HTS platform for identification of regulators of cell adhesion molecule signaling

描述（由申请人提供）：以下提案是对项目公告PA-10-213的回应，该公告标题为“开发用于探针和治疗前发现的高通量筛选试验（R 01）”。“整合素细胞粘附受体在正常生理过程中发挥重要作用，并与肿瘤形成、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。目前基于整合素的治疗剂已被证明在患者的治疗中是无价的，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）组分和细胞表面反受体。它们的活性受到细胞内支架分子如Talin-I和Kindlin的严格调节，所述细胞内支架分子可以结合整联蛋白胞质结构域，并且在称为“由内向外”信号传导的过程中增加整联蛋白胞外域对配体的亲和力。整联蛋白还充当信号传导受体，其中“由外而内”信号传导由整联蛋白胞质结构域与细胞内效应物（例如Syk家族的非受体酪氨酸激酶）之间的直接相互作用介导。我们已经开发了一个无细胞的测定平台，用于高通量筛选（HTS）的小分子化合物库的整合素胞质结构域和细胞内效应分子之间的相互作用的拮抗剂。该系统采用扩增发光邻近均相测定（ALPHA）筛选形式。在我们的第一个目标中，我们建议开发一套主要的HTS检测方法，靶向整合素β 3细胞质结构域与Syk，Kindlin-3和Galpha 13的结合。还将开发二次筛选，以筛选假阳性命中。在第二个目标，我们提出了一些正交试验，从ELISA为基础的方法，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的特异性和选择性。最后 第三个目的是测试拮抗剂在原代细胞中的作用机制，检查Syk的整联蛋白活化、整联蛋白亲和力调节和血小板功能。 所有初级ALPHA筛选将根据NIH化学基因组学中心指导的HTS指标进行优化。将对小分子化合物文库进行中试筛选，以证明初步和二次筛选的易处理性。本文鉴定的小分子拮抗剂可能代表开发新型抗血小板和抗炎药物候选物的良好起点，其可以在细胞内水平靶向整合素功能。此外，小分子化合物来源于HTS检测提出了这里可能是重要的新工具，在分析“由内而外”和“由外而内”的整合素信号转导。 公共卫生相关性：在该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粘附分子治疗剂开发的新方向。此外，小分子拮抗剂的确定将是重要的工具，基础研究的分子机制调节细胞粘附分子的功能，在体外和体内。

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.