Discovery of apoE4 modulators for Alzheimer’s disease therapy

项目摘要 阿尔茨海默氏病（AD）是最常见的痴呆形式，伴有有害的认知功能障碍。 淀粉样蛋白-β（A β）斑块和含tau蛋白的神经元缠结的缺陷和病理学积累。的 大多数AD病例发生在生命的晚期，并且通常在65岁之后发展。最大的遗传风险 晚发性AD（LOAD）的因素是载脂蛋白E（APOE）基因型，ε 4等位基因增加AD风险， 与常见的ε 3等位基因相比，ε 2等位基因具有保护性。越来越多的证据表明，下降- 调节apoE4蛋白水平和/或抑制apoE4聚集不仅减轻淀粉样病变， 也可以防止tau介导的神经变性。在我们的初步研究中，我们开发了一种apoE4 报告基因分析与分裂荧光素酶蛋白片段互补，使我们不仅能够监测apoE4 蛋白水平，而且还通过荧光素酶生物发光信号测量apoE4自身寡聚化/聚集， 高通量筛选格式。在梅奥SBP（Sanford Burnham Prebys）药物的支持下， 发现合作计划，我们进行了约17，000小分子化合物的试点筛选， 令人满意的性能，并发现了一种新的apoE4调节剂，下调apoE4蛋白水平， 抑制来自AD患者的诱导多能干细胞（iPSC）衍生的脑类器官中的tau磷酸化 携带APOE4的小鼠，证明我们的HTS测定是稳健的并且能够鉴定新的apoE4调节剂。 在此，我们提出了一个合作的努力，以确定apoE4调节剂，下调apoE4蛋白水平 和/或抑制apoE4聚集用于AD治疗。Aim 1将完成apoE4报告基因的高通量 在大型化学文库中筛选以鉴定用于抑制apoE4水平的有效且特异性的apoE4调节剂 和/或聚集。目的2将检测apoE4的效力、肌动蛋白模式（MOA）和治疗作用 使用生物化学和基于细胞的测定以及人iPSC衍生的细胞模型来研究细胞因子和细胞因子调节剂。目标3将 对先导化合物进行按目录SAR和有限化学处理，以提高其效力和功效， 确定最有希望的候选化合物在3-D脑类器官模型中的治疗功效 这样我们的发现就可以在人性化的环境中发挥作用。此外，"类毒品"和 药代动力学特性和脑渗透将被表征和基准化，以定位它们， 未来的体内临床前动物研究。所鉴定的apoE4调节剂将是新的有希望的药物先导物。 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.