Elucidation of P2X7 Receptor Signaling and Development of Novel Small Molecule and Aptamer Ligand Therapies

项目概要： P2X嘌呤能受体是三聚体的非选择性阳离子通道，由细胞外ATP激活，以调节细胞内的ATP水平。 在心血管和免疫系统的过程。P2X7受体，在结构和功能上 不同的P2X受体亚型，参与细胞凋亡和炎症的信号传导途径，并预测 在炎症性疾病和动脉粥样硬化之间的联系中发挥关键作用。 动脉疾病，基因敲除P2X7受体缺陷消除动脉粥样硬化，这表明发展 P2X7特异性拮抗剂抑制P2X7信号传导可能导致预防冠状动脉粥样硬化的新疗法。 动脉疾病然而，尽管是积极追求的药理学靶点，但没有FDA批准的 靶向P2X7受体的药物。利用P2X受体作为治疗靶点受到缺乏 定义受体结构和功能分子机制的信息。最近，我的团队 发表了第一个原子分辨率结构的全长P2X7受体使用单粒子低温 电子显微镜，改变了这一前景。我的结构证明了为什么P2X7受体亚型 没有经历脱敏，并揭示了P2X7受体的胞质结构域具有新的折叠， 与蛋白质数据库中的任何折叠结构同源，含有高亲和力（纳摩尔）鸟苷 核苷酸结合位点。这一令人惊讶的发现可能开始解释细胞质结构域的激活是如何发生的。 离子型P2X7受体募集代谢型第二信使系统， 鸟苷酸结合位点揭示了受体与细胞内信号伴侣的界面。 了解这些过程和分子间的相互作用， 在细胞外结构域的P2X7受体的拮抗作用，包括在细胞质 域该基金的主要目的是利用我的P2X7受体结构作为平台，研究其在细胞内的表达。 独特的信号转导途径，并使用基于结构的药物设计，开发新的配体， 调节P2X7受体功能用于治疗目的。这一提议的一个变革方面是扩大 寻找靶向P2X7受体的配体，而不是小分子，包括核酸适体， 配体的鉴定（小分子或 适体）成功抑制P2X7受体活化或调节P2X7受体胞内信号传导， 为研究P2X受体提供了宝贵的研究工具，并有可能开发成治疗方法， 血管炎症和预防动脉粥样硬化。 P2X受体中迄今未探索的有前途的范例。

PROJECT SUMMARY: P2X purinergic receptors are trimeric, non-selective cation channels activated by extracellular ATP to modulate processes in the cardiovascular and immune systems. The P2X7 receptor, the most structurally and functionally distinct P2X receptor subtype, is involved in signaling pathways for apoptosis and inflammation, and is predicted to play a key role in the link between inflammatory disease and atherosclerosis.In a mouse model of coronary artery disease, gene knock-out P2X7 receptor deficiency abolished atherosclerosis, suggesting that development of P2X7-specific antagonists to inhibit P2X7-signaling could result in a novel therapy for the prevention of coronary artery disease. However, despite being an actively pursued pharmacologic target, there are no FDA-approved drugs targeting the P2X7 receptor. Utilizing P2X receptors as a target for therapy has been hampered by a lack of information defining the receptor’s structure and molecular mechanisms of function. Recently, my group published the first atomic-resolution structures of full-length P2X7 receptor using single particle cryogenic electron microscopy, changing this outlook. My structures demonstrated why the P2X7 receptor subtype does not undergo desensitization and revealed that the cytoplasmic domain of P2X7 receptor has a novel fold without structural homology to any fold in the Protein Data Bank, containing a high-affinity (nanomolar) guanosine nucleotide binding site. This surprising finding in the cytoplasmic domain may begin to explain how activation of the ionotropic P2X7 receptor recruits metabotropic secondary messenger systems, with the location of the guanosine nucleotide binding site revealing where the receptor interfaces with intracellular signaling partners. Understanding these processes and molecular interactions wouldexpand pharmaceutical strategies beyond antagonism of P2X7 receptor at the extracellular domain to include modulating signaling at the cytoplasmic domain. The major aim of this grant is to use my structures of P2X7 receptor as a platform to investigate its unique signal transduction pathways and, using structure-based drug design, to develop novel ligands to modulate P2X7 receptor function for therapeutic purposes. A transformative aspect of this proposal is expanding the search of ligands targeting P2X7 receptor beyond small-molecules to include nucleic acid aptamers, a Identification of ligands (either small molecule or aptamer) that successfully inhibit P2X7 receptor activation or modulate P2X7 receptor intracellular signaling will provide invaluable research tools for studying P2X receptors with the potential to be developed into therapies for vascular inflammation and to prevent atherosclerosis. promising paradigm heretofore unexplored in P2X receptors.