Platelet alphaIIbbeta3 activation and its therapeutic targeting

血小板αIIbbeta3激活及其治疗靶向

• 批准号: 10251142
• 负责人: M. AMIN ARNAOUT
• 金额: $ 53.35万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10251142
• 关键词: 3-Dimensional; Adhesions; Affinity; Agonist; Agreement; American; Binding; Binding Sites; Biological Availability; Blood Platelets; Cardiology; Cell membrane; Cessation of life; Collaborations; Complex; Cryoelectron Microscopy; Crystallization; Crystallography; Cytoplasmic Tail; Data; Detergents; Development; Drug Design; Drug Targeting; Early treatment; European; Event; Excision; Face; Guidelines; Hemorrhage; Hemostatic function; Heterogeneity; Hospitals; Immune; Impairment; Integral Membrane Protein; Integrins; Leg; Length; Libraries; Life; Ligand Binding; Ligands; Link; Measures; Membrane; Membrane Potentials; Methodology; Modeling; Molecular Conformation; Monitor; Myocardial Infarction; Nature; Negative Staining; Pathologic; Patients; Peptide Synthesis; Peptides; Pharmaceutical Preparations; Phase; Platelet aggregation; Pre-hospital setting; Preparation; Primates; Prior Therapy; Proteins; Publishing; Receptor Activation; Recombinants; Regulation; Reporting; Resolution; Risk; Role; Signal Transduction; Societies; Solid; Structure; Structure-Activity Relationship; Surface Plasmon Resonance; Technology; Testing; Therapeutic; Thrombocytopenia; Thrombosis; Transmembrane Domain; Water; X-Ray Crystallography; adverse outcome; attack victim; base; clinical efficacy; coronary thrombus; design; eptifibatide; high risk; improved; improved outcome; inhibitor/antagonist; insight; interdisciplinary approach; mimetics; mouse model; novel; percutaneous coronary intervention; prevent; receptor; response; small molecule; therapeutic target; three dimensional structure; tomography; two-dimensional

Abstract Platelet αIIbβ3 is required for normal hemostasis, but is also a validated drug target because of its essential role in pathologic thrombosis. αIIbβ3 is normally kept in an inactive bent conformation on circulating platelets, but rapidly switches to an active (ligand-binding) conformation in response to inside-out signaling. The ligand- occupied receptor then transmits outside-in signals via the αβ transmembrane (TM) and cytoplasmic domains that initiate platelet adhesion, a response inadvertently produced by current orthosteric inhibitory drugs, which has limited their clinical efficacy. The structural basis of bidirectional integrin signaling remains to be clarified. One model suggests that the ligand-binding site directly faces the plasma membrane in the bent conformation, with integrin genuextension required for access to ligand. However, the integrin ectodomain/TM tilt has not been defined experimentally. Further, regulated αβ TM domain association in integrins is driven by the ectodomain, which may explain some of the differences found in the reported NMR structures of the isolated αIIbβ3 TM domains. In preliminary studies, we produce a low-resolution cryo-EM structure of full-length αIIbβ3 in an unexpected orientation relative to the membrane, the result of its complex in cis with a tetraspanner, providing the first experimental definition of an integrin ectodomain/TM tilt, and novel insights into the structural basis of αIIbβ3 activation. We also develop a water soluble, stable and high affinity orthosteric inhibitor of αIIbβ3 that is not a partial agonist, and use it in a structure-guided approach to generate like compounds. We propose to expand on these preliminary data in three specific aims, utilizing multidisciplinary approaches that include electron cryomicroscopy, tomography, protein crystallography, structure-based drug design, new peptide synthesis technology and novel murine models of thrombosis.

抽象的 血小板 αIIbβ3 是正常止血所必需的，但由于其重要作用，它也是经过验证的药物靶点 在病理性血栓形成中的作用。 αIIbβ3 通常在循环血小板上保持非活性弯曲构象， 但响应由内而外的信号传导，迅速切换到活性（配体结合）构象。配体- 然后，占据的受体通过 αβ 跨膜 (TM) 和细胞质结构域传递由外而内的信号 启动血小板粘附，这是当前正构抑制药物无意中产生的反应， 限制了它们的临床疗效。双向整合素信号传导的结构基础仍有待阐明。 一种模型表明配体结合位点直接面向弯曲构象的质膜， 具有接近配体所需的整合素基因延伸。然而，整合素胞外域/TM倾斜并没有 已通过实验定义。此外，整合素中受调节的 αβ TM 结构域关联是由 胞外域，这可以解释所报告的分离的 NMR 结构中发现的一些差异 αIIbβ3 TM 结构域。在初步研究中，我们制作了全长 αIIbβ3 的低分辨率冷冻电镜结构 相对于膜以意想不到的方向，其与四扳手顺式复合的结果， 提供了整合素胞外域/TM倾斜的第一个实验定义，以及对整合素胞外域/TM倾斜的新颖见解 αIIbβ3 激活的结构基础。我们还开发了一种水溶性、稳定和高亲和力的正构 αIIbβ3 的抑制剂，不是部分激动剂，并在结构引导方法中使用它来生成类似的 化合物。我们建议利用多学科，在三个具体目标上扩展这些初步数据 方法包括电子冷冻显微镜、断层扫描、蛋白质晶体学、基于结构的药物 设计、新的肽合成技术和新型小鼠血栓形成模型。