Modulators of granulocyte regulated secretion for control of inflammation

描述（由申请人提供）：中性粒细胞的胞吐作用及其颗粒与吞噬体的融合是炎症和宿主防御的关键事件。中性粒细胞分泌机制的严格调节是必不可少的，因为不受控制地释放其颗粒的有毒货物对宿主有害。例如，包括髓过氧化物酶和丝氨酸蛋白酶弹性蛋白酶的嗜中性粒细胞氧化酶参与关节炎、动脉粥样硬化和败血症的发病机制。此外，在动物模型中，中性粒细胞分泌蛋白的缺陷或嗜天青颗粒胞吐机制的损伤减少了内毒素诱导的全身性炎症和死亡。虽然中性粒细胞分泌蛋白在急性和慢性炎症中起重要作用，但尚未探索干扰中性粒细胞胞吐的分子靶向方法。本申请寻求鉴定新的中性粒细胞胞吐和炎症的小分子抑制剂。在这个应用中，我们专注于小GTP酶及其特异性效应分子，这是膜组织者，调节囊泡运输过程，包括胞吐。该提议的目的是实施高通量筛选以鉴定由小GTdR ab 27 a及其效应物突触结合蛋白样蛋白1（Slp 1）调节的新型分泌途径的小分子抑制剂，这两种蛋白是嗜中性粒细胞中嗜天青颗粒胞吐作用的重要调节剂。 我们的目标还包括通过已建立的二级试验以及使用基于细胞的方法和体内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，以使用分析完整细胞内细胞器上的复合物的活化的创新方法来鉴定小GTdR ab 27 a及其效应物Slp 1之间的相互作用的抑制剂; 2）为了进行正交确认测定，基于细胞的第二方法和活性系列的分子相似性分析，以鉴定和优先化活性探针，以及3）研究囊泡运输的机制，并在体内炎症模型中利用识别的命中。这项研究的意义在于，新的中性粒细胞胞吐的小分子抑制剂将有助于设计新的、有针对性的治疗方法，以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.