HTS for Enhancer of Glycosylation of Alpha Dystroglycan

用于α肌营养不良聚糖糖基化增强剂的 HTS

• 批准号: 7839495
• 负责人: Xiaohua Wu
• 金额: $ 3.6万
• 依托单位: CAROLINAS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7839495
• 关键词: Actins; Binding; Biological Assay; Biological Models; Cell surface; Cells; Chemicals; Congenital Disorders; Cytoskeleton; Disease; Enhancers; Environmental Risk Factor; Extracellular Matrix; Extracellular Matrix Proteins; Fluorescence; Genes; Goals; Human; Infection; Knowledge; Laminin; Ligands; Link; Methodology; Methods; Monitor; Muscle; Muscular Dystrophies; Mutation; Nerve; Organism; Pathogenesis; Pathologic Processes; Pathway interactions; Patients; Performance; Pharmaceutical Preparations; Play; Polysaccharides; Process; Proteins; Public Health; Regulation; Reproducibility; Research; Role; Screening procedure; Stimulus; Surface; System; Testing; Time; Up-Regulation; alpha Dystroglycan; base; drug discovery; dystroglycan 1; effective therapy; environmental change; functional restoration; glycosylation; glycosyltransferase; high throughput screening; innovation; interest; muscular dystrophy mouse model; novel; overexpression; pathogen; public health relevance; research study; response; sugar; tool

DESCRIPTION (provided by applicant): The long term goal of our research is to discover effective treatments for muscular dystrophies. A number of muscular dystrophies (MD) are associated with abnormal O-mannosylation of alpha dystroglycan (1-DG) that plays a central role in linking the extracellular matrix (ECM) to the actin-based cytoskeleton in muscle. Hypoglycosylation of 1-DG impairs its ability to bind to the ECM proteins such as laminins and results in pathogenesis in the muscle and nerve systems and often causes devastating diseases in humans. Currently, there is no specific treatment for any form of MD. Increasing evidence suggests that over-expression of glycosyltransferases enhancing the glycosylation of 1-DG can restore the functions of 1-DG in MD mouse models. We hypothesize that cells can enhance glycosylation of 1-DG in response to certain chemical stimuli. We recently developed a cell-based assay for determination of O-mannosylation of alpha-DG (ADOMA). With ADOMA, we are able to quantitatively monitor the levels of O-mannosylation of 1-DG and its functional binding to the ligands, laminins, on the cell surface with remarkable reliability and reproducibility. This assay has great potential to become a high throughput screening (HTS) platform. A pilot screening of 2000 bioactive compounds with ADOMA has been conducted to test our hypothesis and the performance of ADOMA under HTS conditions. The result has shown that the performance of the pilot screen is acceptable for HTS. Furthermore, we have identified several positive hits which are able to enhance the functional binding of laminins to the cell surface, thus proved our hypothesis. However, the current ADOMA screening procedure requires multiple washing steps and need to be reconfigured to HTS friendly format for large scale HTS campaign. In this project, thus, we will focus on the specific aim: Reconfiguration of ADOMA into a homogeneous, miniaturizable, and wash-free HTS assay. We plan to integrate homogeneous time-resolved fluorescence (HTRF) methodology into our HTS assay to develop HTRF based HTS assay. PUBLIC HEALTH RELEVANCE: The goal of this project is to discover effective treatments for muscular dystrophies. A number of muscular dystrophies (MD) are associated with deficiency of sugar chains synthesis. The abnormality of sugar chain on alpha dystroglycan, a key protein in muscle, results in pathogenesis in the muscle and nerve systems and often causes devastating diseases in humans. Currently, there is no specific treatment for any form of MD. This project aims to develop a drug discovery assay to identify compounds and develop drug for MD.

描述（由申请人提供）：我们研究的长期目标是发现肌营养不良症的有效治疗方法。许多肌营养不良症（MD）与α肌营养不良蛋白聚糖（1-DG）的异常O-甘露糖基化有关，α肌营养不良蛋白聚糖（1-DG）在将细胞外基质（ECM）连接到肌肉中基于肌动蛋白的细胞骨架中起核心作用。1-DG的低糖基化损害了其与ECM蛋白如层粘连蛋白结合的能力，并导致肌肉和神经系统的发病机制，并且经常导致人类的毁灭性疾病。目前，对于任何形式的MD都没有特定的治疗方法。越来越多的证据表明，糖基转移酶的过表达增强1-DG的糖基化可以恢复MD小鼠模型中1-DG的功能。我们假设，细胞可以增强糖基化的1-DG在响应某些化学刺激。我们最近开发了一种基于细胞的测定α-DG（ADOMA）的O-甘露糖基化的方法。使用ADOMA，我们能够定量监测1-DG的O-甘露糖基化水平及其与细胞表面配体层粘连蛋白的功能性结合，具有显著的可靠性和重现性。该测定具有成为高通量筛选（HTS）平台的巨大潜力。已经进行了2000种具有ADOMA的生物活性化合物的中试筛选，以测试我们的假设和ADOMA在HTS条件下的性能。试验结果表明，该试验筛的性能是可以接受的。此外，我们已经确定了几个积极的命中，能够增强层粘连蛋白的功能结合到细胞表面，从而证明了我们的假设。然而，目前的ADOMA筛选程序需要多个洗涤步骤，并且需要重新配置为HTS友好格式以用于大规模HTS活动。因此，在本项目中，我们将专注于特定目标：将ADOMA重新配置为均匀，可重复和免洗涤的HTS检测。我们计划将均相时间分辨荧光（HTRF）方法整合到我们的HTS检测中，以开发基于HTRF的HTS检测。 公共卫生相关性：该项目的目标是发现有效的治疗肌肉萎缩症。许多肌营养不良症（MD）与糖链合成不足有关。α-肌营养不良蛋白聚糖是肌肉中的关键蛋白，其糖链的异常导致肌肉和神经系统的发病，并且常常导致人类的毁灭性疾病。目前，对于任何形式的MD都没有特定的治疗方法。该项目旨在开发一种药物发现检测方法，以识别化合物并开发用于MD的药物。