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Updated: Jun 7, 2026
Prediction and Validation of Gene Regulatory Elements Activated During Retinoic Acid Induced Embryonic Stem Cell Differentiation
Published on: June 21, 2016
Development and validation of a cell-based assay for the nuclear receptor retinoid-related orphan receptor gamma
Jennifer Wilkinson1, Tina Hallis, Spencer Hermanson
1Life Technologies, Madison, Wisconsin 53719, USA.
Abstract:
The nuclear receptor retinoid-related orphan receptor gamma (RORγ) has become an attractive target for drug discovery due to its important role in the development and differentiation of Th17 cells, a subset of T cells that produce interleukin-17 and are involved in the pathogenesis of human inflammatory and autoimmune diseases. To facilitate the drug discovery efforts in this area, we have developed a cellular assay for screening for RORγ inverse agonists. We stably engineered a tetracycline-inducible Gal4 DNA-binding domain/RORγ ligand-binding domain fusion protein into an upstream activation sequence driven-beta-lactamase reporter gene cell line. Due to its constitutive activity, the induced Gal4-RORγ expression leads to increased reporter activity, which can be knocked down using RORγ ligand-binding domain-specific RNA interference oligos. Using this assay, we tested several recently reported ligands for RORγ and observed varying levels of partial inverse agonist activity at μM concentrations. Additionally, we screened a small library of biologically active compounds with this assay and demonstrated its robustness and usefulness in high-throughput screening and follow-up studies for this emerging drug target.
Insights
Researchers developed a new cellular assay to screen for retinoid-related orphan receptor gamma (RORγ) inverse agonists, crucial for targeting Th17 cell-mediated inflammatory diseases. This assay aids in discovering novel therapeutics for autoimmune conditions.
Area of Science:
- Molecular biology
- Immunology
- Drug discovery
Background:
- Retinoid-related orphan receptor gamma (RORγ) is vital for Th17 cell development and interleukin-17 production.
- Th17 cells are implicated in the pathogenesis of human inflammatory and autoimmune diseases.
- Targeting RORγ is a promising strategy for developing novel therapeutics.
Purpose of the Study:
- To develop and validate a cellular assay for screening RORγ inverse agonists.
- To facilitate high-throughput screening for drug discovery targeting RORγ.
Main Methods:
- Engineered a stable cell line with a tetracycline-inducible Gal4-RORγ fusion protein and a beta-lactamase reporter gene.
- Utilized RNA interference to confirm RORγ specificity.
- Tested known RORγ ligands and screened a small compound library.
Main Results:
- The assay demonstrated RORγ inverse agonist activity for several known ligands at micromolar concentrations.
- Screening identified compounds with partial inverse agonist activity.
- The assay proved robust and useful for high-throughput screening.
Conclusions:
- A novel cellular assay for RORγ inverse agonist screening has been successfully developed.
- This assay is valuable for identifying and validating drug candidates targeting RORγ.
- The assay supports drug discovery efforts for RORγ-mediated inflammatory and autoimmune diseases.
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