ABSTRACT Previously thought to be phospholipases, PLD3 and PLD4 have recently been revealed to be endosomal exonucleases that regulate the innate immune response by digesting the ligands of nucleic acid sensors. These enzymes can suppress RNA and DNA innate immune sensors like TLR7-9, TLR13 and an unknown STING sensor. Cells deficient in PLD3 or PLD4 have enhanced responses to certain ligands of TLR9. Additionally, Pld4– /– mice overproduce cytokines and upregulate major histocompatibility complex class II on the surface of resident peritoneal macrophages. These responses are unsurprising considering the powerful immunomodulatory effects of nucleic acid ligands on B cell, macrophages, and dendritic cells, particularly plasmacytoid dendritic cells, which produce significant quantities of type I interferon (IFN). The recently discovered immunoregulatory role of these enzymes helps explain the correlation between their polymorphisms and diseases like rheumatoid arthritis, systemic sclerosis, Alzheimer’s disease, Kidney Fibrosis, and other inflammatory diseases. In addition to therapeutics for these diseases, modulators of PLD3 and PLD4 may serve as adjuvants, stabilizers of oligonucleotide therapies, and immune suppressants and stimulators. Nucleic acid sensors agonists are highly sought with several compounds that target TLRs and STING pathways undergoing undergoing clinical trials as adjuvants for immune responses to cancer and in other vaccines. Imiquimod demonstrates the feasibility of targeting TLRs and is approved for use in humans for the treatment of actinic keratosis, genital warts and non-melanoma skin cancers. PLD3 and PLD4 modulators present a unique strategy of targeting nucleic acid sensors for two reasons. (1) Rather than target specific sensors, PLD3 and PLD4 modulators would affect the availability of the nucleic acid ligands of several immune sensors. (2) Due differences in cell type expression, PLD4 modulators would allow for selective targeting of immune cells as opposed to the boarder effects of PLD3 modulators. To realize any promising therapeutic potential, PLD3 and PLD4 pathways, and their role in disease, must be defined. To this end, we seek to identify small molecule probes that selectively and nonselectively activate or inhibitor PLD3 and PLD4. We have developed a high-throughput fluorescence enzymatic activity assay to identify modulators of PLD3 and PLD4 function. A pilot screen of a diversity library (N = 1056) has identified nonselective and selective activators and inhibitors of PLD3 and PLD4. We seek to explore a broader chemical space by screening a ~100K diversity library. With these data, structural activity relationships will be identified and explored through medicinal chemistry. Cell assays to assess potency, specificity, and toxicity will be employed to vet hits. Finally, binding kinetics, protein isoform activity, and in vitro chemical stability will be evaluated to guide optimization of probe molecules for ...