Discovery of apoE4 modulators for Alzheimer’s disease therapy

项目总结 阿尔茨海默病(AD)是痴呆症中最常见的一种，伴有认知障碍 淀粉样蛋白β(Aβ)斑块和含有tau的神经原纤维缠结的缺失和病理性堆积。这个 大多数AD病例发生在晚年，通常发生在65岁之后。最强的遗传风险 晚发性AD(LOAD)的致病因素是载脂蛋白E(ApoE)基因，ε4等位基因增加了AD的风险， 与常见的ε3等位基因相比，ε2等位基因具有保护作用。越来越多的证据表明- 调节apoE4蛋白水平和/或抑制apoE4聚集不仅减轻淀粉样蛋白病变，而且 还可以防止tau介导的神经退行性变。在我们的初步研究中，我们开发了apoE4 用分裂的荧光素酶蛋白片段互补的报告分析，使我们不仅能够监测apoE4 蛋白质水平，还可以通过荧光素酶生物发光信号测量apoE4的自我寡聚/聚集 高通量筛选格式。在Mayo-SBP(Sanford Burnham Prebys)药物的支持下 发现合作计划，我们对大约17,000种小分子化合物进行了初步筛选 性能令人满意，并发现了一种新型的apoE4调节剂，它下调apoE4蛋白水平并 抑制AD患者诱导多能干细胞(IPSCs)来源的脑器官中tau的磷酸化 携带APOE4，表明我们的HTS检测是可靠的，并能够识别新的apoE4调节子。 在此，我们提出了一项合作努力，以确定下调apoE4蛋白水平的apoE4调节器 和/或抑制用于AD治疗的apoE4聚集。AIM 1将完成apoE4报告器的高通量 在大型化学文库上筛选以确定抑制apoE4水平的有效和特定的apoE4调节剂 和/或聚合。目标2将研究apoE4的效力、肌动蛋白(MOA)的模式和治疗效果 使用生化和基于细胞的分析和人类IPSC衍生的细胞模型的调节器。目标3将 对先导化合物进行逐个目录的和有限的化学处理，以提高其效力和有效性 确定最有希望的候选化合物在三维脑器官模型中的治疗效果 这样我们的发现可能会在人性化的环境中具有相关性。此外，“类毒品”和 将对药代动力学特性和脑渗透率进行表征和基准，以确定它们的位置 未来的活体临床前动物研究。已鉴定的载脂蛋白E4调节剂将成为新的药物先导化合物 AD的治疗策略。

PROJECT SUMMARY Alzheimer's disease (AD) is the most common form of dementia accompanied by detrimental cognitive deficits and pathological accumulation of amyloid-β (Aβ) plaques and tau-containing neurofibrillary tangles. The majority of AD cases occur late in life, and it usually develops after 65 years of age. The strongest genetic risk factor for late-onset AD (LOAD) is apolipoprotein E (APOE) genotype, with the ε4 allele increasing AD risk and the ε2 allele being protective compared with the common ε3 allele. Increasing evidence indicates that down- regulation of apoE4 protein level and/or inhibition of apoE4 aggregation not only alleviate amyloid pathology but also protect against tau-mediated neurodegeneration. In our preliminary studies, we have developed an apoE4 reporter assay with a split luciferase protein-fragment complementation, enabling us to not only monitor apoE4 protein level but also measure apoE4 self-oligomerization/aggregation by luciferase bioluminescent signals in a high-throughput screening format. Under the support of the Mayo-SBP (Sanford Burnham Prebys) Drug Discovery Collaboration Program, we performed a pilot screen of ~17,000 small molecule compounds with satisfactory performance, and discovered a novel apoE4 modulator that down-regulates apoE4 protein level and inhibits tau phosphorylation in induced pluripotent stem cells (iPSCs)-derived cerebral organoids from AD patient carrying APOE4, demonstrating that our HTS assay is robust and capable of identifying novel apoE4 modulators. Herein, we proposed a collaborative effort to identify apoE4 modulators that down-regulate apoE4 protein level and/or suppress apoE4 aggregation for AD therapy. Aim 1 will complete the apoE4 reporter high-throughput screen on a large chemical library to identify potent and specific apoE4 modulators for suppressing apoE4 level and/or aggregation. Aim 2 will examine the potency, modes of actin (MOA), and therapeutic effects of apoE4 modulators using biochemical and cell-based assays and human iPSC-derived cellular models. Aim 3 will perform SAR-by-catalog and limited chemistry on lead compounds to improve their potency and efficacy and determine the therapeutic efficacy of the most promising candidate compounds in 3-D cerebral organoid models such that our findings may have relevance in a humanized setting. Moreover, the “drug-like” and pharmacokinetics properties and brain penetration will be characterized and benchmarked to position them for future in vivo preclinical animal studies. The identified apoE4 modulators will be promising drug leads in novel therapeutic strategies for AD.