A High-throughput Screen of Lytic Granule Exocytosis

裂解颗粒胞吐作用的高通量筛选

• 批准号: 8423897
• 负责人: ADAM ZWEIFACH
• 金额: $ 3.83万
• 依托单位: UNIVERSITY OF CONNECTICUT STORRS
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-30 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8423897
• 关键词: Antibodies; Apoptosis; Autoimmune Diseases; Basic Science; Biological Assay; Biological Models; Cell membrane; Cells; Cellular biology; Chemicals; Chemistry; Cytometry; Cytoplasmic Granules; Cytotoxic T-Lymphocytes; Cytotoxic agent; Data; Dimethyl Sulfoxide; Disease; Dose; Etiology; Exocytosis; Flow Cytometry; Genomics; Goals; Graft Rejection; Granzyme; Health; Human; Immune; Immune response; Immunosuppressive Agents; Institutes; Insulin-Dependent Diabetes Mellitus; Knowledge; Label; Lead; Libraries; Lupus; Lysosomes; Lytic; Manuals; Measures; Medical; Membrane Proteins; Methods; Molecular; Molecular Bank; Monitor; Multiple Sclerosis; Neoplasm Transplantation; New Mexico; Oncogenic Viruses; Organ; Organ Transplantation; Pathogenesis; Performance; Pharmaceutical Preparations; Pharmacologic Substance; Process; Protocols documentation; Readiness; Screening procedure; Signal Pathway; Signal Transduction; Stem cells; Testing; Transplanted tissue; United States National Institutes of Health; Universities; Validation; Virus; base; cell killing; design; extracellular; granulocyte; high throughput screening; inhibitor/antagonist; insight; killings; neoplastic cell; novel; perforin; public health relevance; repository; response; small molecule; small molecule libraries; success

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.

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反应至关重要，与移植排斥反应有关，并有助于自身免疫性疾病的发病。CTL用来杀伤靶细胞的一个关键机制是从专门的溶酶体溶解颗粒中分泌细胞杀伤剂。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的阻滞剂的分析；2)揭示未知信号的存在并为其定义化学探针；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。CTL颗粒胞吐可以用荧光标记的抗体来检测溶酶体相关膜蛋白1(LAMP-1)从溶解颗粒到质膜的外化，这是一种适用于流式细胞术的简单的免清洗检测方法。我们计划利用这一简单的检测方法以及新墨西哥大学分子发现中心独特的HTS流式细胞术能力来建立表型筛查，并发现抑制粒细胞胞吐的化学探针。我们提出了三个具体目标。首先，我们将优化检测条件，确定DMSO耐受性，并应用重复测量测试来评估检测的稳健性。我们将以低通量的形式测量与颗粒胞吐有关的不同信号通路的已知抑制剂的剂量-反应曲线，然后确定在模拟的小规模试验中是否以及在什么浓度下可以可靠地检测到这些抑制剂。在我们的第二个目标中，也将在第一年进行，我们将把化验转移到UNCMD，以高通量格式执行确认化验性能所需的验证步骤，然后筛选Prestwick药物文库、来自分子库小分子存储库(MLSMR)的7板验证集和ICCB文库，其中包含480种已知的生物活性化合物。我们相信，这一目标的完成本身就可以极大地提高我们对控制颗粒胞吐的信号的了解。最后，在第二年(当然要视目标1和目标2的成功情况而定)，我们将在该特派团的MLSMR中对化合物进行全面筛选。 公共卫生相关性：细胞毒性T细胞裂解颗粒胞吐对杀死病毒感染的细胞、肿瘤细胞和移植细胞很重要，也参与了一些自身免疫性疾病的病因学。通过高通量筛选识别颗粒胞吐的小分子抑制剂可能会导致新的临床重要药物，这些药物可以用作免疫抑制剂，也可能为控制这一过程的信号通路提供重要的见解。这项应用旨在确定是否可以建立一种简单而可靠的基于溶酶体相关膜蛋白外化的裂解颗粒胞吐的高通量检测方法。如果可以，我们将使用它来筛选大规模的化合物库中的抑制性化合物。