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)是以脑淀粉样斑块为特征的疾病，这种斑块由不溶性的β-淀粉样蛋白(A2)和含有神经原纤维缠结(NFT)的tau蛋白组成。广告药物发现策略在很大程度上专注于降低大脑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 Inc.开发了许多不同的体外筛选技术和细胞模型，以确定新的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[含有双儿茶酚的小分子(200-400 mW)组成的新化学实体]，其中一些被发现是对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。这一第一阶段项目的初步结果表明，我们的一些新化学实体在体外和相关的创新细胞检测中具有干扰/抑制tau蛋白原纤维形成的能力。[我们假设，我们独特的小分子新化学实体库中的某些小化合物可以作为tau聚集/纤维形成的直接抑制剂，并可能作为AD和其他tau病的新治疗药物具有巨大的潜力。]我们建议的主要目标是通过体外和基于细胞的分析，从ProteoTech独特的文库中鉴定特定的小分子化合物，以了解它们抑制/破坏tau聚集/纤维形成的能力。在[特定目标1]中，我们将使用硫代黄素T荧光法、SDS-PAGE/Western印迹分析和定量、CD光谱和电子显微镜等体外检测方法，筛选ProteoTech(包含多羟基芳香化合物的小分子)开发的一组约50个小分子化合物(全部代表新的化学实体)，以考察它们抑制tau蛋白原纤维形成和破坏预先形成的tau纤维的能力。]在特定目标2中，我们将利用可诱导表达tau重复结构域(TauRD)的细胞系来筛选/验证我们在目标1中确定的先导化合物是否能够影响细胞内tau多聚体/聚集的能力，方法是对可溶/不溶的tau重复结构域聚集体进行Western分析，并对硫黄素S染色和电子显微镜下的tau重复结构域聚集体/纤维进行检测。我们还将测试这些化合物是否可以挽救TauRD诱导的微管网络破坏和细胞毒性，通过免疫染色和定量图像分析来确定。该项目中确定的最具活性的化合物将导致未来的第二阶段SBIR项目，以优化这些关键的先导化合物的药效性、脑渗透性、药代动力学和无毒性，并在相关转基因动物模型中测试它们的有效性，这些动物模型模仿AD和相关tauopathy患者NFT中观察到的tau蛋白聚集的许多特征。 公共卫生相关性： 这个第一阶段的SBIR项目将导致开发新的小分子药物，用于治疗阿尔茨海默病神经原纤维缠结中发现的tau蛋白聚集。神经原纤维缠绕形成是阿尔茨海默病的病理特征之一，阿尔茨海默病是导致老年人痴呆的主要原因。

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.