High throughput tau oligomer assay for drug screening for Alzheimer's disease

描述（由申请人提供）：对于预防或改善阿尔茨海默病（AD）的分子发病机制的新药存在关键的未满足的需求。AD导致进行性记忆丧失、个性和行为改变以及执行复杂甚至简单任务的能力下降，结果是严重受影响的个体需要持续的护理。疾病病理学的特征在于不溶性纤维和缠结以及淀粉样斑块的细胞外和细胞内积累。这些不溶性蛋白质沉积物由与神经元缠结（NFT）相关的tau蛋白或与老年斑相关的淀粉样蛋白（A-<$）组成。组装了一个体外系统，其中tau蛋白错误折叠和寡聚化的关键事件在生理条件下使用小的、高度结构化的RNA伴侣（ShsRNA）实现。Tau在这些条件下形成寡聚体和细丝，这取决于反应条件和所使用的shRNA的序列。形成的寡聚体和细丝在生物化学上和结构上与阿尔茨海默病（AD）中发现的相似，因此是淀粉样蛋白。所提出的计划的中心目标是采用tau体外寡聚化测定并将其转化为适合于筛选化学文库的高通量形式，以用于命中和先导物优化研究。具体目标是：优化和表征tau蛋白体外寡聚体测定？利用噬菌体展示技术分离和鉴定tau寡聚体特异性抗体？开发时间分辨荧光免疫测定法（TRFIA）来检测tau蛋白开花？将tau寡聚体特异性测定（TOSA）转换为高通量a-筛选形式将使用工具化合物测试完成TOSA的初步验证。在第二阶段，tau寡聚体特异性抗体将用于阿尔茨海默病患者的脑切片中，以显示其与神经原纤维缠结或其前体反应，以验证该平台作为药物靶标。TOSA将作为阿尔茨海默病和其他适当的tau蛋白病药物发现的工具推向制药和生物制药行业。拟议的计划将利用Q-RNA在使用分子促进剂产生tau寡聚体方面的专业知识，并将其与ASU的Michael Sierks博士在筛选噬菌体文库以分离寡聚体特异性scFv方面的专业知识结合联合收割机，以开发快速，低成本，体外药物筛选平台，该平台将用于鉴定抑制或改善tau寡聚化的化合物。

DESCRIPTION (provided by applicant): There is a critical unmet need for novel drugs that prevent or ameliorate the molecular pathogenesis of Alzheimer's disease (AD). AD results in progressive memory loss, changes in personality and behavior, and decreasing ability to perform complex and even simple tasks, with the result that severely affected individuals require constant care. The disease pathology is characterized by extracellular and intracellular accumulations of insoluble fibrils and tangles, and amyloid plaques. These insoluble protein deposits are composed of either tau protein associated with neurofibrillary tangles (NFTs), or ¿-amyloid (A-¿) protein associated with senile plaques. An in vitro system was assembled, in which key events of the tau protein misfolding and oligomerization were achieved under physiological conditions using small, highly structured RNA chaperones (shsRNA). Tau formed oligomers and filaments under these conditions depending on the reaction conditions, and the sequence of the shsRNA used. The oligomers and filaments that formed were biochemically, and structurally similar to those found in Alzheimer's disease (AD) and were therefore amyloid-like. The central aim of the proposed program is to take the tau in vitro oligomerization assay and convert it to a high throughput format suitable for screening chemical libraries for hits, and for lead optimization studies. The specific aims that will be accomplished are: Optimize and characterize the tau in vitro oligomer assay? Isolate and characterize tau oligomer specific antibodies using phage display? Develop a time resolved fluorescence immunoassay (TRFIA) for the detection of tau bloomers? Convert tau oligomer specific assay (TOSA) to high throughput a-screen format Preliminary validation of the TOSA will be accomplished using tool compound testing. During phase II, the tau oligomer specific antibody will be used in brain slices from Alzheimer's disease patients, to show that it reacts with neurofibrillar tangles or their precursors to validate the platform as a drug target. The TOSA will be marketed as a tool for drug discovery for Alzheimer's disease and other appropriate taupathies to the pharmaceutical and biopharmaceutical industries. The proposed program will take Q-RNA's expertise in generating tau oligomers using molecular facilitator's and combine this with the expertise of Dr. Michael Sierks at the ASU in screening phage libraries to isolate oligomer specific scFvs to develop a rapid, low cost, in vitro drug screening platform that will be used for identifying compounds that inhibit or ameliorate tau oligomerization.