Antitumor studies on celastrol and its derivatives as RORα agonists and RORγ inhibitors based on Alphafold reverse docking strategy

Celastrol (CSL), an active compound extracted from the root bark of Tripterygium wilfordii, has been studied for its outstanding efficacy in anti-cancer and cerebral neurology. We have obtained a series of derivatives with reduced toxicity through biotransformation. Here, 23391 proteins of homo sapiens from AlphaFold DB and Schrödinger’s Glide were used for reverse docking with the basic scaffold of CSL to discover the pharmacological activity of its derivatives. Based on the drug target database, the targets selected for the study were the RORα and RORγ of the Retinoic Acid Related-Orphan Receptors family (RORs). The series of compounds were filtered through QikProp, docked for dynamics stimulation 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, found binding sites Gln19, Arg97, Arg100 for RORα-Ligand binding domain (LBD) and Gln25, Leu26, Arg103, Arg106 for RORγ-LBD, 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, while CSLs had the weak inhibitory effect on RORγ-LBD, and these compounds also demonstrated a good apoptotic effect on the KG-1a tumor cell.