Identification of New Lupane-Type Triterpenoids as Inverse Agonists of RAR-Related Orphan Receptor Gamma (RORγ)

Abstract

Targeting retinoic acid-related orphan receptor γ (RORγ) with inverse agonists presents a promising therapeutic strategy for treating autoimmune diseases, including psoriasis, rheumatoid arthritis, and multiple sclerosis. Through structure-based virtual screening, we identified a lupane-type pentacyclic triterpenoid, (2Z)-2-(2-furanylmethylene)-3-oxolup-20(29)-en-28-oic acid (<b>15</b>), as a new inverse agonist of RORγ. The compound exhibited IC₅₀ values of 0.4 μM and 0.9 μM in Gal4-RORγ and full-length RORγ luciferase assays, respectively. Compound <b>15</b> showed improved potency and efficacy compared to a structurally related known inverse agonist, betulinic acid. Among the four additional analogues tested (<b>15.1</b>-<b>15.4</b>), two (<b>15.2</b> and <b>15.3</b>) also demonstrated RORγ inverse agonist activity with low micromolar IC₅₀ values in Gal4-RORγ luciferase assay. Real-time quantitative polymerase chain reaction experiments confirmed that compounds <b>15</b>, <b>15.2</b>, and <b>15.3</b> downregulated RORγ target genes. Thermal shift assays showed that both betulinic acid and <b>15</b> stabilized the RORγ ligand-binding domain. Molecular docking and structure-activity relationship analysis revealed distinct binding modes within the RORγ ligand-binding domains, further supported by site-directed mutagenesis. These findings expand the repertoire of RORγ inverse agonists based on the pentacyclic triterpenoid scaffolds.