment.
More Related Videos
07:41A Kinetic Fluorescence-based Ca2+ Mobilization Assay to Identify G Protein-coupled Receptor Agonists, Antagonists, and Allosteric Modulators
Published on: February 20, 2018
11:49Characterization of G Protein-coupled Receptors by a Fluorescence-based Calcium Mobilization Assay
Published on: July 28, 2014
Related Concept Videos
01:31Transducer Mechanism: Nuclear Receptors
About 48 different soluble family members of nuclear receptors are identified that can be divided into two main classes:
02:04Co-activators and Co-repressors