Munc13-4–STX7 inhibitors impair endosomal TLR activation and systemic inflammation

Endosomal function is essential for pattern recognition receptor signaling, through endosomal Toll-like receptor (TLR) sensing of nonself RNA and DNA. The specific interaction of the calcium sensor Munc13-4 with syntaxin 7 (STX7) regulates endosomal flux and Munc13-4 depletion decreases the systemic inflammatory response to unmethylated DNA. Using high-throughput screening and orthogonal cell-based validation, we identified small-molecule inhibitors of the Munc13-4–STX7 interaction, ENDOtollins (ENDOs). ENDOs inhibit extracellular signal-regulated kinase signaling in neutrophils and interferon (IFN) regulatory factor signaling in plasmacytoid dendritic cells (DCs) in response to endosomal TLR ligands but not to plasma membrane agonists, highlighting specificity for the endocytic pathway. Mechanistically, ENDOs inhibit endolysosomal flux and decrease endolysosomal cargo degradation. Chemical optimization identified ENDO12 as the most potent inhibitor. ENDO12 inhibited primary DC responses to TLR3, TLR7 and TLR9 and reduced CpG-induced systemic inflammation, manifested as decreased levels of the proinflammatory mediators myeloperoxidase, interleukin 6 and IFNγ. Our findings have significant implications for immunodeficiency, inflammation and innate immunity. Endosomal Toll-like receptors (TLRs) are central to the innate immune response but their overactivation can cause systemic inflammation and disease. Now, new small molecules targeting the interaction between Munc13-4 and syntaxin 7 essential for endosomal maturation impair overactivation of endosomal TLR-dependent pathways and inflammation.