Inhibition of CDC25B phosphatase by targeting protein-protein interactions

项目摘要 CDC25s磷酸酶家族是细胞周期的重要调节因子，在细胞周期中起重要作用 激活CDK-细胞周期蛋白复合体，促进细胞周期进程。CDC25A和CDC25B 磷酸酶是经典的原癌基因，其功能的改变促进了肿瘤的发生。 体内转化。CDC25磷酸酶在多种癌症中经常过表达，这些癌症 与预后不良有关。重要的是，小干扰RNA对CDC25的下调已经被 证实CDC25具有抑制肿瘤生长的作用，是非常重要的治疗靶点。 使用小分子靶向蛋白磷酸酶通常是困难的。尽管做了很多努力，但是 到目前为止，CDC25的验证和非共价抑制剂还没有被描述。这些生物的活性部位 酶很浅，缺乏明确的结合袋。此外，CDC25还具有很高的反应性 半胱氨酸与其他蛋白酪氨酸磷酸酶的活性部位阻碍了筛选和设计工作。在……里面 结果，CDC25被证明很难被小分子靶向，并且是最有效的抑制剂之一 到目前为止，所描述的是最有可能代表磷酸酶的共价抑制剂的苯醌衍生物。 在这个项目中，我们提出了一种新的方法来开发针对CDC25B磷酸酶的小分子 抑制CDC25B和CDK2/CyCA复合体之间的蛋白质相互作用。CDK2/Cyca 代表CDC25B的天然底物，可通过远离活性物质的界面识别 地点。我们建议在这个界面上鉴定与CDC25B结合的小分子化合物以抑制 与CDK2/Cyclin A的相互作用我们的建议代表了一种非常创新的方法来识别有效和非 CDC25磷酸酶的共价抑制剂。我们已经开发了高质量的检测方法来评估蛋白质- CDC25B与CDK2/CyCA的蛋白质相互作用及其高通量筛选 这种相互作用的小分子抑制剂。放映将在大学CCG中心进行 来自密歇根州。随后，我们将使用一组正交分析来消除假阳性和 生物物理和结构生物学方法选择直接非共价抑制剂。已确定的活动 化合物将在细胞实验中进行评估。我们项目的长期目标是开发出强大的细胞-- 作为新型抗癌药物的周期抑制剂。

Project Summary The family of CDC25s phosphatases represents important regulators of the cell cycle that are required for activation of the CDK-cyclin complexes to promote the cell cycle progression. The CDC25A and CDC25B phosphatases are classical proto-oncogenes, and alteration in their function promotes oncogenic transformation in vivo. CDC25 phosphatases are frequently overexpressed in a variety of cancers, which correlates with poor prognosis. Importantly, downregulation of CDC25s by small interfering RNAs has been shown to inhibit tumor growth validating CDC25s as very important therapeutic targets. Targeting protein phosphatases using small molecules is generally difficult. Despite numerous efforts, well validated and non-covalent inhibitors of CDC25s have not been described to date. The active site of these enzymes is very shallow and lacks well defined binding pocket. Furthermore, CDC25s share highly reactive active site cysteine with other protein tyrosine phosphatases hampering screening and design efforts. In consequence, CDC25s proved difficult to target by small molecules and one of the most potent inhibitors described to date are quinone derivatives which most likely represent covalent inhibitors of phosphatases. In this project, we propose a novel approach to develop small molecules targeting CDC25B phosphatase by inhibiting the protein-protein interaction between CDC25B and CDK2/CycA complex. The CDK2/CycA represents a natural substrate of CDC25B, which is recognized via an interface that is distant from the active site. We propose to identify small molecule compounds binding to CDC25B at this interface to inhibit the interaction with CDK2/Cyclin A. Our proposal represents a very innovative approach to identify potent and non- covalent inhibitors of CDC25 phosphatases. We have developed high quality assay for assessing the protein- protein interaction between CDC25B and CDK2/CycA and will use high-throughput screening to identify potent small molecule inhibitors of this interaction. The screen will be carried out in the CCG center at the University of Michigan. Subsequently we will employ a panel of orthogonal assays to eliminate false positives and biophysical and structural biology methods to select direct non-covalent inhibitors. The activities of identified compounds will be assessed in cellular experiments. The long term goal of our project is to develop potent cell- cycle inhibitors as novel anti-cancer agents.