Identification of Novel Small Molecules as Tau Protein Aggregation Inhibitors for

描述（由申请人提供）： 阿尔茨海默病（AD）的特征在于由不溶性β-淀粉样蛋白（A2）和含tau蛋白的神经元缠结（NFT）组成的脑淀粉样蛋白斑块。AD药物发现策略主要集中在降低脑A2水平，而对tau导向方法的重视程度较低。在过去的十年中，ProteoTech公司已经开发了许多不同的体外筛选技术和细胞模型，以确定新的和有效的淀粉样变性抑制剂。这些技术使我们设计和合成了一类[由含有双儿茶酚的小分子（200-400 MW）组成的新化学实体]，发现其中一些是对AD、帕金森病和系统性AA淀粉样变性至关重要的蛋白质/肽聚集体的有效抑制剂。该I期项目的初步结果表明，我们的一些新化学实体能够在体外和相关的创新性基于细胞的测定中破坏/抑制tau蛋白原纤维形成。[We假设我们独特小分子新化学实体文库中的某些小化合物可以用作tau聚集/原纤维形成的直接抑制剂，且可能具有作为AD和其它tau蛋白病的新治疗剂的巨大潜力。]我们建议的主要目标是使用体外和基于细胞的测定从ProteoTech的独特文库中鉴定特定的小分子化合物，以确定其抑制/破坏tau聚集/纤维形成的能力。在[具体目标1]中，我们将筛选一组约50种小分子化合物（所有代表新的化学实体）在ProteoTech（含有多羟基化芳族化合物的小分子）开发，用于它们抑制tau蛋白原纤维形成和破坏预形成的tau原纤维的能力，使用体外测定，包括硫磺素T荧光测定、SDS-PAGE/蛋白质印迹分析和定量、CD光谱和电子显微镜。在具体目标2中，我们将利用诱导表达tau重复结构域（TauRD）的细胞系来筛选/验证我们在目标1中鉴定的先导化合物，其使用可溶性/不溶性TauRD聚集体的Western分析和TauRD聚集体/原纤维的硫磺素S染色和电子显微镜检查影响细胞内tau多聚化/聚集的能力。我们还将测试这些化合物是否可以拯救TauRD诱导的微管网络破坏和细胞毒性，如通过免疫染色和定量图像分析所确定的。在该项目中鉴定的最具活性的化合物将导致未来的II期SBIR项目，用于优化这些关键先导化合物的可药用性、脑渗透性、药代动力学和非毒性，并用于测试它们在相关转基因动物模型中的功效，所述转基因动物模型模拟AD和相关tau蛋白病中NFT中观察到的tau蛋白聚集的许多特征。 公共卫生相关性： 该I期SBIR项目将导致开发新型小分子药物，用于治疗阿尔茨海默病神经元缠结中发现的tau蛋白聚集。神经系统缠结的形成是阿尔茨海默病的病理标志之一，阿尔茨海默病是老年痴呆症的主要原因。

DESCRIPTION (provided by applicant): Alzheimer's disease (AD) is characterized by brain amyloid plaques consisting of insoluble beta-amyloid (A2) and tau protein containing neurofibrillary tangles (NFTs). AD drug discovery strategies have been largely focused on reducing brain A2 levels, with substantially less emphasis on tau-directed approaches. In the past decade, ProteoTech Inc has developed a number of different in vitro screening technologies and cellular models that identified new and potent inhibitors of amyloidosis. The techniques led us to design and synthesize a class of [new chemical entities consisting of small molecules (200-400 MW) containing bis-catechols,] some of them found to be potent inhibitors of protein/peptide aggregates that are of central importance for AD, Parkinson disease and systemic AA amyloidosis. Preliminary results for this Phase I project demonstrated that some of our new chemical entities have abilities to disrupt/inhibit tau protein fibril formation in vitro and in relevant innovative cell-based assays. [We hypothesize that certain small compounds in our unique small molecule new chemical entity library can serve as direct inhibitors of tau aggregation/fibrillogenesis, and may have great potential as new therapeutic agents for AD and other tauopathies.] The major objective our proposal is to identify specific small molecule compounds from ProteoTech's unique library for their ability to inhibit/disrupt tau aggregation/fibrillogenesis using both in vitro and cell-based assays. In [Specific Aim 1 we will screen a group of ~50 small molecule compounds (all representing new chemical entities) developed at ProteoTech (small molecules containing polyhydroxylated aromatic compounds) for their ability to inhibit tau protein fibril formation and to disrupt pre-formed tau fibrils using in vitro assays including Thioflavin T fluorometry, SDS-PAGE/western blot analysis and quantitation, CD spectroscopy, and electron microscopy.] In Specific Aim 2 we will utilize cell lines that inducibly express tau repeat domains (TauRD) to screen/validate our lead compounds identified in aim 1 for their ability to affect intracellular tau multimerization/ aggregation using Western analysis for soluble/insoluble TauRD aggregates, and Thioflavin S staining and electron microscopy for TauRD aggregates/fibrils. We will also test whether these compounds can rescue TauRD-induced microtubule network disruption and cytotoxicity as determined by immunostaining and quantitative image analysis. The most active compounds identified in this project will lead to a future Phase II SBIR project for optimizing these key lead compounds for drugability, brain penetration, pharmacokinetics and non-toxicity, and for testing their efficacy in relevant transgenic animal models that mimic many of the hallmarks of tau protein aggregation as observed in NFTs in AD and related tauopathies. PUBLIC HEALTH RELEVANCE: This Phase I SBIR project will lead to the development of novel small molecule drugs for the treatment of tau protein aggregation found in the neurofibrillary tangles of Alzheimer disease. Neurofibrillary tangle formation is one of the pathological hallmarks of Alzheimer's disease, the leading cause of dementia in the elderly.