Development and Validation of a Cell-Based Assay for the Nuclear Receptor Retinoid-Related Orphan Receptor Gamma

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.