Cholesterol Unbound RORγt Protein Enables a Sensitive Inverse Agonist Screening.

The retinoic acid-related orphan receptor gamma T (RORt) plays an important role in Th17 cell proliferation and functionality. Thus, RORt 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 RORt has constitutive activity, it is recognized that the receptor is physiologically regulated by various cholesterol derivatives. In this study, we sought to identify RORt inverse agonists through a high-throughput screening campaign. To this end, we compared an apo-RORt protein from Escherichia coli and a cholesterol-bound RORt protein from insect cells. The IC50 of the known RORt inverse agonist TO901317 was significantly lower for the apoprotein than for the cholesterol-bound RORt. Through high-throughput screening using a fluorescence-based cholesterol binding assay with the apoprotein, we identified compound 1 as a novel cholesterol-competitive RORt inverse agonist. Compound 1 inhibited the RORt-TopFluor cholesterol interaction, coactivator recruitment, and transcriptional activity of RORt. 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 RORt protein is suitable for sensitive high-throughput screening to identify RORt inverse agonists.