Identification and characterization of novel antithrombotic PDI inhibitors

项目总结（见说明）： 使用抗体抑制蛋白质二硫键异构酶（PDI）可防止血栓形成小鼠模型中的血小板积聚和纤维蛋白形成。这一观察结果表明，抑制PDI可能是控制病理性血栓形成的可行策略。然而，有效的，选择性的小分子抑制剂，以测试这一假设目前还没有。我们已经开始高吞吐量 筛选以鉴定抑制PDI的化合物。对约5000种化合物的初步筛选鉴定出PDI抑制剂，命中率为0.3%。在活性化合物中有几种黄酮类化合物，包括广泛使用的营养补充剂槲皮素-3-芸香糖苷。槲皮素-3-芸香糖苷在小鼠模型中具有显著的抗血栓形成作用。该PDI抑制剂在体内耐受良好且有效抗血栓形成的事实支持抑制PDI用于抗血栓形成治疗的可行性。然而，需要具有改善的生物利用度的更具选择性的有效化合物。我们将进行大规模的高通量筛选，以确定新的PDI抑制剂。该项目的目的是表征一组有效的和选择性的PDI抑制剂， 探针研究PDI在血栓形成中的作用，并鉴定可开发为抗血栓药的先导化合物。PDI在血管系统中表现出多种功能，包括氧化还原酶/异构酶、分子伴侣和脱亚硝化活性。在目标1中，我们将根据其阻断这些不同活性的能力来表征PDI抑制剂。在目标2中进行的研究将使用NMR光谱法来确定PDI抑制剂活性的结构基础。PDI抑制剂对 血小板活化和内皮细胞功能将在目标3中确定。然后使用活体显微镜检查法在血栓形成的小鼠模型中测试所选化合物的抑制活性（目的4）。 在酶和基于细胞的测定中评价PDI抑制剂将能够鉴定对PDI抑制剂的抗血栓形成活性至关重要的特征。这些信息对于进一步开发PDI抑制剂作为一类新型抗血栓药物至关重要。

PROJECT SUMMARY (See instructions): Inhibition of protein disulfide isomerase (PDI) using antibodies prevents both platelet accumulation and fibrin formation in murine models of thrombus formation. This observation indicates that inhibition of PDI could represent a viable strategy for control of pathological thrombus formation. However, potent, selective small molecule inhibitors to test this hypothesis are not presently available. We have begun high throughput screening to identify compounds that inhibit PDI. A preliminary screen of ~5000 compounds identified PDI inhibitors with a hit rate of 0.3%. Among the active compounds were several flavonoids, including the widely used nutritional supplement quercetin-3-rutinoside. Quercetin-3-rutinoside was markedly antithrombotic in murine models. The fact that this PDI inhibitor is well-tolerated and potently antithrombotic in vivo supports the feasibility of inhibition of PDI for antithrombotic therapy. However, more selective, potent compounds with improved bioavailability are required. We will perform a large scale high throughput screen to identify novel PDI inhibitors. The objective of this project is to characterize a set of potent and selective PDI inhibitors as probes to study the role of PDI in thrombus formation and identify lead compounds that could be developed as antithrombotics. PDI demonstrates multiple functions in the vasculature including oxidoreductase/isomerase, chaperone, and denitrosation activities. In Aim 1, we will characterize PDI inhibitors on the basis of their ability to block these different activities. Studies performed in Aim 2 will use NMR spectroscopy to determine the structural basis of PDI inhibitor activity. The effect of PDI inhibitors on platelet activation and endothelial cell function will be detennined in Aim 3. Select compounds will then be tested for their inhibitory activity in a mouse model of thrombus formation using intravital microscopy (Aim 4). Evaluation of PDI inhibitors in enzymatic and cell-based assays will enable the identification of characteristics that are essential for the antithrombotic activity of PDI inhibitors. Such information will be critical for further development of PDI inhibitors as a novel class of antithrombotics.