Platelet alphaIIbbeta3 activation and its therapeutic targeting

摘要 血小板αIIbβ3是正常止血所必需的，但也是一个经过验证的药物靶点，因为它具有重要的 病理性血栓形成的作用。αIIbβ3通常在循环血小板上保持无活性的弯曲构象， 但响应于由内向外的信号传导而迅速转变为活性（配体结合）构象。配体- 被占据的受体然后通过αβ跨膜（TM）和胞质结构域传递由外向内的信号 其引发血小板粘附，这是由目前的正构抑制药物无意中产生的反应， 限制了其临床疗效。双向整合素信号传导的结构基础仍有待澄清。 一种模型认为，配体结合位点在弯曲构象中直接面对质膜， 其中整合素β延伸是接近配体所必需的。然而，整联蛋白胞外域/TM倾斜并没有 被实验定义。此外，整联蛋白中调节的αβ TM结构域缔合是由αβ TM结构域驱动的。 胞外域，这可以解释在报道的分离的化合物的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， 在相对于膜的意想不到的方向上，其与四跨膜剂顺式复合的结果， 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倾斜的新见解。 αIIbβ3激活的结构基础。我们还开发了一种水溶性的、稳定的、高亲和力的正构 非部分激动剂αIIbβ3抑制剂，并将其用于结构指导的方法以产生类似 化合物.我们建议在三个具体目标中扩展这些初步数据，利用多学科 方法包括电子低温显微镜、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的小鼠血栓形成模型。

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.