Fluorescence polarization-based discovery of natural RORγ orthosteric inhibitors.

Retinoic acid receptor-related orphan receptor γ (RORγ) is a key transcriptional regulator of the T helper 17 cell and has emerged as a promising therapeutic target for autoimmune diseases. To identify novel orthosteric inhibitors of RORγ, we designed and synthesized ten fluorescent probes targeting the RORγ orthosteric site. Among them, probe 19g exhibited the best performance (Kd = 252 nM, fluorescence quantum yield = 29.7%) and was subsequently employed to establish a fluorescence polarization (FP)-based assay for screening natural orthosteric binders. Using this optimized FP system, the natural polyphenolic compounds tannic acid (FP Ki = 250 ± 13 nM) and epigallocatechin gallate (EGCG, FP Ki = 506 ± 8 nM) were identified as RORγ ligands. Both compounds exhibited significant inhibitory activity against RORγ, as assessed by RORγ-Gal4 reporter, qPCR and CESTA assays. Furthermore, in the psoriatic-like mouse model, EGCG can effectively alleviate psoriatic-like skin lesions. Collectively, the fluorescent probes and FP-based screening platform provide convenient tools for the discovery and mechanistic investigation of RORγ orthosteric inhibitors. Moreover, the identification of RORγ as a molecular target of tannic acid and EGCG offers new insights into the anti-inflammatory mechanisms of these natural compounds.