High Throughput Screen for Novel Inhibitors of Platelet Integrin alphaIIb-beta3

描述（由申请人提供）：拟议筛选项目的长期目标是鉴定具有比现有药物更有利特性的整合素 IIb 3 新型抑制剂。第一个具体目标是通过高通量筛选鉴定其他新型拮抗剂，第二个目标是评估其对 IIb 3 的特异性，第三个目标是表征其抑制机制。新抑制剂的开发代表了重要的临床需求，因为目前的 IIb 3 拮抗剂已被证明会导致一些患者出现血小板减少症，而口服活性 IIb 3 抑制剂在 III 期试验中反而导致死亡率增加。所有口服活性药物以及 FDA 批准的 2 种小分子肠胃外药物均通过竞争性阻断 IIb 3 的配体结合位点发挥作用；因此，这些试剂充当配体模拟物。众所周知，IIb 3 与配体的结合会导致受体胞外结构的构象变化，并且这些变化会暴露受体上的新表位，称为配体诱导的结合位点 (LIBS)。这些构象变化可能是血小板减少症和口服药物矛盾的促血栓形成作用的基础。因此，缺乏配体模拟活性的 IIb 3 拮抗剂可以提高治疗安全性和疗效。具体目标 1：使用血小板与纤维蛋白原的粘附作为筛选试验，鉴定血小板与纤维蛋白原粘附的新型抑制剂。我们已经使用该测定法筛选了 33,000 多种化合物。具体目标 2：通过测试初级筛选中确定的任何抑制剂对各种其他细胞粘附分子的影响来评估其特异性，这些分子包括但不限于：高度同源的整合素 V 3 与玻连蛋白的相互作用、整合素 2 1 与胶原蛋白的相互作用以及 GPIb 与冯·维勒布兰德因子的相互作用。还将评估化合物对细胞活力的影响。具体目标 3：表征化合物抑制血小板与纤维蛋白原粘附的机制。将采用以下测定来确定化合物是否与IIb 3直接相互作用：1)添加外源激活剂后血浆中血小板的聚集； 2)纤维蛋白原与纯化的IIb 3结合； 3)表达IIb 3的细胞与固定化纤维蛋白原的粘附； 4)用IIb 3 激活剂处理后可溶性纤维蛋白原与血小板的结合。那些直接与 IIb 3 相互作用的化合物将被进一步表征，以评估它们对 IIb 3 构象的影响，方法是：1) 测量血小板上配体诱导的结合位点 (LIBS) 表位的暴露，2) 测试化合物瞬时暴露“引发”纯化的 IIb 3 使其与纤维蛋白原结合的能力。最后，将选择抑制 IIb 3 但不诱导受体构象变化的化合物进行额外的计算机对接研究、血栓形成和止血动物模型的体内测试以及化学修饰，作为潜在治疗开发的前奏。

DESCRIPTION (provided by applicant): The long term goal of the proposed screening project is the identification of novel inhibitors of the integrin IIb 3 with more favorable characteristics than currently available agents. The first specific aim is the identification of additional novel antagonists via high throughput screening, the second is the assessment of their specificity for IIb 3, and the third is the characterization of their mechanisms of inhibition. The development of new inhibitors represents a significant clinical need because current IIb 3 antagonists have been shown to cause thrombocytopenia in some patients, and the orally active IIb 3 inhibitors paradoxically caused increased mortality in Phase III trials. All of the orally active agents, as well as the 2 FDA-approved small molecule parenteral agents, function by competitively blocking the ligand binding site of IIb 3; thus, these agents act as ligand-mimetics. It is known that the binding of ligand by IIb 3 results in conformational changes in the receptor extracellular structure, and that these changes expose neo-epitopes on the receptor known as ligand-induced binding sites (LIBS). These conformational changes may underlie both the thrombocytopenia and the paradoxical prothrombotic effects of the oral agents. Thus, IIb 3 antagonists that lack ligand-mimetic activity may improve both therapeutic safety and efficacy. Specific Aim 1: To identify novel inhibitors of platelet adhesion to fibrinogen using platelet adhesion to fibrinogen as the screening assay. We have already used this assay to screen more than 33,000 compounds. Specific Aim 2: To assess the specificity of any inhibitors identified in the primary screen by testing their effects on various other cell adhesion molecules including but not limited to: interaction of the highly homologous integrin V 3 with vitronectin, interaction of integrin 2 1 with collagen, and interaction of GPIb with von Willebrand factor. The effect of the compounds on cell viability will also be assessed. Specific Aim 3: To characterize the mechanism by which the compounds inhibit adhesion of platelets to fibrinogen. The following assays will be employed to determine whether compounds interact directly with IIb 3: 1) aggregation of platelets in plasma following the addition of exogenous activators; 2) binding of fibrinogen to purified IIb 3; 3) adhesion of cells expressing IIb 3 to immobilized fibrinogen; 4) binding of soluble fibrinogen to platelets following treatment with IIb 3 activators. Those compounds that interact directly with IIb 3 will be characterized further to assess their effects on the conformation of IIb 3 by 1) measuring the exposure of ligand-induced binding site (LIBS) epitopes on platelets, and 2) testing the ability of transient exposure of the compounds to "prime" purified IIb 3 so that it binds fibrinogen. Finally, compounds that inhibit IIb 3 but do not induce conformational changes in the receptor will be selected for additional in silico docking studies, in vivo testing in animal models of thrombosis and hemostasis, and chemical modifications as a prelude to potential therapeutic development.