Cholesterol Unbound RORγt Protein Enables a Sensitive Inverse Agonist Screening

The retinoic acid-related orphan receptor gamma T (RORγt) plays an important role in Th17 cell proliferation and functionality. Thus, RORγt inverse agonists are thought to be potent therapeutic agents for Th17-mediated autoimmune diseases, such as rheumatoid arthritis, asthma, inflammatory bowel disease, and psoriasis. Although RORγt has constitutive activity, it is recognized that the receptor is physiologically regulated by various cholesterol derivatives. In this study, we sought to identify RORγt inverse agonists through a high-throughput screening campaign. To this end, we compared an apo-RORγt protein from Escherichia coli and a cholesterol-bound RORγt protein from insect cells. The IC 50 of the known RORγt inverse agonist TO901317 was significantly lower for the apoprotein than for the cholesterol-bound RORγt. Through high-throughput screening using a fluorescence-based cholesterol binding assay with the apoprotein, we identified compound 1 as a novel cholesterol-competitive RORγt inverse agonist. Compound 1 inhibited the RORγt-TopFluor cholesterol interaction, coactivator recruitment, and transcriptional activity of RORγt. Cell-based reporter gene assay demonstrated that compound 1 showed higher potency by lipid depletion treatment. Collectively, our findings indicate that eliminating cholesterol from the RORγt protein is suitable for sensitive high-throughput screening to identify RORγt inverse agonists.