PROJECT SUMMARY More than 40% of patients with diabetic retinopathy (DR) are refractory to direct intraocular injection of anti- VEGF antibodies, the standard of care. The inability to effectively treat DR puts a heavy burden on the health care system and limits the quality of life for those affected. The need for novel, clinically relevant targets that can be exploited for new therapies is very clear. Diabetes is well-known to induce retinal mitochondrial dysfunction that leads to DNA damage and cytosolic release. Cytosolic DNA activates the cGAS-STING pathway, inducing inflammation. Although the cGAS-STING signaling axis is essential for sensing foreign DNA and thus contributing to innate immunity, aberrant activation of this pathway by self-DNA is a significant contributor to autoinflammatory and autoimmune diseases. Activation of cGAS-STING due to chronic diabetes-induced inflammation leads to activation of circulating monocytes, eventually giving rise to leukostasis. Leukostasis causes cell death and breakdown of the blood-retinal barrier, leading to retinal vascular leakage, a major cause of diabetic macular edema and leading cause of vision loss in diabetic patients. We have shown that 1) the cGAS-STING pathway is activated in ischemic retinopathy; 2) inhibition of STING attenuates monocyte activation and alleviates retinal leukostasis and angiogenesis; 3) STING knockout significantly reduces retinal leakage, number of leukocytes in the retina, and DR relevant inflammatory factors in an oxygen-induced retinopathy murine model; and 4) small molecule agonism of peroxisome proliferator-activated receptor alpha (PPARa) inhibits the cGAS-STING signaling axis and cytosolic mitochondrial DNA release. The objective of this exploratory R21 proposal is to assess two mechanistically differentiated approaches to inhibit STING with bifunctional small molecules in the context of DR. In Aim 1 we will assess small molecule induced STING degradation through proteolysis-targeting chimeras (PROTACs) as a strategy for STING inhibition in the context of DR. The PROTAC strategy is unexplored in the eye but especially well-suited for ocular conditions. In Aim 2 we will leverage our success in developing selective PPARa agonists as new therapeutic leads for DR to develop first-in-class bifunctional probes that simultaneously agonize PPARa (neuronal protection) and inhibit STING (retinal vascular homeostasis and inflammation) to explore the potential benefits of polypharmacology in retinal disease. The heterobifunctional modulators of these two targets are expected to provide a spatiotemporal benefit affecting multiple cell types and pathological events. This work is conceptually and technically innovative and is expected to provide the fundamental data needed to enable proof-of-concept for the PROTAC and multi-action compound strategies in ocular contexts (new approaches), a foundation for targeting STING in DR and related retinal diseases (new target), and new c...