HTS platform for identification of regulators of cell adhesion molecule signaling

用于鉴定细胞粘附分子信号传导调节因子的 HTS 平台

基本信息

批准号：
8295318
负责人：
DARREN G WOODSIDE
金额：
$ 38.25万
依托单位：
TEXAS HEART INSTITUTE
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-02-07 至 2015-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8295318
关键词：
Adhesions Adhesives Adverse effects Affinity Animals Anti-Inflammatory Agents Anti-inflammatory Antibodies Artificial Membranes Atherosclerosis Autoimmune Process Autoimmunity Basic Science Binding Biological Assay Blood Platelets Calorimetry Cardiovascular Diseases Cell Adhesion Cell Adhesion Molecules Cell Membrane Permeability Cell surface Cells Chemicals Clinical Clot retraction Collagen Cytoplasmic Tail Development Disease Dose Drug Delivery Systems Drug Kinetics Epitopes Extracellular Domain Extracellular Matrix Family Fibrinogen Fibronectins Future GTP-Binding Proteins Genomics Goals Hemorrhage Hemostatic function Homologous Gene Immunosuppression Immunosuppressive Agents In Vitro Industry Inflammation Inflammatory Integrin Binding Integrin beta3 Integrins Lead Left Libraries Ligand Binding Ligands Mediating Metric Molecular Molecular Bank Mutation NIH Program Announcements Neoplasms Pathogenesis Patients Peptides Pharmaceutical Preparations Pharmacologic Substance Physiological Physiological Processes Platelet aggregation Play Process Production Protein Subunits Protein Tyrosine Kinase Reaction Receptor Cross-Talk Receptor Signaling Regulation Research Role Screening procedure Serum Signal Transduction Signaling Molecule Specificity Structure Surface Plasmon Resonance System Talin Technology Testing Therapeutic Thrombocytopenia Titrations Triage United States National Institutes of Health Validation Vascular Cell Adhesion Molecule-1 acute coronary syndrome adhesion receptor assay development atherothrombosis base drug candidate extracellular high throughput screening in vivo leukocyte activation meetings microcalorimetry mimetics novel novel strategies prevent public health relevance receptor response scaffold small molecule tirofiban tool

项目摘要

DESCRIPTION (provided by applicant): The following proposal is in response to the program announcement PA-10-213 titled "Development of Assays for High-Throughput screening for use in Probe and Pre-therapeutic Discovery (R01)." Integrin cell adhesion receptors play essential roles in normal physiologic process and have been implicated in neoplasia, autoimmunity, inflammation, and cardiovascular disease. Indeed, they are validated drug targets. Present integrin-based therapeutics has proven invaluable in the treatment of patients but is limited by side-effects related to immunosuppression and the induction of autoimmune reactions. There is need for a new strategy to target integrins that would eliminate side-effects. Integrins bind a variety of extracellular ligands such as serum fibrinogen, extracellular matrix (ECM) components, and cell-surface counter receptors. Their activity is tightly regulated by intracellula scaffolding molecules such as Talin-1 and Kindlins that can bind to integrin cytoplasmic domains and in a process termed "inside-out" signaling increase integrin ectodomain affinity for ligand. Integrins also serve as signaling receptors, where "outside-in" signaling is mediated by direct interactions between integrin cytoplasmic domains and intracellular effectors such as the Syk family of nonreceptor tyrosine kinases. We have developed a cell-free assay platform for high throughput screening (HTS) of small molecule compound libraries for antagonists of the interactions between integrin cytoplasmic domains and intracellular effector molecules. This system utilizes the amplified luminescent proximity homogenous assay (ALPHA) screen format. In our first aim, we propose to develop a suite of primary HTS assays targeting the binding of integrin beta3 cytoplasmic domains with Syk, Kindlin-3, and Galpha13. A secondary screen to triage false positive hits will also be developed. In a second aim, we propose a number of orthogonal assays ranging from Elisa-based approaches to isothermal titration calorimetry and surface plasmon resonance, which will serve to validate hit specificity and selectivity. Finally, a third aim is proposed to test antagonist mechanism of action in primary cells, examining integrin activation of Syk, integrin affinity regulation, and platelet function. All primary ALPHA screens will be optimized according to HTS metrics as guided by the NIH Chemical Genomics Center. Pilot screens of a small molecule compound library will be performed to demonstrate primary and secondary screen tractability. Small molecule antagonist identified here may represent excellent starting points for the development of novel classes of anti-platelet and anti-inflammatory drug candidates, which could target integrin function at the intracellular level. Furthermore, small molecule compounds derived from the HTS assays proposed here could be important novel tools in the analysis of both "inside-out" and "outside-in" integrin signal transduction. Public Health Relevance: The screening platforms developed in this proposal will lead to discovery of novel small molecule antagonists that could represent a new direction in the development of adhesion molecule therapeutics for cardiovascular diseases and others. Also, small molecule antagonists identified will be important tools for basic research into the molecular mechanisms regulating cell adhesion molecule function both in vitro and in vivo.

