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：
8420443
负责人：
DARREN G WOODSIDE
金额：
$ 35.96万
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/8420443
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 Kinetics Drug Targeting 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 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 screening 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.

描述（由申请人提供）：以下提案是对计划公告PA-10-213的响应，标题为“开发用于用于探针和治疗前发现（R01）的高通量筛选的测定法”。整联蛋白细胞粘附受体在正常生理过程中起着至关重要的作用，并且与肿瘤，自身免疫性，炎症和心血管疾病有关。确实，它们是经过验证的药物靶标。目前基于整合素的治疗剂在患者的治疗中已证明是无价的，但受到与免疫抑制和诱导自身免疫反应有关的副作用的限制。需要一种新的策略来靶向整合素，以消除副作用。整联蛋白结合多种细胞外配体，例如血清纤维蛋白原，细胞外基质（ECM）成分和细胞表面反对受体。它们的活性受到细胞内支架分子的严格调节，例如talin-1和Kindlins，它们可以与整联蛋白的细胞质结构域结合，并且在称为“内部”信号传导的过程中增加了整联蛋白外节域对配体的亲和力。整联蛋白还用作信号受体，其中“外部”信号传导是由整联蛋白细胞质结构域与细胞内效应子（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。我们已经开发了一个无细胞的测定平台，用于用于小分子化合物库的高吞吐量筛选（HTS），用于整合素细胞质结构域与细胞内效应分子之间相互作用的拮抗剂。该系统利用了放大的发光近端同质测定法（Alpha）屏幕格式。在我们的第一个目标中，我们建议开发针对整联蛋白beta3细胞质结构域与SYK，Kindlin-3和Galpha13结合的主要HTS测定套件。还将开发一个二级屏幕，即伪造积极命中。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，这些分析将有助于验证HIT特异性和选择性。最后，一个提出了第三个目的，以测试原代细胞中作用机理，检查SYK的整联蛋白激活，整联蛋白亲和调节和血小板功能。 NIH化学基因组学中心指导的HTS指标将根据HTS指标进行优化所有原发性α屏幕。将执行一个小分子化合物库的试验屏幕，以证明原发性和次级筛选性。这里确定的小分子拮抗剂可能代表了新型抗血清和抗炎药候选物的出色起点，这些抗血压和抗炎药候选者可以靶向整合素在细胞内水平。此外，从这里提出的HTS分析得出的小分子化合物可能是分析“内部”和“外部”整合素信号转导的重要新工具。