Rational design of RORγT small molecule modulators results in tunable LXRα pleiotropic effects in Th17 cells 2267479

Abstract

Abstract Introduction RAR-related orphan receptor gamma T (RORγT), a nuclear receptor highly expressed in immune cells, is the master regulator for Th17 cell fate. RORγT dysregulation drives pathogenic Th17 phenotypes and autoimmune diseases. Directly targeting RORγT with small molecule inverse agonists has been largely unsuccessful. Therefore, we utilized rational design to improve RORγT inverse agonist effects while simultaneously tuning a pleiotropic target, Liver X Receptor (LXRα). LXR agonism suppresses Th17 inflammatory responses, resulting in new small molecules with potentially better therapeutic profiles. Methods We used Promega’s Dual-Glo luciferase assay in 293T cells for primary screening, generating EC50/IC50 values via serial dilution. Human primary CD4+ T-cells, differentiated into Th17 cells, were treated with 1 μM compound for 72h. RNA was harvested, and IL-17 ELISA was performed on supernatants. Structural studies used recombinant protein for radioligand competition and HDX-MS (Q-Exactive, automated PAL system). Results Novel compounds at 1 and 5 μM suppressed RORγT transcriptional response more effectively than parent compounds. This led to decreased RORγT transcription and IL-17 secretion, concurrent with increased LXRα activity. Although LXRα transcript decreased, its target genes were elevated, suggesting prolonged downstream activation. Structural modeling showed the ligands bind in similar LBD positions but vary in LXR pocket depth. We validated these in silico findings using recombinant RORγT and LXRα in radioligand competition assays (to determine Kd) and HDX-MS (to confirm binding sites) Conclusion Rational design of small molecule modulators enhanced RORγT inverse agonism and tuned LXRα agonism. These compounds better suppressed the pathogenic Th17 phenotype, reducing IL-17 secretion and downstream gene expression. Given LXRα’s anti-inflammatory benefits, these modulators are promising novel therapeutics. In vivo data is pending Funding Source NIH, Synkine Therapeutics Inc. Topic Categories Immune Response Regulation: Molecular Mechanisms (IRM)