Chemical, Structural and Cell-Signaling Interrogation of 15-Prostanglandin Dehydrogenase in Tissue Repair and Regeneration

抽象的。前列腺素E2（PGE 2）调节多个器官的组织生长和修复。的保守 合成和降解机制调节PGE 2水平，以响应创伤、炎症和炎症。 疾病特别地，酶15-前列腺素脱氢酶（15-PGDH）是主要的PGE 2降解酶。 酶，因此是组织修复和再生的关键调节剂。15-PGDH是一个有吸引力的药物靶点 以组织损伤为特征的疾病。我们的团队成功开发出第一个小分子 具有体内活性的15-PGDH抑制剂。在啮齿类动物中，我们的抑制剂1）加速骨损伤后的恢复 骨髓移植，2）加速溃疡性结肠炎的恢复或预防，3）加速溃疡性结肠炎的再生， 部分肝切除术后的肝组织，4）改善博莱霉素诱导的疾病中的肺纤维化 模型，5）增强成年小鼠中新海马神经元的存活，以及6）保持认知功能， 最小化小鼠创伤性脑损伤后的神经元损伤。独立报道称， 15-PGDH抑制剂在肾脏疾病和肺纤维化模型中的有益作用。 我们现在提出了一个合作的化学，结构和细胞信号的作用和活动的审问， 15-PGDH。我们的专业知识包括药物化学，生物化学，神经科学，药理学， 结构生物学在目标1中，我们将定义和开发小分子抑制15-PGDH的结构基础。 分子。这一目标建立在15-PGDH的第一个cryoEM结构（2.3 μ m分辨率）和两个不相关的 15-PGDH的低nM抑制剂的支架。拟议的研究旨在解决15-PGDH的结构， 与新的小分子抑制剂或底物的复合物。将采用计算方法来 询问底物/抑制剂结合和酶机制。在目标2中，我们将定义蜂窝， 由15-PGDH调节并由15-PGDH参与的蛋白质和细胞因子信号网络 抑制剂以增强组织再生和修复。这一目标的基础包括第一个 证明脑中的15-PGDH活性，鉴定巨噬细胞和小胶质细胞是主要的 外周组织和脑中15-PGDH表达的储存库，以及细胞和 对抑制15-PGDH有反应的细胞因子网络。我们现在建议使用单细胞RNA测序， 确定表达15-PGDH的细胞类型。类似的方法将识别以下的细胞信号网络： 诱导的细胞因子和细胞类型被激活以表达它们。这些研究将在小鼠中进行 从已经用15-PGDH抑制剂治疗的损伤中恢复，沿着适当的对照。最后， 我们将设计靶向巨噬细胞和小胶质细胞的15-PGDH敲除，以定义15-PGDH的作用 在巨噬细胞和小胶质细胞中的表达介导对PGE 2和15- PGDH抑制剂。该数据集将为靶向15- 20的治疗方法的未来发展提供基础。 PGDH和其他调节组织再生的药物靶点。

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 foundation for future advancement of therapeutics targeting 15- PGDH and additional drug targets that modulate tissue regeneration.