Celastrol Derivatives: Targeting RORα and RORγ for Anti-Inflammatory and Anti-Tumor Effects

Celastrol (CSL) is a pentacyclic triterpenoid extracted from the root bark of Tripterygium wilfordii, which has shown effectiveness in treating cancer and cerebral neurology. However, its toxicity limits its clinical use. Through biotransformation, we have obtained a series of derivatives with reduced toxicity and increased efficacy. In this study, we used AlphaFold DB and the Schrödinger platform Small Molecule Drug Discovery to perform reverse docking of CSL derivatives on 23391 proteins of homo sapiens to discover their pharmacological activity. Based on the drug target database, we selected the RORα and RORγ of the Retinoic Acid Related-Orphan Receptors (RORs) family as targets for study. We filtered the series of compounds through QikProp, docked them for dynamics simulation, and calculated the molecular mechanics-generalized born surface area (MMGBSA) binding energy. We also performed fluorescence polarization assay (FP assay), luciferase reporter gene assay, and the CCK8 assay.

Our study discovered the key targets RORα and RORγ for CSLs to explain their anti-inflammatory and anti-tumor effects. We found binding sites for RORα-Ligand binding domain (LBD) and RORγ-LBD and screened for the derivatives with the highest affinity. Our luciferase reporter gene assay showed that 2 μM 18-OH-CSL and 28-OH-CSL had the strongest agonistic effect on RORα-LBD, and 12-OH-CSL and a-ring aromatized 15-OH-CSL had the strongest inhibitory effect on RORγ-LBD. The positive and negative coulomb energy in MMGBSA supported our experimental results.