Celastrol Derivatives: Unveiling Pharmacological Activity and Targeting RORα/RORγ for Anti-inflammatory and Anti-tumor Effects

Celastrol (CSL) is a pentacyclic triterpenoid terpene extracted from the root bark of Tripterygium wilfordii. It's known for its exceptional efficacy in anti-cancer and cerebral neurology, but its toxicity limits clinical use. We've developed a series of derivatives with reduced toxicity and increased efficacy through biotransformation.

To explore the pharmacological activity of these derivatives, we employed a reverse docking approach using 23391 proteins from AlphaFold DB and Schrödinger's Small Molecule Drug Discovery platform. Focusing on the Retinoic Acid Related-Orphan Receptors (RORs) family, we specifically targeted RORα and RORγ.

The derivatives were filtered through QikProp, subjected to dynamics simulations, and analyzed for binding energy using MMGBSA calculations. We also conducted fluorescence polarization (FP) assays, luciferase reporter gene assays, and CCK8 assays.

Our findings reveal that CSL derivatives exhibit significant interactions with RORα and RORγ. We identified key binding sites: GLN19, ARG97, ARG100 for RORα-Ligand binding domain (LBD) and GLN25, LEU26, ARG103, ARG106 for RORγ-LBD. This discovery led to the identification of derivatives with the highest affinity for these targets.

Luciferase reporter gene assays demonstrated that 2 μM 18-OH-CSL and 28-OH-CSL exert the strongest agonistic effect on RORα-LBD, while 12-OH-CSL and a-ring aromatized 15-OH-CSL exhibit the strongest inhibitory effect on RORγ-LBD. These findings are supported by the positive and negative coulomb energy observed in MMGBSA calculations.

In conclusion, this study provides valuable insights into the pharmacological activity of CSL derivatives and their potential as therapeutic agents targeting RORα and RORγ for anti-inflammatory and anti-tumor applications.