Live-Cell Assays for Lysosomal Enzyme Activity

描述(由申请人提供)：这个小型企业创新研究第二阶段项目旨在开发能够测量活细胞和组织中溶酶体酶活性的新的靶向荧光底物。如果成功，这项拟议的研究将提供所需的突破，以推进溶酶体储存疾病和相关医疗应用的有前景的新疗法和调节药物的发现。在该项目的第一阶段，Marker gene Technologies，Inc.通过为溶酶体酶制备新的荧光糖苷酶、酯酶、磷酸酶、脂肪酶和硫酸酶底物，确立了该技术的可行性，并在正常或疾病来源的活细胞中显示了差异染色，或在诱导抑制溶酶体酶活性的情况下进行了差异染色。在第二阶段，这些和其他新的底物将在体外测试它们在活细胞中测量特定的和局部的抑制或诱导溶酶体酶的能力，以及以细胞或组织特异性的方式区分单个酶的活性。这些新系统将被验证用于药物发现的高通量筛选，用于临床诊断以评估疾病的发生和发展，并用于监测这些疾病的现有或新出现的治疗干预措施的有效性。新的底物和由此产生的检测系统还将提供定量测定溶酶体酶功能和筛选二次给药或蛋白质给药的影响的创新方法，使它们成为各种重要的生化和医学应用的有用医学研究工具。该公司已经与几个著名的研究实验室和机构以及这一领域的主要制药公司进行了合作，这些公司渴望在现有的临床应用中测试这些方法和系统。由此产生的分析和产品将向研究、制药、生物技术和诊断行业销售。 与公共卫生相关：该项目的成功为桑德霍夫病、泰-萨克斯氏综合症、克拉比氏病和高谢病等溶酶体储存障碍的医疗干预领域开辟了巨大的商业可能性，因为它发现了用于调节这些疾病和其他相关疾病的溶酶体酶活性的新蛋白质和药物。这些分析还将使临床医生能够确定当前和新兴疗法的有效性，并为患者评估增加一套新的临床诊断分析方法。此外，它还将在这些领域带来商业化和可许可的产品。

DESCRIPTION (provided by applicant): This Small Business Innovation Research Phase II project aims to develop new targeted fluorogenic substrates capable of measuring lysosomal enzyme activity in living cells and tissues. If successful, the proposed research will provide breakthroughs needed to advance the discovery of promising new therapies and modulating drugs for lysosomal storage diseases and allied medical applications. In Phase I of this project, Marker Gene Technologies, Inc. established the feasibility of the technology by preparing new fluorogenic glycosidase, esterase, phosphatase, lipase and sulfatase substrates for lysosomal enzymes and demonstrated differential staining in living cells that were from normal or were of disease origin or upon induction of inhibition of lysosomal enzyme activities. In Phase II, these and additional new substrates will be assayed in vitro for their ability to measure specific and localized inhibition or induction of lysosomal enzymes in living cells as well as differentiate individual enzyme activities in a cell- or tissue-specific manner. These new systems will be validated for use in high-throughput screening for drug discovery, for use in clinical diagnostics to evaluate the occurrence and progression of disease, and for use in monitoring the effectiveness of existing or emerging therapeutic interventions for these disorders. The new substrates and the resulting detection systems will also provide innovative methods to quantitate lysosomal enzyme function and to screen for the influence of secondary drug or protein administration, making them useful medical research tools for a variety of significant biochemical and medical applications. The company has engaged the collaboration of several noted research laboratories and institutions as well as major pharmaceutical companies in this arena, who are eager to test the methods and systems in their existing clinical applications. The resulting assays and products will be marketed to the research, pharmaceutical, biotechnology and diagnostic industries. PUBLIC HEALTH RELEVANCE: The success of this project opens up enormous commercial possibilities in the fields of medical intervention in lysosomal storage disorders such as Sandhoff's disease, Tay-Sachs syndrome, Krabbi's disease and Gaucher's disease through discovery of new proteins and drugs for use in modulating lysosomal enzyme activity in these and other related diseases. These assays will also enable clinicians to determine the efficacy of current and emerging therapies as well as add a new set of clinical diagnostic assays for patient assessment. In addition, it will lead to commercial and licensable products in these areas.