Alzheimer's Drug Discovery Using Unique Nanotechnology Platform

描述(申请人提供)：阿尔茨海默病(AD)是导致老年人痴呆症的主要原因，在美国发病率为540万，每年花费1830亿美元。目前还没有有效的治疗方法。这项建议是对NIA提出的R21高风险/高回报建议的回应，以产生新的AD药物发现战略。它侧重于目前的知识表明，AD中显著的神经元损伤可归因于毒性抗体寡聚体的影响。这些可扩散的小分子不同于淀粉样斑块，被认为通过其靶向突触和破坏突触可塑性的能力而引发AD记忆丧失。目前预防AD的努力主要集中在阻断抗体单体积累的代谢抑制剂和去除单体或其有毒组件的被动疫苗上。这些努力到目前为止还没有成功。这项建议引入了另一种战略，重点放在低聚物毒性机制的第一步。为了引起损伤，寡聚体必须首先与细胞受体结合。这些受体介导低聚物与特定神经元的联系，并将结合转化为突触毒性反应。虽然受体的同一性对于阐明毒性机制是有价值的，但即使在没有这种知识的情况下，使用寡聚体-受体结合作为药物发现的靶点也是可行的。需要一种适合于结合拮抗剂的大规模高通量筛选(HTS)的方法。我们提出了一种新的策略来实现这一目标，使用一种产生人造纳米级膜(纳米盘)的方法。这是一种成熟的方法，适用于提供无偏倚和功能性的突触质膜蛋白的可溶性制剂。低聚物与突触质膜(SPM)纳米盘的结合已经被证实，并表现出预期的配体-受体相互作用的特征。结合反应已适用于适用于低聚物结合拮抗剂的均相化学发光分析。与高含量、基于细胞的分析不同，与可溶性受体结合的生化分析具有对非常大的化合物文库进行初步筛选所需的带宽和精度。与该项目分开，对这些扫描探针纳米盘的平行研究有望确定受体蛋白(S)。我们的药物发现方法遵循筛选树，在筛选树中，使用SPM纳米盘进行初步分析的命中结果在基于细胞的分析中得到结合和毒性验证，首先是合成低聚物，然后是脑源性低聚物。NanoDisk HTS和二次屏幕将首先使用一个小库进行优化，然后进行大幅扩展。西北分子创新和药物发现中心的Hit-to-Lead资源将根据需要实施。这一目标预计将在两年内实现，目的是为目前在国家卫生研究院主持下提供的超大型图书馆的HTS制定一个全面运作的战略。结果最终有很大的潜力发现先导化合物，目标是AD发病机制中一个未被充分利用但重要的方面。 公共卫生相关性：这项建议是对NIA提出的R21高风险/高回报建议的回应，以产生新的阿尔茨海默病(AD)药物发现战略。AD是导致老年人痴呆症的主要原因，在美国的发病率为540万，每年花费1830亿美元。目前还没有有效的治疗方法。该项目有望有很强的潜力识别以突触毒素抗体寡聚体与其受体结合为靶点的先导化合物，这是AD发病机制中未被充分利用但关键的早期步骤。

DESCRIPTION (provided by applicant): Alzheimer's disease (AD) is the major cause of dementia in the elderly, with a US incidence of 5.4 million and an annual cost of $183 billion. There is no effective treatment. This proposal is in response to an NIA request for R21 high risk/high reward proposals to generate new AD drug discovery strategies. It focuses on current knowledge indicating that significant neuron damage in AD can be attributed to the impact of toxic Ab oligomers. These small diffusible molecules are distinct from amyloid plaques and are thought to instigate AD memory loss through their ability to target synapses and disrupt synaptic plasticity. Current efforts to prevent AD largely focus on metabolic inhibitors that block accumulation of the Ab monomer and on passive vaccines that remove either the monomer or its toxic assemblies. These efforts have not as yet been successful. This proposal introduces an alternative strategy that focuses on the first step in the mechanism of oligomer toxicity. To elici damage, oligomers must first bind to cellular receptors. These receptors mediate association of oligomers with particular neurons and transduce binding into synaptotoxic responses. Although receptor identity would be valuable for elucidating the mechanism of toxicity, it is feasible even without this knowledge to use oligomer-receptor binding as a target for drug discovery. What is required is an assay suitable for large- scale high-throughput screening (HTS) of binding antagonists. We propose a novel strategy to achieve this goal using an approach that generates artificial nanoscale membranes (Nanodiscs). This is a well-established methodology that has been adapted here to provide unbiased and functional soluble preparations of synaptic plasma membrane proteins. Binding of oligomers to synaptic plasma membrane (SPM) Nanodiscs has been demonstrated and exhibits characteristics expected of ligand-receptor interaction. The binding reaction has been adapted to a homogeneous chemiluminescence assay well-suited to HTS for antagonists of oligomer binding. Unlike high content, cell-based assays, the biochemical assay for binding to soluble receptors has the bandwidth and precision required for the primary screening of very large libraries of compounds. Parallel investigations of these SPM-Nanodiscs, separate from this project, are expected to identify the receptor protein(s). Our Approach to drug discovery follows a screening tree in which hits from the primary assay using SPM-Nanodiscs are validated in cell-based assays for binding and toxicity, first with synthetic oligomers and then with brain-derived oligomers. The Nanodisc HTS and secondary screens will be optimized at first using a small library and then greatly expanded. Hit-to-lead resources o the Northwestern Center for Molecular Innovation and Drug Discovery will be implemented as needed. The Aim, expected to be achieved by the end of two years, is to establish a fully functioning strategy for HTS of the very large libraries now available under the auspices of NIH. Results ultimately have strong potential for discovering lead compounds that target an underexploited but significant aspect of AD pathogenesis. PUBLIC HEALTH RELEVANCE: This proposal is in response to an NIA request for R21 high risk/high reward proposals to generate new Alzheimer's disease (AD) drug discovery strategies. AD is the major cause of dementia in the elderly, with a US incidence of 5.4 million and an annual cost of $183 billion. There is no effective treatment. This project is expected to have strong potential to identify lead compounds that target binding of synaptotoxic Ab oligomers to their receptors, an underexploited but critical early-step in AD pathogenesis.