Integrin alphaIIbbeta3 Structure, Activation, and Ligand Binding

整合素 αIIbbeta3 结构、激活和配体结合

• 批准号: 8722585
• 负责人: Barry Coller
• 金额: $ 60.94万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1976
• 资助国家: 美国
• 起止时间: 1976-09-01 至 2017-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8722585
• 关键词: 3-Dimensional; Accounting; Address; Adhesions; Algorithms; Applications Grants; Aspirin; Binding; Binding Sites; Biological Assay; Biology; Blood Platelets; Blood Vessels; C-terminal; Cardiovascular Diseases; Caring; Cell Line; Cells; Chemicals; Clinical; Complementary DNA; Computer Simulation; Computing Methodologies; Cryoelectron Microscopy; Crystallography; Cytoplasmic Tail; DNA; DNA Double Strand Break; DNA Repair; Data; Detection; Detergents; Development; Diagnosis; Disease; Docking; Drug Targeting; Electron Microscopy; Family; Fibrinogen; Genes; Glycoproteins; Goals; Grant; Head; Health; Hemostatic function; Hospitals; Human; Hybrids; Inflammation; Integrins; Knowledge; Lead; Ligand Binding; Ligands; Lipid Bilayers; Mass Spectrum Analysis; Mediating; Medical; Methods; Modeling; Modification; Molecular; Molecular Conformation; Monitor; Monoclonal Antibodies; Mus; Mutate; Mutation; Myocardial Infarction; National Heart, Lung, and Blood Institute; Natural Immunity; Negative Staining; Neoplasm Metastasis; Pathway interactions; Patients; Pattern; Pharmaceutical Chemistry; Phase; Phenotype; Physiology; Plasma; Platelet Glycoprotein GPIIb-IIIa Complex; Platelet Glycoproteins; Platelet aggregation; Play; Prenatal Diagnosis; Proteins; RGD (sequence); Radial; Resolution; Role; Science; Side; Signal Transduction; Stroke; Structure; Surface; Talin; Techniques; Technology; Testing; Therapeutic; Thrombasthenia; Thrombocytopenia; Thrombosis; Translating; Translations; Wound Healing; Zinc Fingers; abciximab; base; combinatorial chemistry; design; filamin; flexibility; glycocalicin; high throughput screening; improved; inhibitor/antagonist; insight; meetings; molecular dynamics; mutant; nanodisk; novel; novel therapeutics; nuclease; prevent; programs; public health relevance; receptor; reconstruction; screening; simulation; three dimensional structure; virtual; zinc finger nuclease

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.

描述（由申请人提供）：血小板在止血和血栓形成中起核心作用，并导致广泛的相关现象，包括炎症和转移形成。这项拨款提案的最终目标是了解血小板与血管壁以及通过其表面上的糖蛋白受体与其他血小板相互作用的方式，并利用这些知识来改善人类健康。尽管在分析<$IIb <$3受体的结构和功能方面取得了进展，这对正常止血和抗血栓治疗的有效靶点至关重要，但在理解配体结合及其对血小板生理学的影响方面仍存在重大差距。 此外，需要改进的有效抗血小板疗法，其可以在心肌梗塞的院前阶段施用。 获得的数据 这些研究将为开发比现有药物具有治疗优势的新型<$IIb <$3抑制剂的尝试提供信息。 在具体目标1中，我们建议通过以下方式提高我们对配体结合的理解：a）使用功能性配体结合数据和<$IIb <$3的新晶体结构作为最先进的计算方法的输入，以确定纤维蛋白原？模块和<$IIb <$3除了那些由纤维蛋白原？链C端十二肽（？C-12），B）使用电子低温显微镜（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）的柔性拟合和转向MD，以在不存在去污剂的情况下获得纳米盘脂质双层中完整III B 3的原子分辨率三维（3D）表示。 非活性受体， 将分别研究在不存在配体的情况下由talin头部结构域（THD）激活的受体和在存在纤维蛋白原的情况下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的<$3突变，而不是缺乏血小板信号传导机制的细胞系中的突变，首先关注我们假设的突变将增强细丝蛋白与<$3细胞质尾的结合，从而降低血小板对激活的敏感性。 d）使用增强的MD技术来表征 ² IIb ² 3激活途径，并测试在过去的授权期内鉴定的新的<$IIb <$3拮抗剂（RUC-1，RUC-2，MSSM-1，MSSM-2）稳定封闭的， 无活性构象，因此与Arg-Gly-Asp（RGD）细胞识别序列上的<$IIb <$3拮抗剂相比，它们激活<$IIb <$3的能力降低。 在具体目标2中，我们提出确定新的化合物，这些化合物将提供对<$IIb <$3结构-功能的深入了解，并可能通过表征最有效抑制<$IIb <$3介导的血小板粘附和聚集的57种化合物（在126，000种测试化合物中）而具有治疗潜力。 我们还将进行一项新的筛选，以选择性地鉴定靶向辅助纤维蛋白原结合位点的化合物，因为这些可能导致 更安全更有效的新疗法