Live-Cell Assays of ER and Golgi Enzyme Activities

ER 和高尔基体酶活性的活细胞测定

• 批准号: 9405346
• 负责人: JOHN J NALEWAY
• 金额: $ 63.14万
• 依托单位: MARKER GENE TECHNOLOGIES
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-01 至 2019-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9405346
• 关键词: Alzheimer' s Disease; Amyotrophic Lateral Sclerosis; Area; Basic Science; Biochemical; Biological Assay; Biological Markers; Biological Monitoring; Biotechnology; Cell Culture System; Cells; Cellular biology; Chemicals; Clinical; Collaborations; Communities; Cytolysis; Defect; Detection; Development; Diagnostic; Diagnostic tests; Disease; Disease Progression; Dose; Drug usage; Endoplasmic Reticulum; Enzyme Induction; Enzymes; Evaluation; Flow Cytometry; Fluorescence; Fluorescence Microscopy; Fluorescence Spectroscopy; Fluorogenic Substrate; Funding; Galactosidase; Gaucher Disease; Genes; Globoid cell leukodystrophy; Glycoside Hydrolases; Glycosphingolipids; Goals; Golgi Apparatus; Huntington Disease; Image Analysis; Individual; Industrialization; Industry; Institution; Intervention; Laboratories; Laboratory Research; Lead; Legal patent; Lethal Dose 50; Libraries; Licensing; Literature; Lysosomal Storage Diseases; Mannosidase; Measurement; Measures; Medical; Metabolic; Methods; Monitor; Morphologic artifacts; Neurodegenerative Disorders; Neurologic; Non-Insulin-Dependent Diabetes Mellitus; Oculocerebrorenal Syndrome; Oregon; Organelles; Parkinson Disease; Pathway interactions; Patients; Peer Review; Peptide Hydrolases; Pharmaceutical Preparations; Pharmacologic Substance; Pharmacology; Phase; Process; Proteins; Protocols documentation; Publications; Reporting; Reproducibility; Research; Sampling; Small Business Innovation Research Grant; Specificity; Staining method; Stains; System; Techniques; Technology; Testing; Therapeutic; Therapeutic Effect; Tissues; Treatment Efficacy; Treatment Protocols; United States National Institutes of Health; Universities; Validation; Work; base; beta-n-acetylhexosaminidase; cell immortalization; chemical standard; clinical diagnostics; commercialization; diagnostic assay; drug candidate; drug discovery; efficacy testing; endoplasmic reticulum stress; enzyme activity; enzyme substrate; fluorophore; genetic analysis; glucosidase; high throughput screening; human disease; immortalized cell; inhibitor/antagonist; innovation; meetings; multiplex detection; nervous system disorder; novel drug class; novel therapeutics; programs; protein folding; protein transport; research clinical testing; response; scale up; screening; small molecule; small molecule therapeutics; success; symposium; tool; trafficking

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.

项目摘要/摘要 这个小型企业创新研究阶段II阶段旨在开发新的目标氟化 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。该项目的最终目标和整体影响是提供理解 人类疾病中细胞内酶和蛋白质运输缺陷中发生的动态过程 并获取将为更有效的治疗选择提供途径的信息。如果成功， 拟议的研究将为进步发现有希望的新疗法提供突破 并调节药物为神经退行性疾病，包括高刺病和其他溶酶体储存 疾病，帕金森氏病（PD），阿尔茨海默氏病（AD），肌萎缩性侧索硬化症（ALS），2型 糖尿病，Lowe综合征，亨廷顿氏病和盟军医疗状况。在这个项目的第一阶段， Marker Gene Technologies，Inc。通过准备新的氟化物来确定技术的可行性 糖苷酶和肽酶底物用于在高尔基和ER中具有局部活性的酶 证明来自正常或疾病起源的活细胞中的差异染色。在第二阶段， 这些和其他新的底物将被评估，以衡量特定和局部化的能力 抑制或诱导活细胞中各个细胞器中的酶活性以及阐明 与疾病进展有功能关系的贩运机制。新的底物和 结果检测系统将提供创新的方法来量化次级药物的影响或 细胞器特异性溶酶体，高尔基体或ER酶活性的蛋白质给药。此外，它们的使用 在筛选潜在候选药物的库中，以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铅。这使得合并 作为基础研究工具有用的系统，可用于多种重要的细胞生物学，生化和医学 申请。该公司与几个著名的学术研究实验室合作 以及该领域的研究机构以及主要的制药公司，他们渴望测试 现有的临床，诊断或药物发现应用中的方法和系统。最终的测定法 产品将销售用于研究，制药，生物技术和诊断行业的销售。