P2Y6R在动脉粥样硬化中的作用及先导化合物的靶向干预机制研究

Pro-atherogenic stimuli-induced abnormal behavior of vascular endothelial cells, mononuclear macrophages and vascular smooth muscle cells play key roles in the disease course of atherosclerosis (AS), nevertheless, the regulatory mechanisms are poorly understood. P2Y6R activates Ca2+-dependent signaling pathways through phospholipase Cβ by coupling Gq/11 protein, which is highly expressed in cardiovascular system. Deficiency of P2Y6R could improve high cholesterol diet-induced AS and plaque inflammation, which prompted us that P2Y6R was associated with AS, but the mechanisms were not clear. Since Ca2+ is closey related to signaling pathways involved in pyroptosis, foaming, proliferation of cells, we put forward a hypothesis that P2Y6R regulates behavior of core cells participated in the pathogenesis of AS through Gq/11-PLCβ-Ca2+ pathways, which will be confirmed by three perspectives of cells, animals and clinical samples. EBBC is a lead compound first synthesized by our research group as a potent P2Y6R inhibitor, which could attenuate AS in mice. We plan to establish in vivo and in vitro models to reveal the molecular mechanisms by which the lead compound improves AS targeting P2Y6R, that is the lead compound blocks pyroptosis of vascular endothelial cells macrophages, inhibits foaming and pyroptosis of mononuclear macrophages, regulates proliferation and pyroptosis of vascular smooth muscle cells.

动脉粥样硬化（AS）病程中，促AS刺激物介导的内皮细胞、单核巨噬细胞、血管平滑肌细胞的异常行为起关键作用，但其调控机制不清。嘌呤受体P2Y6R在心血管系统中高度表达，该受体与Gq/11蛋白偶联，通过磷脂酶Cβ（PLCβ）途径，激活Ca2+依赖的信号通路调控细胞生理功能，其缺失能够缓解高胆固醇饮食诱导的AS和斑块炎症，提示P2Y6R与AS病程相关，但确切机制不明。基于Ca2+与细胞焦亡、泡沫化、增殖相关信号通路的联系，申请人提出P2Y6R通过Gq/11-PLCβ-Ca2+途径调控AS核心参与细胞行为而影响AS病程的假说，拟从细胞、动物和临床样本三个层面进行确证。EBBC是本课题组首次合成的具有P2Y6R抑制活性的先导化合物，能显著缓解小鼠AS。本项目拟构建体内外模型，揭示其靶向P2Y6R阻断血管内皮细胞焦亡、抑制单核巨噬细胞泡沫化和焦亡、调控血管平滑肌细胞增殖和焦亡，从而防治AS的分子机制。

动脉粥样硬化以脂质代谢障碍为病变基础，是促动脉粥样硬化刺激物介导内皮细胞、单核巨噬细胞、血管平滑肌细胞等发生交互作用而形成的一种慢性炎症性疾病，除降血脂药物外缺少理想的治疗药物，故探索动脉粥样硬化治疗的原创靶标，寻求全新的治疗策略成为了重点。本研究基于嘌呤受体家族成员P2Y6受体（P2Y6R）的异常表达与动脉粥样硬化的临床相关性，聚焦P2Y6R在巨噬细胞和内皮细胞异常行为调控中的潜在作用，围绕提出的假说展开了系统研究。结果显示，P2Y6R的全身敲除能够显著抑制高脂饮食诱导的小鼠动脉粥样硬化，巨噬细胞特异性P2Y6R缺失同样能够缓解小鼠的血管病变。具体机制上，P2Y6R依赖PLCβ信号调控钙库操纵的钙内流（SOCE），继而通过钙网蛋白（CALR）干预清道夫受体SR-A的表达，在ox-LDL介导的巨噬细胞泡沫化过程中发挥关键作用。另一方面，P2Y6R依赖钙离子（Ca2+）信号调控NLRP3炎症小体的组装，干预ox-LDL导致的巨噬细胞和内皮细胞焦亡。在证明了P2Y6R可能是动脉粥样硬化的潜在治疗靶标基础上，研究采用多构象整合预测模型在老药新用库和天然产物库中进行了筛选，发现了焦磷酸硫胺素（TPP）和木通苯乙醇苷B等高活性的P2Y6R潜在抑制剂。与此同时，对从小分子化合物库中筛选得到的苗头化合物进行结构修饰和成药性优化，得到了若干高效低毒、结构新颖的全新P2Y6R抑制剂，通过体内外模型的验证，阐明了先导化合物治疗动脉粥样硬化的分子机制。本研究为动脉粥样硬化的治疗提供了原创的潜在治疗靶标，同时为动脉粥样硬化及其他P2Y6R相关疾病治疗药物的研发奠定了基础。