描述（由申请人提供）：以下提案是对标题为“用于探针和治疗前发现（R01）的高通量筛选测定的开发”的计划公告 PA-10-213 的回应。整合素细胞粘附受体在正常生理过程中发挥重要作用，并与肿瘤、自身免疫、炎症和心血管疾病有关。事实上，它们是经过验证的药物靶点。目前基于整合素的疗法已被证明在治疗患者方面具有无价的价值，但受到与免疫抑制和诱导自身免疫反应相关的副作用的限制。需要一种针对整合素的新策略来消除副作用。整合素结合多种细胞外配体，例如血清纤维蛋白原、细胞外基质 (ECM) 成分和细胞表面反受体。它们的活性受到细胞内支架分子（例如 Talin-1 和 Kindlins）的严格调节，这些分子可以与整合素胞质结构域结合，并在称为“由内而外”信号传导的过程中增加整合素胞外域对配体的亲和力。整合素还充当信号传导受体，其中“由外向内”信号传导是通过整合素胞质结构域与细胞内效应器（例如非受体酪氨酸激酶的 Syk 家族）之间的直接相互作用介导的。我们开发了一个无细胞测定平台，用于高通量筛选（HTS）小分子化合物库，以寻找整合素胞质结构域和细胞内效应分子之间相互作用的拮抗剂。该系统采用放大发光邻近均质测定 (ALPHA) 屏幕格式。我们的第一个目标是开发一套针对整合素 beta3 胞质结构域与 Syk、Kindlin-3 和 Galpha13 结合的初级 HTS 检测。还将开发对误报进行分类的辅助屏幕。在第二个目标中，我们提出了许多正交测定，从基于 Elisa 的方法到等温滴定量热法和表面等离子体共振，这将有助于验证命中特异性和选择性。最后，一个第三个目标是测试原代细胞中拮抗剂的作用机制，检查 Syk 的整合素激活、整合素亲和力调节和血小板功能。所有初级 ALPHA 筛选都将根据 NIH 化学基因组中心指导下的 HTS 指标进行优化。将进行小分子化合物库的中试筛选，以证明初级和二级筛选的易处理性。这里鉴定的小分子拮抗剂可能代表了开发新型抗血小板和抗炎候选药物的良好起点，这些候选药物可以在细胞内水平靶向整合素功能。此外，源自此处提出的 HTS 测定的小分子化合物可能是分析“由内而外”和“由外而内”整合素信号转导的重要新工具。公共健康相关性：该提案中开发的筛选平台将导致新型小分子拮抗剂的发现，这可能代表心血管疾病和其他疾病的粘附分子疗法开发的新方向。此外，所鉴定的小分子拮抗剂将成为调节体外和体内细胞粘附分子功能的分子机制基础研究的重要工具。