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

Celastrol (CSL) is a pentacyclic triterpenoid terpene extracted from the root bark of Tripterygium wilfordii, which has been studied for its efficacy in anti-cancer and cerebral neurology. However, its toxicity limits its clinical use. Through biotransformation in the early stage, we have obtained a series of derivatives with reduced toxicity and increased efficacy. 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. The targets selected for study were the RORα and RORγ of Retinoic Acid Related-Orphan Receptors (RORs) family based on the drug target database. 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.

In summary, we performed reverse docking of CSLs to find its key targets RORα and RORγ to explain its anti-inflammatory and anti-tumor effects. We found binding sites GLN19, ARG97, ARG100 for RORα-Ligand binding domain (LBD) and GLN25, LEU26, ARG103, ARG106 for RORγ-LBD and screened for the highest affinity derivatives. The 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. Meanwhile, the positive and negative coulomb energy in MMGBSA supported this experimental result.