Live-Cell Assays of ER and Golgi Enzyme Activities

项目总结/摘要 这个小企业创新研究第二阶段项目旨在开发新的靶向荧光 能够测量高尔基体和内质网（ER）中酶活性的底物 活细胞和组织。这个项目的最终目标和总体影响是提供一个理解 发生在细胞内酶和蛋白质运输缺陷的人类疾病的动态过程 并获得将提供更有效治疗选择的途径的信息。如果成功， 拟议中的研究将提供必要的突破，以促进有前途的新疗法的发现 以及用于神经变性疾病包括戈谢病和其它溶酶体贮积症的调节药物 疾病、帕金森病（PD）、阿尔茨海默病（AD）、肌萎缩侧索硬化（ALS）、2型 糖尿病、Lowe综合征、亨廷顿氏病和相关医学病症。在该项目的第一阶段， Marker基因技术公司通过制备新型荧光染料，确立了该工艺的可行性 在高尔基体和ER中具有局部活性的酶的糖苷酶和肽酶底物， 表明在来自正常或疾病来源的活细胞中的差异染色。在第二阶段， 将对这些和其他新基质进行评估，以确定它们测量特定和局部的能力。 抑制或诱导活细胞中单个细胞器内的酶活性，以及阐明 与疾病进展有功能关系的贩运机制。新的基质和 由此产生的检测系统将提供创新的方法来量化次级药物或 蛋白质施用对细胞器特异性溶酶体、高尔基体或ER酶活性的影响。此外，其使用 在筛选潜在药物候选物文库中调节酶功能和定位的能力 将根据这些参数确定新的小分子治疗先导物。这使得组合 系统可用作各种重要的细胞生物学、生物化学和医学的基础研究工具， 应用.该公司与几个著名的学术研究实验室合作， 和研究机构以及主要的制药公司在这个竞技场，谁是渴望测试的 在其现有的临床、诊断或药物发现应用中的方法和系统。所得到的分析 产品将销售给研究，制药，生物技术和诊断行业。

PROJECT SUMMARY/ABSTRACT This Small Business Innovation Research Phase II project aims to develop new targeted fluorogenic substrates capable of measuring enzyme activities in the Golgi apparatus and Endoplasmic Reticulum (ER) of living cells and tissues. The ultimate goal and the overall impact of this project is to provide an understanding of the dynamic processes that occur in intracellular enzyme and protein trafficking defects in human disease and to obtain information that will provide a pathway to more efficacious treatment options. If successful, the proposed research will provide breakthroughs needed to advance the discovery of promising new therapies and modulating drugs for neurodegenerative disorders including Gaucher disease and other lysosomal storage diseases, Parkinson's disease (PD), Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), Type 2 diabetes, Lowe syndrome, Huntington's disease and allied medical conditions. In Phase I of this project, Marker Gene Technologies, Inc. established the feasibility of the technology by preparing new fluorogenic glycosidase and peptidase substrates for enzymes with localized activity in the Golgi and ER and demonstrating differential staining in living cells that are from normal or are of disease origin. In Phase II, these and additional new substrates will be evaluated for their ability to measure specific and localized inhibition or induction of enzyme activities inside individual organelles in living cells as well as to elucidate the trafficking machinery that has a functional relationship to disease progression. The new substrates and the resulting detection systems will provide innovative methods to quantitate the influence of secondary drug or protein administration on organelle-specific lysosomal, Golgi or ER enzyme activities. Furthermore, their use in screening libraries of potential drug candidates for their ability to modulate enzyme function and localization will identify new small molecule therapeutic leads based on these parameters. This makes the combined systems useful as basic research tools for a variety of significant cell biology, biochemical and medical applications. The company has engaged the collaboration of several noted academic research laboratories and research institutions as well as major pharmaceutical companies in this arena, who are eager to test the methods and systems in their existing clinical, diagnostic or drug discovery applications. The resulting assays and products will be marketed to the research, pharmaceutical, biotechnology and diagnostic industries.