Discovery of betulinaldehyde as a natural RORγt agonist

Retinoic Acid Receptor-Related Orphan Receptor γt (RORγt) is a dual-functional therapeutic target. The agonists and inhibitors of RORγt are potential agents for tumor immunotherapy and autoimmune diseases, respectively, and sometimes share similar scaffolds. Although the widely distributed triterpenoid ursolic acid (UA) has been identified as a RORγt inhibitor, the report of a triterpenoid RORγt agonist is still absent. By screening an in-house triterpenoid library, we uncovered a novel RORγt agonist, betulinaldehyde (1), together with an inhibitor (2, 3β, 28-Dihydroxy-lupan-29-oic acid). Compound 1 showed a good RORγt activating effect with the EC₅₀ of 11.4 μM in Alpha Screen assay, and altered the thermal stability of RORγt by directly binding to the protein in vitro. Combined with the SPR assay, the Kd value of compound 1 was examined as 2.99 μM. The modulation mechanism of triterpenoid agonists and inhibitors were discussed by molecular docking. Herein, we firstly discovered compound 1 as a triterpenoid agonist of RORγt. The co-distribution of triterpenoid RORγt agonist and inhibitors in the same plant, might be related to the anti-inflammatory and anti-cancerous bioactivity of the plant extract.