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.