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目前可用的药物更有利的特性的整合素IIb 3的新抑制剂。第一个目标是通过高通量筛选发现新的拮抗剂，第二个目标是评估它们对IIb3的特异性，第三个目标是表征它们的抑制机制。新的抑制剂的开发具有重要的临床需求，因为目前的IIb3拮抗剂已被证明会导致一些患者的血小板减少，而在III期试验中，口服活性的IIb3抑制剂矛盾地导致死亡率增加。所有的口服活性药物，以及FDA批准的两种小分子注射用药物，都是通过竞争性地阻断IIb 3的配体结合部位发挥作用的；因此，这些药物起到了配体模拟剂的作用。已知的是，IIb3与配体的结合会导致受体胞外结构的构象变化，这些变化暴露了受体上的新表位，称为配体诱导结合位点(LIBs)。这些构象变化可能是口服药物引起的血小板减少和矛盾的血栓前反应的基础。因此，缺乏配体模拟活性的IIb3拮抗剂可能会提高治疗的安全性和有效性。具体目的1：以血小板与纤维蛋白原的黏附为筛选指标，寻找新型的抑制血小板黏附纤维蛋白原的药物。我们已经用这种方法筛选了33,000多种化合物。特异目的2：通过检测在初步筛选中发现的任何抑制剂对其他多种细胞黏附分子的作用，包括但不限于：高度同源的整合素V3与玻璃连素的相互作用，整合素21与胶原的相互作用，以及GPIB与von Willebrand因子的相互作用，来评价这些抑制剂的特异性。还将评估这些化合物对细胞活性的影响。具体目的3：研究这些化合物抑制血小板与纤维蛋白原黏附的机制。这些化合物是否直接与IIb3相互作用：1)加入外源性激活剂后血浆中的血小板聚集；2)纤维蛋白原与纯化的IIb3结合；3)表达IIb3的细胞与固定化纤维蛋白原的粘附性；4)IIb3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)测量配体诱导结合位点(LIBS)表位在血小板上的暴露；2)测试化合物瞬时暴露于“优质”纯化的IIb 3以与纤维蛋白原结合的能力。最后，抑制IIb 3但不会引起受体构象变化的化合物将被选择用于进一步的电子对接研究，体内血栓形成和止血动物模型的测试，以及作为潜在治疗开发的前奏的化学修饰。

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.