HTS platform for identification of regulators of cell adhesion molecule signaling

描述(由申请人提供)：以下建议是对PA-10-213题为“用于探针和治疗前发现(R01)的高通量筛查分析的发展”的计划公告的回应。整合素细胞黏附受体在正常的生理过程中起着重要的作用，并与肿瘤、自身免疫、炎症和心血管疾病有关。事实上，它们是有效的药物靶标。目前基于整合素的治疗方法已被证明对患者的治疗非常有价值，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。整合素结合多种细胞外配体，如血清纤维蛋白原、细胞外基质(ECM)成分和细胞表面受体。它们的活性受到细胞内支架分子如Talin-1和Kindlins的严格调控，这些分子可以与整合素细胞质结构域结合，并在一个被称为“内向外”的信号传递过程中增加整合素与配体的胞外亲和力。整合素也可以作为信号受体，其中“由外而内”的信号是通过整合素细胞质结构域和细胞内效应分子(如Syk家族的非受体酪氨酸激酶)之间的直接相互作用来介导的。我们开发了一个用于高通量筛选小分子化合物文库的无细胞分析平台，用于整合素细胞质结构域和细胞内效应分子之间相互作用的拮抗剂。该系统使用放大发光接近均相分析(Alpha)屏幕格式。在我们的第一个目标中，我们建议建立一套针对整合素Beta3细胞质结构域与Syk、Kindlin-3和Galpha13结合的初级HTS分析方法。还将开发一种辅助筛查，以对假阳性命中进行分类。在第二个目标中，我们提出了一些正交分析方法，从基于ELISA的方法到等温滴定量热法和表面等离子体共振，这些方法将用于验证HIT的特异性和选择性。最后，一个 第三个目的是测试拮抗剂在原代细胞中的作用机制，检测Syk的整合素激活、整合素亲和力调节和血小板功能。在NIH化学基因组中心的指导下，所有初级Alpha屏幕将根据HTS指标进行优化。将进行小分子化合物文库的中试筛选，以展示初级和次级筛选的可操作性。发现的小分子拮抗剂可能是开发新型抗血小板和抗炎药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，从本文提出的HTS分析中衍生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。 公共卫生相关性：本提案中开发的筛选平台将导致发现新的小分子拮抗剂，这可能代表心血管疾病和其他疾病的黏附分子疗法发展的新方向。此外，识别的小分子拮抗剂也将是在体外和体内调节细胞黏附分子功能的分子机制基础研究的重要工具。

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.