Celastrol Derivatives: Unlocking Anti-Cancer and Anti-Inflammatory Potential by Targeting RORα and RORγ

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

Our findings revealed that CSLs bind to RORα and RORγ to explain its anti-inflammatory and anti-tumor effects, and we identified the binding sites GLN19, ARG97, ARG100 for RORα-Ligand binding domain (LBD) and GLN25, LEU26, ARG103, ARG106 for RORγ-LBD. We also screened for the highest affinity derivatives, and our luciferase reporter gene assay showed that 2 μM 18-OH-CSL and 28-OH-CSL had the strongest agonistic effect on RORα-LBD, while 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 these experimental results.