PROJECT SUMMARY The innate immune system is the first line of defense against invading pathogens and intimately collaborates with the adaptive immune system to maintain physiological homeostasis. However, components of the immune response can sometimes become dysfunctional, failing in this protective role and even directly causing a variety of autoimmune diseases. Immune dysfunction arises from an interplay of genetic and environmental factors, however a mechanistic understanding of the various proteins and pathways that drive these conditions remains incomplete. In particular, it is known that immune sensors, which are typically dedicated to protection against infection, are sometimes usurped, and instead initiate and propagate autoimmune diseases such as systemic lupus erythematosus (SLE) and Crohn’s disease. Specifically, self-induced signaling by nucleic acid-sensing endosomal Toll-like receptors (TLRs 7 and 9) and the unchecked production of pro-inflammatory cytokines (e.g. type I interferons;; IFN-I) in plasmacytoid dendritic cells (pDCs) are key events in the pathogenesis of numerous autoimmune conditions. Thus, compounds that can suppress the production of these cytokines in pDCs would be clinically useful agents for the treatment of such diseases. Recently, loss-of-function studies of the poorly characterized endolysosomal solute carrier gene family 15 member 4 (SLC15A4) in lupus mouse models revealed significantly reduced disease manifestation as well as near complete suppression of TLR7/9-mediated production of IFN-I and other proinflammatory cytokines. In this application, we have leveraged our lab’s innovative chemoproteomic fragment-based ligand discovery platform to develop a suite of chemical probes that engage SLC15A4 in human pDCs, block SLC15A4 mediated transport, and suppress IFN-I production in human and mouse primary pDCs. We will utilize an interdisciplinary strategy that draws upon the fields of chemical biology, immunology and mass spectrometry to illuminate how SLC15A4 controls TLR-mediated production of IFN-I in primary human and mouse immune cells and evaluate pharmacological inhibition in vivo. Specifically, we will investigate the role of SLC15A4 in regulating endolysosomal homeostasis, map the protein interaction network of SLC15A4 and broadly characterize pharmacological inhibition of SLC15A4 in immune cells. We will also evaluate and optimize chemical probes to assess the therapeutic potential of SLC15A4 in mouse models of lupus. The chemical tools generated, and knowledge gained from these studies are certain to greatly advance our understanding of SLC15A4 biology and the endolysosomal regulation of TLR signaling, enabling the identification of novel strategies to treat human autoimmune diseases.