Platelet alphaIIbbeta3 activation and its therapeutic targeting

摘要 血小板αIIbβ3是正常止血所必需的，但也是有效的药物靶点，因为它是必不可少的 在病理性血栓形成中的作用。αIIbβ3通常保持在循环血小板上的非活性弯曲构象中， 但响应内向外信号，迅速转换为活性(配体结合)构象。配基- 然后，被占领的受体通过αβ跨膜(TM)和细胞质结构域传递由外向内的信号 这种反应是由目前的正构体抑制药物无意中产生的，它 限制了它们的临床疗效。双向整合素信号转导的结构基础尚不清楚。 一种模型认为，在弯曲构象中，配体结合位置直接面对质膜， 具有获得配体所需的整合素真延伸。然而，整合素的胞外结构域/TM倾斜并没有 是通过实验定义的。此外，整合素中受调控的αβTM结构域关联是由 胞外结构域，这可能解释了已报道的分离的核磁共振结构中的一些差异 αIIbβ3 TM域。在初步研究中，我们生产了全长αIIbβ3的低分辨率低温EM结构 在相对于膜的意想不到的方向上，其结果是它的顺式与四叶刀的复合体， 提供了第一个整合素胞外结构域/TM倾斜度的实验定义，并对 αIIbβ3激活的结构基础。我们还开发了一种水溶性、稳定性和高亲和力的正构体 不是部分激动剂的αIIbβ3的抑制剂，并在结构指导的方法中使用它来产生LIKE 化合物。我们建议在三个具体目标上扩展这些初步数据，利用多学科 方法包括电子冷冻显微镜、断层扫描、蛋白质结晶学、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.