Identification of a novel selective inverse agonist probe and analogs for the Retinoic acid receptor-related Orphan Receptor Gamma (RORγ)

Several nuclear receptors (NRs) are still characterized as orphan receptors because endogenous ligands have not yet been identified for these proteins. Evidence is growing suggesting the retinoic acid receptor-related orphan receptors (RORs) bind to, and are modulated by oxysterols. Recently, we discovered that the synthetic LXRα agonist T0901317 (ML125) was a potent inverse agonist of RORα and RORγ. Structure activity relationship (SAR) studies quickly revealed a strategy to remove the LXRα activity from the ML125 chemical scaffold which led to ML124. ML124 represented the first synthetic RORα/RORγ dual inverse agonist devoid of LXRα activity. While there appear to be clear non-overlapping roles for RORα and RORγ, chemical probes that are isoform selective are needed to dissect this biology. We described the identification of a selective RORα synthetic ligand, ML176, which directly binds to RORα, but not other RORs, and functions as a selective inverse agonist of RORα in cell-based assays. Here we describe the identification of a selective RORγ synthetic ligand, ML310, which functions as an inverse agonist. We show that ML310 can displace T1317 in a binding assay and does interact with RORγ protein to stabilize the protein in hydrogen-deuterium exchange (HDX)-based experiments. In cotransfection assays, ML310 suppresses transcription activity in both GAL4-RORγ ligand binding domain (LBD) and full-length RORγ contexts. Furthermore, treatment of EL-4 cells with ML310 results in suppression of gene expression and production of IL-17. These data strongly suggest that ML310 is a potent and efficacious RORγ modulator and represses its activity. Thus, we have identified the first synthetic RORγ selective inverse agonist, and this compound can be utilized as a chemical tool to probe the function of this receptor both and . Additionally, our data suggests that RORγ inverse agonists may hold utility for the treatment of autoimmune disorders.