Identification of Betulinic Acid Derivatives as Inverse Agonists of RAR-related Orphan Receptor Gamma (RORγ)

Retinoic acid-related orphan receptor γ (RORγ) is crucial for the differentiation of CD4+ T cells into pro-inflammatory T helper 17 (Th17) cells. These cells are implicated in various autoimmune diseases, including psoriasis, rheumatoid arthritis, and multiple sclerosis. Inhibiting Th17 cell differentiation through RORγ inverse agonists presents a viable therapeutic strategy for treating some of these diseases. In this study, we identified a novel pentacyclic triterpenoid inverse agonist of RORγ, (2Z)-2-(2-furanylmethylene)-3-oxolup-20(29)-en-28-oic acid (15), via structure-based virtual screening. IC50 values of 0.4 µM and 0.6 µM were determined for 15 in a RORγ-Gal4 assay and a RORγ full-length luciferase assay, respectively. Compound 15 is a betulinic acid derivative demonstrating higher potency and efficacy than betulinic acid. In follow-up experiments with 15.1-15.4, two out of four tested betulinic acid derivatives (15.2 and 15.3) were identified as RORγ inverse agonists, with IC50 values in the low micromolar range. Real-time quantitative polymerase chain reaction (RT-qPCR) experiments showed that 15, 15.2, and 15.3 down-regulated various RORγ target genes. Furthermore, both betulinic acid and 15 exhibited significant stabilization of hRORγ ligand-binding domains (LBD) in the thermal stability assay. The structure-activity relationships and the binding modes within the RORγ ligand binding domain, as predicted by molecular docking for all six compounds, including betulinic acid, are discussed and experimentally validated using a site-directed mutagenesis approach. The newly identified compounds feature distinct substitutions on the pentacyclic triterpenoid ring system, expanding the repertoire of RORγ inverse agonists.