Identification and characterization of novel antithrombotic PDI inhibitors

项目总结(见说明)： 使用抗体抑制蛋白二硫键异构酶(PDI)可防止血栓形成小鼠模型中的血小板聚集和纤维蛋白形成。这一观察表明，抑制PDI可能是控制病理性血栓形成的一种可行的策略。然而，目前还没有有效的、选择性的小分子抑制剂来测试这一假说。我们已经开始了高吞吐量 筛选出抑制PDI的化合物。初步筛选出约5000种化合物，鉴定出PDI抑制剂，命中率为0.3%。在活性化合物中有几个黄酮类化合物，包括广泛使用的营养补充剂-3-芦丁糖苷。在小鼠模型中，槲皮素-3-芦丁糖苷具有明显的抗血栓作用。事实上，这种PDI抑制剂耐受性好，在体内具有有效的抗血栓作用，这支持了抑制PDI用于抗血栓治疗的可行性。然而，需要更具选择性、更有效、生物利用度更高的化合物。我们将进行大规模的高通量筛选，以确定新的PDI抑制剂。该项目的目标是表征一组有效的和选择性的PDI抑制剂，如 探索研究PDI在血栓形成中的作用，并鉴定可开发为抗血栓药物的先导化合物。PDI在血管系统中具有多种功能，包括氧化还原酶/异构酶、伴侣和反硝化活性。在目标1中，我们将根据PDI抑制剂阻断这些不同活性的能力来描述它们的特征。在AIM 2中进行的研究将使用核磁共振波谱来确定PDI抑制剂活性的结构基础。PDI抑制剂对血管内皮细胞生长的影响 在AIM 3中将测定血小板激活和内皮细胞功能。然后将使用活体显微镜(AIM 4)在小鼠血栓形成模型中测试它们的抑制活性。 在酶分析和基于细胞的分析中对PDI抑制剂的评价将使我们能够确定对PDI抑制剂的抗血栓活性至关重要的特性。这些信息将对PDI抑制剂作为一类新的抗血栓药物的进一步开发至关重要。

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.