A High-throughput Screen of Lytic Granule Exocytosis

描述（由申请方提供）：细胞毒性T淋巴细胞（CTL）对靶细胞的杀伤对于病毒和肿瘤的免疫应答至关重要，参与移植排斥反应，并导致自身免疫性疾病的发病机制。CTL用于杀死靶细胞的关键机制是从特化的溶酶体溶解颗粒分泌细胞杀伤剂。筛选抑制溶解性颗粒胞吐作用的化合物的小分子文库将1）用作控制该过程的已知信号的阻断剂的测定; 2）揭示未知信号的存在并定义化学探针; 3）导致新型免疫调节药物，其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。可以使用荧光标记的抗体监测CTL颗粒胞吐作用，以检测溶酶体相关膜蛋白1（LAMP-1）在适用于流式细胞术的简单无洗涤测定方案中从裂解颗粒到质膜的外化。我们计划利用这种简单的检测方法以及新墨西哥州大学分子发现中心（UNMCMD）独特的HTS流式细胞术能力，建立表型筛选并发现抑制粒细胞胞吐的化学探针。我们提出三个具体目标。首先，我们将优化试验条件，确定DMSO耐受性，并应用重复测量试验来评估试验耐用性。我们将以低通量形式测量参与颗粒胞吐作用的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什么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，我们将把检测转移到UNMCMD，执行以高通量形式确认检测性能所需的验证步骤，然后筛选Prestwick药物库，这是一个来自分子库小分子库（MLSMR）的7板验证集，以及480种已知生物活性化合物的ICCB库库。我们相信，完成这一目标本身可以大大提高我们的知识控制颗粒胞吐的信号。最后，在第二年（当然，取决于目标1和目标2的成功与否），我们将在联合国排雷行动中心对MLSMR中的化合物进行全面筛选。 公共卫生相关性：细胞毒性T细胞溶解性颗粒的胞吐作用对于杀死病毒感染的细胞、肿瘤细胞和移植物是重要的，并且还参与一些自身免疫性疾病的病因学。通过高通量筛选确定颗粒胞吐的小分子抑制剂可能会导致新的临床重要药物，可用作免疫抑制剂，也可以提供控制该过程的信号通路的重要见解。本申请旨在确定是否可以开发基于溶酶体相关膜蛋白外化的溶解颗粒胞吐的简单且稳健的高通量测定。如果可以的话，我们将用它来筛选大规模的化合物库中的抑制性化合物。

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.