Live-Cell Assays of ER and Golgi Enzyme Activities

项目摘要/摘要 该小型企业创新研究第二阶段项目旨在开发新的靶向荧光 用底物测定猪高尔基体和内质网中的酶活性 活细胞和活组织。这个项目的最终目标和总体影响是为了提供一个理解 人类疾病中细胞内酶和蛋白质运输缺陷的动态过程 并获得将为更有效的治疗选择提供途径的信息。如果成功，则 拟议中的研究将提供所需的突破，以推进有前景的新疗法的发现 以及调节神经退行性疾病的药物，包括高谢病和其他溶酶体储存 疾病，帕金森氏病(PD)，阿尔茨海默病(AD)，肌萎缩侧索硬化症(ALS)，2型 糖尿病、洛韦综合征、亨廷顿氏病和相关的医疗条件。在这个项目的第一阶段， Marker gene Technologies，Inc.通过制备新的荧光物质证实了该技术的可行性 高尔基体和内质网中具有局部活性的酶的糖苷酶和肽酶底物 在正常或疾病来源的活细胞中显示不同的染色。在第二阶段， 这些和其他新的底物将被评估其测量特定和本地化的能力 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器。新的衬底和 由此产生的检测系统将提供创新的方法来量化二次药物或 蛋白质给药对细胞器特异性溶酶体、高尔基体或ER酶活性的影响。此外，它们的使用 在筛选潜在候选药物的库中，了解它们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线索。这使得组合 可用作各种重要细胞生物学、生化和医学的基本研究工具的系统 申请。该公司与几个著名的学术研究实验室进行了合作 和研究机构以及这一领域的主要制药公司，他们渴望测试 方法和系统在其现有的临床、诊断或药物发现应用中。由此产生的化验结果 产品将销往研究、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.