Modulators of granulocyte regulated secretion for control of inflammation

描述（由申请人提供）：中性粒细胞的胞吐及其颗粒与吞噬体的融合是炎症和宿主防御的关键事件。严格调节中性粒细胞的分泌机制是必不可少的，因为不受控制地释放它们颗粒的有毒货物对宿主有害。例如，中性粒细胞氧化酶包括髓过氧化物酶和丝氨酸蛋白酶弹性酶参与关节炎、动脉粥样硬化和败血症的发病。此外，在动物模型中，中性粒细胞分泌蛋白缺乏或嗜氮颗粒胞吐机制受损可减少内毒素诱导的全身炎症和死亡。虽然中性粒细胞分泌蛋白在急性和慢性炎症中发挥重要作用是显而易见的，但干扰中性粒细胞胞吐的分子靶向方法尚未被探索。本应用程序旨在识别中性粒细胞胞吐和炎症的新小分子抑制剂。在这个应用中，我们关注小gtpase和它们的特定效应分子，它们是调节包括胞吐在内的囊泡运输过程的膜组织者。本提案的目的是实施高通量筛选，以鉴定由小GTPase Rab27a及其效应物synaptotagin样蛋白1 （Slp1）调节的新型分泌途径的小分子抑制剂，这两种途径是中性粒细胞中嗜氮颗粒胞吐的重要调节因子。我们还旨在通过建立二级分析以及使用基于细胞和体内的方法来验证这些化合物。我们的具体目标是：1)实施高通量筛选，以鉴定小GTPase Rab27a及其效应物Slp1之间相互作用的抑制剂，使用一种创新的方法，分析复合物在完整细胞内细胞器上的激活；2)通过正交验证试验、基于细胞的二次方法和活性序列的分子相似性分析来鉴定和优先考虑活性探针；3)研究水泡运输的机制，并利用鉴定的命中炎症的体内模型。这项研究的意义在于，新的小分子中性粒细胞胞吐抑制剂将有助于设计新的、有针对性的治疗方法来预防和治疗与炎症过程相关的人类疾病。

DESCRIPTION (provided by applicant): Exocytosis in neutrophils and the fusion of their granules with the phagosome are crucial events in inflammation and host defense. Strict regulation of the secretory mechanism in neutrophils is essential because uncontrolled release of the toxic cargo of their granules is harmful to the host. For example, neutrophil oxidative enzymes including myeloperoxidase and the serine protease elastase are involved in the pathogenesis of arthritis, atherosclerosis and sepsis. Furthermore, deficiencies in neutrophil secretory proteins or impairment in the mechanism of azurophilic granule exocytosis decreases endotoxin-induced systemic inflammation and death in animal models. Although it is clear that neutrophil secretory proteins play an essential role in acute and chronic inflammation, molecular targeted approaches to interfere with neutrophil exocytosis have not been explored. This application seeks to identify new small molecule-inhibitors of neutrophil exocytosis and inflammation. In this application, we focus on small GTPases and their specific effector molecules, which are membrane organizers that regulate vesicular transport processes including exocytosis. The objective of this proposal is to implement high-throughput screenings to identify small- molecule-inhibitors of the novel secretory pathway regulated by the small GTPase Rab27a and its effector synaptotagmin-like protein 1 (Slp1), two important regulators of azurophilic granule exocytosis in neutrophils. We also aim to validate these compounds through established secondary assays as well as using cell-based and in vivo approaches. Our specific Aims are: 1) To implement high-throughput screenings to identify inhibitors of the interaction between the small GTPase Rab27a and its effector Slp1 using an innovative approach that analyzes the activation of the complex on intact intracellular organelles; 2) To perform orthogonal confirmation assays, cell-based secondary approaches and analysis of the molecular similarity of the active series to identify and prioritize active probes and 3) To investigate mechanisms of vesicular trafficking and utilize the identified hits in vivo models of inflammation. The significance of the research proposed is that new small molecule-inhibitors of neutrophil exocytosis will help design novel, targeted, therapeutic approaches to prevent and treat human diseases associated with inflammatory processes.