A High-throughput Screen of Lytic Granule Exocytosis

描述（由申请人提供）：细胞毒性T淋巴细胞（ctl）杀伤靶细胞对病毒和肿瘤的免疫应答至关重要，参与移植排斥反应，并有助于自身免疫性疾病的发病。ctl杀伤靶细胞的一个关键机制是由专门的溶酶体溶解颗粒分泌细胞杀伤剂。筛选抑制溶解颗粒胞吐的化合物的小分子文库将1)作为控制这一过程的已知信号阻阻剂的测定；2)揭示和定义未知信号的化学探针的存在，3)导致新的免疫调节药物，可以增强我们移植器官和干细胞以及治疗自身免疫性疾病的能力。使用荧光标记抗体检测溶酶体相关膜蛋白1 （LAMP-1）从溶解颗粒到质膜的外化，可以监测CTL颗粒的胞外分泌，这是一种适用于流式细胞术的简单免洗检测方案。我们计划利用这一简单的检测方法和新墨西哥大学分子发现中心（UNMCMD）独特的HTS流式细胞术能力来建立表型筛选，并发现抑制粒细胞胞外分泌的化学探针。我们提出三个具体目标。首先，我们将优化分析条件，确定DMSO耐受性，并应用重复测量测试来评估分析稳健性。我们将以低通量形式测量颗粒胞吐过程中不同信号通路的已知抑制剂的剂量-反应曲线，然后确定这些抑制剂是否以及在何种浓度下可以在模拟小规模试验中可靠地检测到。在我们的第二个目标中，也将在第一年进行，我们将把分析转移到UNMCMD，执行高通量格式确认分析性能所需的验证步骤，然后筛选Prestwick药物库，分子库小分子库（MLSMR）的7个板验证集和ICCB文库的480个已知生物活性化合物。我们相信，完成这一目标本身就可以大大提高我们对控制颗粒胞吐的信号的认识。最后，在第二年（当然取决于目标1和目标2的成功与否），我们将在UNMCMD的MLSMR中对化合物进行全面筛选。

DESCRIPTION (provided by applicant): Target cell killing by cytotoxic T lymphocytes (CTLs) is critical for the immune response to viruses and tumors, is involved in transplant rejection and contributes to autoimmune disease pathogenesis. A key mechanism CTLs use to kill target cells is secretion of cell-killing agents from specialized lysosomal lytic granules. Screening a small molecule library for compounds that inhibit lytic granule exocytosis would 1) serve as an assay for blockers of the known signals that control the process; 2) reveal the presence of and define chemical probes for as-yet-unknown signals and 3) lead to novel immunomodulatory drugs that could enhance our ability to transplant organs and stem cells and to treat autoimmune disorders. CTL granule exocytosis can be monitored using fluorescently-labeled antibodies to detect the externalization of lysosome-associated membrane protein 1 (LAMP-1) from lytic granules to the plasma membrane in a simple no-wash assay protocol suitable for flow cytometry. We plan to capitalize on this simple assay together with the unique HTS flow cytometry capabilities at the University of New Mexico Center for Molecular Discovery (UNMCMD) to establish a phenotypic screen and to discover chemical probes that inhibit granulocyte exocytosis. We propose three specific aims. In the first, we will optimize assay conditions, determine DMSO tolerance, and apply repeated measures tests to assess assay robustness. We will measure in a low-throughput format dose-response curves for known inhibitors of different signaling pathways involved in granule exocytosis, then determine whether and at what concentrations these inhibitors can reliably be detected in a mock small-scale trial of the assay. In our second aim, also to be conducted in Year 1, we will transfer the assay to the UNMCMD, perform the validation steps needed to confirm assay performance in a high-throughput format, then screen the Prestwick Library of pharmaceuticals, a 7-plate validation set from the Molecular Libraries Small Molecule Repository (MLSMR) and the ICCB library library of 480 known bioactive compounds. We believe that completion of this aim by itself could substantially advance our knowledge of the signals controlling granule exocytosis. Finally, in year 2 (and of course contingent on success in aims 1 and 2) we will conduct the full screen of compounds in the MLSMR at the UNMCMD. PUBLIC HEALTH RELEVANCE: Cytotoxic T cell lytic granule exocytosis is important for killing virus infected cells, tumor cells and transplants, and is also involved in the etiology of some autoimmune disorders. Identifying small molecule inhibitors of granule exocytosis via high-throughput screening could lead to new clinically-important drugs that could be used as immunosuppressive agents, and could also provide important insights into the signaling pathways that control the process. This application is designed to determine whether a simple and robust high-throughput assay of lytic granule exocytosis based on the externalization of lysosome-associated membrane protein can be developed. If it can, we will use it to screen a large scale compound library for inhibitory compounds.