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αIIbβ3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 这些研究将为开发与现有药物相比具有治疗优势的αIIbβ3新型抑制剂的尝试提供信息。在特定目标1中，我们建议通过以下方式提高我们对配体结合的理解：a)使用功能配体结合数据和αIIbβ3的新晶体结构作为最先进的计算方法的输入，以确定纤维蛋白原之间的相互作用 γ-模块和αIIbβ3除了由纤维蛋白原γ链C-末端十二肽(γC-12)制成的那些外，b)使用电子冷冻显微镜(Cryo-EM)和负染色EM结合随机锥形倾斜重建，基于分子动力学(MD)的灵活拟合和引导MD获得在没有洗涤剂的情况下完整的αIIbβ3在纳米盘脂双层中的原子分辨率三维(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中β3的突变，而不是在缺乏血小板信号机制的细胞系中，最初专注于一种突变，我们假设该突变将增强细丝蛋白与β3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αIIbβ3激活通路，并测试在过去的授权期中发现的新的αIIbβ3拮抗剂(RUC-1、RUC-2、MSSM-1、MSSM-2)稳定闭合的、 与基于精氨酸-甘氨酸-天冬氨酸(α-Gly-Asp)细胞识别序列的β3拮抗剂相比，它们激活αIIbβ3的能力降低。 在特定目标2中，我们建议通过鉴定最有效地抑制αIIbβ3介导的血小板黏附和聚集的57个化合物来鉴定新的化合物，这些化合物将为αIIbβ3的结构功能提供洞察力，并可能具有治疗潜力。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 αIIbβ3 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 αIIbβ3 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 αIIbβ3 as inputs to state-of-the art computational methods to identify interactions between the fibrinogen γ-module and αIIbβ3 in addition to those made by the fibrinogen γ-chain C-terminal dodecapeptide (γC-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 αIIbβ3 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 β3 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 β3 cytoplasmic tail and thus diminish platelet sensitivity to activation. d) Using enhanced MD techniques to characterize αIIbβ3 activation pathways and testing the hypothesis that the new αIIbβ3 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 αIIbβ3 compared to αIIbβ3 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 αIIbβ3 structure-function and may have therapeutic potential by characterizing the 57 compounds (out of 126,000 tested) that most potently inhibit αIIbβ3-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.