Novel screening platform for O-GlcNAc transferase substrate-specific inhibitors to combat aging-related diseases

项目摘要 O-连接的β-N-乙酰葡萄糖胺（O-GlcNAc）是一种重要的翻译后修饰 在参与广泛的重要生物过程的数千种蛋白质中发现， 包括表观遗传学、代谢和蛋白质降解。O-GlcNAc修饰是 通过单对人类酶O-GlcNAc转移酶动态添加和去除 (OGT)和O-GlcNAcase（OGA）。O-GlcNAc酰化的失调已经被发现， 在许多与衰老有关的疾病中检测到，包括癌症、糖尿病、阿尔茨海默氏症和 亨廷顿氏病。到目前为止，已经报道了几种有效的活性位点抑制剂用于OGT 和OGA，但它们可能会引起不希望的副作用，因为这些必需的酶调节 O-GlcNAc在数千种蛋白质底物上的动力学。目前，没有关于 OGT或OGA的底物特异性抑制剂，主要是由于缺乏合适的测定。这里我 建议开发一种新的高通量筛选测定法，用于鉴定小分子， 可以特异性结合OGT的不同区域，并调节特定的O-GlcNAc 在不干扰细胞中的总体O-GlcNAc水平的情况下，从建议的屏幕 以及以下体外和细胞表征和优化过程，我希望 确定几种化合物作为第一代底物特异性抑制剂候选物， OGT可以调节与衰老疾病相关的特定蛋白质上的O-GlcNAc酰化。这 新的测定平台可以潜在地适用于其他蛋白质底物，并促进 抗衰老疾病的治疗进展。

Project Summary The O-linked β-N-acetylglucosamine (O-GlcNAc) is an essential post-translational modification found in thousands of proteins involved in a broad range of important biological processes, including epigenetics, metabolism, and protein degradation. The O-GlcNAc modification is dynamically added and removed by a single pair of human enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), respectively. Dysregulation of O-GlcNAcylation has been detected in many aging-related diseases including cancer, diabetes, Alzheimer’s and Huntington’s disease. To date, several effective active-site inhibitors have been reported for OGT and OGA, but they are likely to induce undesired side effects as these essential enzymes regulate O-GlcNAc dynamics on thousands of protein substrates. Currently, there is no report on substrate-specific inhibitors of OGT or OGA, largely due to the lack of suitable assays. Here, I propose to develop a novel high-throughput screening assay for identifying small molecules that can specifically bind to different regions of OGT and modulate O-GlcNAcylation of a particular protein of interest without perturbing the global O-GlcNAc level in cells. From the proposed screen and the following in vitro and cellular characterization and optimization processes, I expect to identify several compounds as the first generation of substrate-specific inhibitor candidates of OGT that can modulate O-GlcNAcylation on particular proteins related to aging diseases. This new assay platform can be potentially adapted for other protein substrates and facilitate the therapeutic development to combat aging disorders.