Abstract. Prostaglandin E2 (PGE2) regulates tissue growth and repair in multiple organs. A conserved mechanism of synthesis and degradation modulates PGE2 levels in response to trauma, inflammation and disease. In particular, the enzyme 15-prostaglandin dehydrogenase (15-PGDH) is the main PGE2-degrading enzyme and therefore a key regulator of tissue repair and regeneration. 15-PGDH is an attractive drug target for diseases characterized by tissue damage. Our team successfully developed the first small molecule inhibitors of 15-PGDH with in vivo activities. In rodents, our inhibitors 1) accelerate recovery following bone marrow transplantation, 2) accelerate recovery from, or prevent, ulcerative colitis, 3) accelerate regrowth of liver tissue following partial hepatectomy, 4) ameliorate pulmonary fibrosis in a bleomycin-induced disease model, 5) enhance survival of new hippocampal neurons in adult mice, and 6) preserve cognitive function and minimize neuronal damage in mice following traumatic brain injury. Independent reports have described beneficial effects of 15-PGDH inhibition in models of renal disease and pulmonary fibrosis. We now propose a collaborative chemical, structural and cell-signaling interrogation of the role and activity of 15-PGDH. Our expertise includes medicinal chemistry, biochemistry, neuroscience, pharmacology, and structural biology. In Aim 1, we will define and exploit the structural basis for inhibition of 15-PGDH by small molecules. This aims builds on the first cryoEM structure (2.3 Å resolution) of 15-PGDH and two unrelated scaffolds of low-nM inhibitors of 15-PGDH. Proposed research aims to solve the structure of 15-PGDH in complex with new small molecule inhibitors or substrate. Computational approaches will be employed to interrogate substrate/inhibitor binding and the enzymatic mechanism. In Aim 2, we will define the cellular, protein and cytokine signaling networks that are regulated by 15-PGDH and that are engaged by 15-PGDH inhibitors to potentiate tissue regeneration and repair. The foundation of this aim includes the first demonstration of 15-PGDH activity in the brain, the identification of macrophages and microglia as major reservoirs of 15-PGDH expression in peripheral tissues and brain, respectively, and the discovery of cell and cytokine networks that respond to inhibiting 15-PGDH. We now propose to use single-cell RNA sequencing to determine the cell types that express 15-PGDH. Similar approaches will identify the cell-signaling network of induced cytokines and the cell types activated to express them. These studies will be performed in mice recovering from injury that have been treated with 15-PGDH inhibitors, along with appropriate controls. Finally, we will engineer macrophage- and microglia-targeted 15-PGDH knockouts to define the role of 15-PGDH expression in macrophages and microglia in mediating a conserved, cross-tissue response to PGE2 and 15- PGDH inhibitors. This data set will provide a foun...