Integrin alphaIIbbeta3 Structure, Activation, and Ligand Binding

描述(申请人提供)：血小板在止血和血栓形成中发挥中心作用，并有助于广泛的相关现象，包括炎症和转移的形成。这项赠款计划的最终目标是了解血小板与血管壁以及通过其表面的糖蛋白受体与其他血小板相互作用的方式，并利用这一知识改善人类健康。尽管在分析IIb受体的结构和功能方面取得了进展，但在了解配体结合及其对血小板生理的影响方面仍存在重大差距。此外，有必要改进有效的抗血小板治疗，使其可以在心肌梗死的院前阶段使用。从以下来源获得的数据 这些研究将为开发比现有药物具有治疗优势的新型IIb抑制剂的尝试提供信息。在具体目标1中，我们建议通过以下方法提高我们对配体结合的理解：a)使用功能性配体结合数据和新的IIb晶体结构作为最新计算方法的输入，以确定纤维蛋白原模块与IIb模块之间的相互作用，以及纤维蛋白原C-末端十二肽(C-12)所产生的相互作用，b)使用电子冷冻显微镜(Cryo-EM)和负染色EM结合随机锥体倾斜重建，基于分子动力学(MD)的灵活拟合和操纵MD，在没有洗涤剂的情况下，获得完整的纳米盘状脂质双层中完整IIb的三维(3D)表示。不活跃的受体， 分别研究在没有配体的情况下由talin头域(THD)激活的受体，以及在纤维蛋白原存在的情况下由THD激活的受体。C)使用锌指核酸酶介导的基因编辑来评估小鼠血小板而不是缺乏血小板信号机制的细胞系中？3的突变，最初专注于一种突变，我们假设该突变将增强细丝蛋白与？3细胞质尾部的结合，从而降低血小板对激活的敏感性。D)使用增强的MD技术来表征 3激活途径，并检验在过去的授权期内发现的新的IIb拮抗剂(RUC-1，RUC-2，MSSM-1，MSSM-2)稳定闭合的， 与根据Arg-Gly-Asp(RGD)细胞识别序列构型的IIb拮抗剂相比，它们激活IIb的能力降低。在具体目标2中，我们建议通过鉴定最有效地抑制IIb介导的血小板黏附和聚集的57种化合物来鉴定新的化合物，这些化合物将提供对IIb结构功能的洞察，并可能具有治疗潜力(在测试的126,000种化合物中)。我们还将进行一项新的筛选，以选择性地识别以辅助纤维蛋白原结合位点为靶点的化合物，因为这些化合物可能会导致 更安全、更有效的新疗法。

DESCRIPTION (provided by applicant): Platelets play a central role in both hemostasis and thrombosis, and contribute to a wide range of related phenomena, including inflammation and metastasis formation. The ultimate goal of this grant proposal is to understanding the way in which platelets interact with the blood vessel wall and with other platelets via the glycoprotein receptors on their surface, and to use that knowledge to improve human health. Despite advances in analyzing the structure and function of the ¿IIb¿3 receptor, which is crucial for normal hemostasis and a validated target of antithrombotic therapy, major gaps remain in understanding ligand binding and its impact on platelet physiology. Moreover, there is a need for improved potent antiplatelet therapies that cn be administered in the pre-hospital phase of myocardial infarction. The data obtained from these studies will inform attempts to develop novel inhibitors of ¿IIb¿3 that have therapeutic advantages over existing agents. In Specific Aim 1 we propose to improve our understanding of ligand binding by: a) Using functional ligand binding data and new crystal structures of ¿IIb¿3 as inputs to state-of-the art computational methods to identify interactions between the fibrinogen ?-module and ¿IIb¿3 in addition to those made by the fibrinogen ?- chain C-terminal dodecapeptide (?C-12), b) Using electron cryomicroscopy (cryo-EM) and negative stain EM in conjunction with random conical tilt reconstructions, molecular dynamics (MD)-based flexible fitting and steered MD to obtain atomic resolution 3-dimensional (3D) representations of intact ¿IIb¿3 in a nanodisc lipid bilayer in the absence of detergent. The inactive receptor, the receptor activated by talin head-domain (THD) in the absence of ligand, and THD-activated receptor in the presence of fibrinogen will each be studied. c) Using zinc finger nuclease-mediated gene editing to evaluate mutations of ¿3 in murine platelets rather than in cell lines that lack the platelet's signaling machinery, focusing initially on a mutation we hypothesize will enhance the binding of filamin to the ¿3 cytoplasmic tail and thus diminish platelet sensitivity to activation. d) Using enhanced MD techniques to characterize ¿IIb¿3 activation pathways and testing the hypothesis that the new ¿IIb¿3 antagonists identified in the past grant period (RUC-1, RUC-2, MSSM-1, MSSM-2) stabilize a closed, inactive conformation, thus accounting for their reduced ability to activate ¿IIb¿3 compared to ¿IIb¿3 antagonists patterned on the Arg-Gly-Asp (RGD) cell recognition sequence. In Specific Aim 2 we propose to identify new compounds that will provide insights into ¿IIb¿3 structure-function and may have therapeutic potential by characterizing the 57 compounds (out of 126,000 tested) that most potently inhibit ¿IIb¿3-mediated platelet adhesion and aggregation. We will also perform a new screen to selectively identify compounds that target ancillary fibrinogen binding sites since these may lead to new therapeutics that are safer and more efficacious.