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

用于对抗衰老相关疾病的 O-GlcNAc 转移酶底物特异性抑制剂的新型筛选平台

• 批准号: 10475192
• 负责人: Arielis Estevez Davila
• 金额: $ 7.19万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10475192
• 关键词: Active Sites; Address; Affect; Aging; Alzheimer' s Disease; Binding; Biological Assay; Biological Process; Cells; Chemicals; Diabetes Mellitus; Disease; Drug Targeting; Enzymes; Epigenetic Process; Fluorescence Resonance Energy Transfer; Generations; Homeostasis; Human; Huntington Disease; In Vitro; Label; Lead; Libraries; Link; Malignant Neoplasms; Metabolism; Modeling; Modification; O-GlcNAc transferase; Performance; Play; Post-Translational Protein Processing; Proteins; Rapid screening; Reporting; Role; Series; Structure; base; combat; experimental study; glycosylation; high throughput screening; inhibitor; innovation; interest; new therapeutic target; novel; peptide O-linked N-acetylglucosamine-beta-N-acetylglucosaminidase; process optimization; protein degradation; screening; side effect; small molecule; therapeutic development; tool

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.

项目摘要 O连接的β-N-乙酰葡萄糖胺（O-GLCNAC）是必不可少的翻译后修饰 在参与广泛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修改为 通过一对人酶动态添加和去除O-GLCNAC转移酶 （OGT）和O-Glcnacase（OGA）。 O-Glcnacylation的失调一直是 在许多与衰老有关的疾病中发现，包括癌症，糖尿病，阿尔茨海默氏症和 亨廷顿氏病。迄今为止，已经报道了OGT的几种有效的活跃位点抑制剂 和OGA，但由于这些基本酶调节 数千种蛋白质底物上的O-GLCNAC动力学。目前，没有关于 OGT或OGA的底物特异性抑制剂很大程度上是由于缺乏合适的测定法。在这里，我 提出开发一种新型的高通量筛选测定法，以识别小分子 可以特异性地与OGT的不同区域结合，并调节特定的O-Glcnacylation 感兴趣的蛋白质不会扰动细胞中全局O-GLCNAC水平。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 将几种化合物识别为第一代底物特异性抑制剂候选者 可以调节与衰老疾病有关的特定蛋白质O-Glcnacylation的OGT。这 新的测定平台可能会适应其他蛋白质底物，并促进 治疗性发育以对抗衰老疾病。