Chirality-dependent toxicity decoupling: Discovery of a resibufogenin-based L-configured STING inhibitor with superior therapeutic profile for ulcerative colitis.

STING, a central component of the cGAS-STING innate immune signaling pathway, is implicated in various autoimmune and inflammatory disorders when aberrantly activated. In this study, an L-configured homoproline derivative Z55 was obtained through structural optimization of the natural product resibufogenin (RBG), which exhibited approximately threefold greater cellular inhibitory activity against STING than RBG (IC50 = 0.40 ± 0.04 μM for Z55 vs. 1.42 ± 0.11 μM for RBG), and maintained favorable in vitro safety. Surface plasmon resonance (SPR) analysis confirmed high-affinity binding of Z55 to hSTING (KD = 2.31 μM), and a cellular thermal shift assay (CETSA) further demonstrated that Z55 directly engages endogenous STING in living cells, with a thermal stabilization of 4.31 ± 0.7 °C. Mechanistic studies, including DTT stability and SPR reversibility assays, support a non covalent interaction mode, while molecular docking provided structural insight into the binding interface. Mechanistically, Z55 inhibited STING phosphorylation and downstream activation of p-TBK1 and p-IRF3, leading to decreased levels of key inflammatory cytokines (IL-1β, IL-6, and TNF-α) in both colon tissue and serum of mice with ulcerative colitis. Importantly, this series of compounds exhibited a marked chiral-toxicity separation, with the L-configuration identified as the optimal pharmacophore for both efficacy and safety. Collectively, these findings highlight Z55 as a promising STING-targeting lead candidate for the treatment of inflammatory diseases.