Hyperphosphorylated tau aggregation-based Alzheimer’s disease early drug discovery

基于过度磷酸化tau聚集的阿尔茨海默病早期药物发现 阿尔茨海默病(AD)是一种不可逆转的神经退行性疾病，全球有4700万人患病， 每年要花费六千零四十亿美元的医疗费。到目前为止，还没有治愈或预防的方法 广告。AD的两个明显特征是Aβ斑块和神经原纤维缠结，它们由 过度磷酸化的tau。虽然AD药物发现领域一直被抗A-β措施所主导， 一再失败的临床试验有力地证明，需要重新调整药物靶点和策略，以 在AD治疗方面取得突破性进展。事实上，多条证据表明，前- 过度磷酸化的tau聚集体的缠结阶段导致扩散的细胞毒性，这可能是 神经退行性变。防止过度磷酸化tau形成细胞毒性的化合物的筛选 因此，聚集体为AD药物的发现提供了一条更可行的途径。 以缠结为中心的药物设计并未取得成果。一种广谱的化合物已经被 在多个筛查中被鉴定为缠结形成的抑制剂，但后来发现是假阳性。一个共享 这些筛查的问题是使用未经修饰的tau蛋白，它需要诱导剂，例如肝素，才能有效 减少环境中的聚合。检测对象(Tau)缺乏本病病理标志 过度磷酸化，诱导物与疾病的相关性尚未得到证实。要克服这些障碍 障碍，我们已经开发了PIMAX技术，在大肠杆菌中产生过度磷酸化的tau(p-tau)。 提纯的p-tau纤维自主化(没有诱导剂)，并引起不同细胞的凋亡，包括 神经母细胞瘤细胞系。这种无诱导剂的p-tau聚集试验的Z‘值为0.699，系数为 变异系数(CV)8.3%。这些参数使我们的p-tau聚集分析成为一个强大的HTS平台。vbl.使用 在这项试验中，我们对1,280种化合物进行了初步筛选，以了解它们调节p-DNA聚集的能力。 陶先生。然后，我们使用新的生化和基于细胞的二次分析来验证候选化合物。我们 研究发现，一种有效的神经药物是一种有效的p-tau聚集抑制剂，它也可以保护细胞免受p-tau的伤害。 Tau的细胞毒性。这些初步研究为p-tau在AD研究中的价值提供了确凿的证据 治疗学。这个R01项目是第一个使用病理生理相关的过度磷酸化的项目 用于AD药物发现的Tau。我们将按照我们的试点筛选协议进行高通量筛选 100,000种化合物，以其抑制p-tau聚集和保护细胞的能力。这个早期发现项目 将以两个主要产品结束：(1)一系列证实具有p-tau聚集抑制作用的化学物质 和细胞保护活动，以及(2)具有全面计划的后续建议，以确定最佳 AD药物开发的化学铅。

Hyperphosphorylated tau aggregation-based Alzheimer’s disease early drug discovery Alzheimer’s disease (AD) is an irreversible neurodegenerative disease affecting 47 million people worldwide, and costs 604 billion US dollars every year for medical expenses. To date, there is no cure or prevention for AD. The two defining features of AD are Aβ plaques and neurofibrillary tangles that are composed of hyperphosphorylated tau. While the AD drug discovery landscape has been dominated by anti-Aβ measures, recurring failures of clinical trials argue strongly that a realignment of the drug target and strategies is needed to make a breakthrough in AD therapeutics development. Indeed, multiple lines of evidence suggest that the pre- tangle stage of hyperphosphorylated tau aggregates cause diffusible cytotoxicity that likely underlies neurodegeneration. Screening for compounds that prevent hyperphosphorylated tau from forming the cytotoxic aggregates thus affords a more viable route for AD drug discovery. The tangle-centric drug design has not come to fruition. A wide spectrum of compounds have been identified in multiple screens as inhibitors of tangle formation, but later found to be false-positive. One shared issue for these screens is the use of an unmodified tau protein that requires an inducer, e.g., heparin, for efficient aggregation in a reducing environment. The assay subject (tau) lacks the pathological mark of hyperphosphorylation, and the disease relevance of the inducer has not been substantiated. To overcome these hurdles, we have developed the PIMAX technology that produces hyperphosphorylated tau (p-tau) in E. coli. Purified p-tau fibrillizes autonomously (without an inducer), and causes apoptosis of different cells including a neuroblastoma cell line. This inducer-free, p-tau aggregation assay has a Z’ value of 0.699, and a coefficient of variation (CV) of 8.3%. These parameters qualify our p-tau aggregation assay as a robust HTS platform. Using this assay, we conducted a pilot screen for 1,280 compounds for their ability to modulate the aggregation of p- tau. We then used novel biochemical and cell-based secondary assays to verify the candidate compounds. We found that an active neurological drug is a potent p-tau aggregation inhibitor, which also protects cells from p- tau cytotoxicity. These preliminary studies afford solid evidence for the values of p-tau in the quest for AD therapeutics. This R01 project is the very first that uses the pathophysiologically relevant hyperphosphorylated tau for AD drug discovery. We will follow our pilot screen protocols to conduct a high-throughput screen of 100,000 compounds for their ability to inhibit p-tau aggregation and to protect cells. This early discovery project will conclude with two major products: (1) A collection of chemical hits with confirmed p-tau aggregation inhibitory and cytoprotective activities, and (2) a follow-up proposal with comprehensive plans to identify the optimal chemical lead for AD drug development.