Identification of an inhibitor of PDI-GPIbalpha signaling as a novel antithromboinflammatory agent

项目摘要 血栓炎症性疾病，包括动脉粥样硬化血栓形成、中风和外周血管炎，导致30% 全球所有死亡病例。血栓性炎症的病理基础是血小板粘附性增加和 白细胞。虽然许多抗血小板和抗炎疗法已被用于疾病治疗， 这些药物会增加大出血或损害免疫反应的风险。利用蛋白质二硫键异构酶 (PDI)条件性基因敲除(CKO)小鼠和抑制剂，我们和其他人已经证明细胞外PDI是至关重要的 动脉血栓形成中的血小板血栓形成和中性粒细胞向炎症的内皮细胞聚集 血管发炎。然而，用功能阻断抗体抑制细胞外PDI会延长尾巴 小鼠的出血时间，这引起了人们的担忧，即PDI的特异性抑制可能会扰乱止血功能。我们的 最近的研究表明，血小板释放的PDI促进糖蛋白的配体结合功能 IBα(GPIBα)和增强GPIBα介导的血小板黏附、血小板-中性粒细胞聚集和血管 血栓炎症状态下的闭塞。这些结果表明，阻断PDI-GPIBα的抑制剂 Signal Axis可能是一种新型的抗血栓炎症药物。使用高通量筛选，我们有 确定了一种特定地抑制PDI-GPIBα结合和GPIBα介导的血小板聚集的化合物。 我们发现，小鼠静脉注射这种化合物可以消除血小板-中性粒细胞的相互作用，并改善 血栓炎症状态下微血管的血流速度。不同于传统的PDI或 GPIBα，用我们的化合物治疗不会延长小鼠的尾巴出血时间。这些结果提供了 识别针对特定PDI信号通路的小分子抑制剂的可行性的证据。在……里面 目的1、利用计算机辅助药物设计技术和一系列体外研究，我们将识别小分子药物。 特异性阻断PDI-GPIBα信号和GPIBα介导的血小板功能的分子化合物。在AIM 2、我们将合成HITS的衍生物，在动物实验中测试它们，并检查选定的DMPK图谱 化合物。这项拟议的研究将证明，与传统的抑制PDI或GPIBα相比，特定的 抑制pDi-GPIBα信号轴可能是一种更安全有效的治疗策略 血栓炎症性疾病。

Project Summary Thromboinflammatory diseases, including atherothrombosis, stroke and peripheral vasculitis, result in >30% of all deaths globally. Underlying the pathology of thromboinflammation is increased adhesiveness of platelets and leukocytes. Although many antiplatelet and anti-inflammatory therapies have been used for disease treatment, these drugs increase the risk of major bleeding or impair immune responses. Using protein disulfide isomerase (PDI) conditional knockout (CKO) mice and inhibitors, we and others have shown that extracellular PDI is crucial for platelet thrombus formation in arterial thrombosis and neutrophil recruitment to inflamed endothelium in vascular inflammation. However, inhibition of extracellular PDI with a function-blocking antibody prolongs tail bleeding times in mice, raising a concern that specific inhibition of PDI may perturb hemostatic function. Our recent studies have demonstrated that platelet-released PDI promotes the ligand-binding function of glycoprotein Ibα (GPIbα) and enhances GPIbα-mediated platelet adhesiveness, platelet-neutrophil aggregation and vascular occlusion under thromboinflammatory conditions. These results suggest that inhibitors blocking the PDI-GPIbα signaling axis may be a novel antithromboinflammatory drug. Using high throughput screening, we have identified one compound that specifically inhibits PDI-GPIbα binding and GPIbα-mediated platelet aggregation. We have found that iv injection of the compound into mice abolishes platelet-neutrophil interactions and improves blood flow rates in microvessels under thromboinflammatory conditions. Unlike a conventional inhibitor of PDI or GPIbα, treatment with our compound does not prolong tail bleeding times in mice. These results have provided evidence for the feasibility of identifying small-molecule inhibitors targeting a specific PDI signaling pathway. In Aim 1, using the computer-aided drug design technique and a series of in vitro studies, we will identify small- molecule compounds that specifically block PDI-GPIbα signaling and GPIbα-mediated platelet functions. In Aim 2, we will synthesize derivatives of hits, test them in animal studies and examine DMPK profiles of the selected compounds. The proposed study will prove that compared to conventional inhibition of PDI or GPIbα, specific inhibition of the PDI-GPIbα signaling axis might be a safer and effective therapeutic strategy for treating thromboinflammatory disease.