Screening for enhancers of sAPPalpha

 描述(申请人提供)：阿尔茨海默病(AD)目前在美国困扰着540多万人，估计每年的成本超过2000亿美元。目前批准的药物只能提供短期的症状缓解，但不能改变疾病的进展。神经性斑块是AD脑的标志，是由淀粉样前体蛋白(APP)产生的淀粉样β多肽(Aβ)生成增加和/或清除减少所致。APP可能会有两条路径进行处理。在一种情况下，它经历β-分泌酶和γ-分泌酶的顺序切割，产生可溶性淀粉样前体蛋白β(SAPβ)和Aβ；在另一种情况下，它被α-分泌酶切割，产生可溶性淀粉样前体蛋白α(SAPα)，这是一种可能预防AD的前认知多肽。这个项目建议推进我们之前的研究，成功地确定了SAPα增强子托烷司琼(F03)，目标是确定新的SAPα增强子(NCE)为新的化学实体(NCE)，构成了一类治疗AD的新药物。我们雄厚的初步数据基础表明，这个项目的具体目标很有可能实现。我们已经利用我们的迭代、分级化学遗传学筛选方法从一个小型临床化合物库中鉴定了F03-一种用于术后恶心和呕吐的药物--已取得初步成功。在体外和体内AD小鼠模型中，F03被证明持续但温和地增加SAPPα，并在口服治疗四周后显著改善AD模型的记忆。F03目前正在进行轻度认知障碍(MCI)的临床试验 将AD作为一种改变用途的药物。在这里，我们建议利用我们的高通量筛选试验、迭代筛选流程方案和加州大学洛杉矶分校的大型化合物库来鉴定具有大于F03的SAPPα增强效应的化合物。在主要的高温超导屏幕中，将识别增加Sappα的“点击”。在第二个屏幕中，使用第二个体外模型验证命中。然后，将通过对AD模型脑的体外器官型培养和平行人工膜通透性分析(PAMPA)和体内药代动力学(PK)分析确定的脑渗透率来进一步验证优先命中。最后，我们将使用CEREP Bioprint Profile、药物亲和力反应靶标稳定性(DARTS)、使用相似性集成方法和新的凝胶增强靶标识别方法(GET)来确定HITS诱导SAPPα增强的机制。虽然其他识别AD和MCI治疗方法的新方法正在开发中，但它们的临床结果仍然不确定。通过鉴定具有比F03更强的SAPPα增强作用的化合物，我们可能会发现有希望的NCEs进一步的临床开发。也许最重要的是，我们对这些促进剂的机制研究可能会进一步阐明SAPPα在AD病因中的作用，为治疗开发提供新的方向。

 DESCRIPTION (provided by applicant): Alzheimer's disease (AD) currently afflicts more than 5.4 million people in the US at an estimated cost greater than $200 billion per year. Currently approved drugs offer only short-term symptomatic relief but do not alter disease progression. The neuritic plaques that are a hallmark of AD brain result from increased generation and/or decreased clearance of the amyloid beta peptide (Aβ) which originates from amyloid precursor protein (APP). APP may be processed by two pathways. In one, it undergoes sequential β-secretase and γ-secretase cleavages to generate soluble amyloid precursor protein beta (sAPPβ) and Aβ; in the other, it is cleaved by an α-secretase to generate soluble amyloid precursor protein alpha (sAPPα), a pro-cognitive peptide that may prevent AD. This project proposes to advance our previous studies that successfully identified the sAPPα-enhancer tropisetron (F03) with the goal of identifying new sAPPα-enhancers as new chemical entities (NCEs) constituting a novel class of therapeutics for AD. Our strong foundation of preliminary data indicates a high probability that the specific aims of this project can be achieved. We have had early success utilizing our iterative, hierarchical chemical-genetics screening approach in the identification of F03 - a drug used for post- operative nausea and vomiting - from a small clinical compound library. F03 was shown to consistently but modestly increase sAPPα in vitro and in vivo in a mouse model of AD, and to significantly improve memory in the AD model after four weeks of oral treatment. F03 is now in clinical trials for mild cognitive impairment (MCI) due to AD as a repurposed drug. Here, we propose utilizing our high-throughput screening (HTS) assay, iterative screening flowscheme, and the large UCLA compound library to identify compounds with sAPPα-enhancing effects greater than F03. In the primary HTS screen, "hits" that increase sAPPα will be identified. In the secondary screen, hits are validated using a second in vitro model. Prioritized hits will then be further validated by ex vivo organotypic cultue of AD model brain and brain penetrance determined by parallel artificial membrane permeability analysis (PAMPA) and in vivo pharmacokinetic (PK) analysis. Finally, we will determine the mechanisms by which hits induce sAPPα enhancement using the CEREP Bioprint profile, drug affinity responsive target stability (DARTS), in silico target ID using the similarity ensemble approach, and by a new gel-enhanced target identification approach (GET). While other new approaches to identify therapeutics for AD and MCI are under development, their clinical outcomes remain uncertain. By identifying compounds with sAPPα enhancing effects greater than F03, we may find promising NCEs for further clinical development. Perhaps most importantly, our mechanistic studies of such enhancers may further elucidate the role of sAPPα in the etiology of AD, providing new direction for therapeutic development.