Cholinesterase Inhibitors in Alzheimer's Disease

描述(申请人提供)：阿尔茨海默病(AD)的特征是胆碱能功能障碍和由AB前体蛋白(APP)衍生的淀粉样β-肽(AB)的沉积。目前FDA批准的四种治疗AD的药物属于胆碱酯酶抑制剂(ChEI)类别。这些药物对治疗轻到中度的AD很有用，但在晚期使用有限。我们的目标是测试新型乙酰胆碱酯酶抑制剂(AChEIs)或丁酰胆碱酯酶抑制剂(BChEIs)对AD病理生理学中的关键步骤的作用，以更好地了解这些药物的效果。我们的重点是测试新的治疗策略，以验证和新的疾病靶点。我们的假设是，由于某些ChEI具有降低AB的特性，因此具有独立于其胆碱能活性的神经保护活性。我们建议研究ChEI药物在淀粉样变通路上的机制，这是基于我们的结果，即某些ChEI显著降低培养细胞和动物的AB水平。具体目的是：1]研究新型AChEI和BChEI对APP通路步骤的影响。我们将考察新的CHEI对APP通路的影响：AB和BACE水平，II)官能团取代的影响，III)APPmRNA5‘-非编码区，以及IV)突触蛋白。[2]检测一组新型CHEI对AB体内沉积的影响。我们将研究不同剂量的选择性AChEIs和BChEIs对APPswe-Tau淀粉样斑块加缠结双转基因小鼠APP和AB的影响。3]探讨胆碱酯酶在AChE基因敲除小鼠中的作用。我们将从新的AChE基因敲除小鼠的脑组织样本中研究AChE酶对APP、AB肽和突触蛋白标记物的作用。[4]探讨CHEIs对人类(存档)标本分子标记的影响。为了验证动物研究，我们将测试选定的CHEIs治疗对人血浆和/或脑脊液样本中APP和AB水平的影响。主要终点是定量的和功能性的：细胞存活、酶测定和APP途径蛋白/肽和突触蛋白标记物的水平。我们将机械地选择与ChE的外周变构结合结构域以及与酯类和阴离子结合结构域相互作用的ChEI，并在细胞培养和动物模型中测试它们。这项工作将表明CHEIs对AB和突触蛋白的独特影响，与它们对酶的选择性无关。这一特性将有助于最大限度地发挥其对淀粉样蛋白和突触蛋白的有益影响，可用于设计更好的阿尔茨海默病治疗剂。与公共卫生相关：阿尔茨海默病(AD)的特点是胆碱能系统的突触前标志物减少，特别是在大脑中与记忆和学习相关的区域，以及由AB前体蛋白(APP)衍生的淀粉样β蛋白(AB)的沉积。目前FDA批准的治疗AD的五种药物中有四种是胆碱酯酶抑制剂(ChEI)，它通过抑制胆碱酯酶(ChE)酶来增加大脑乙酰胆碱(ACh)的供应，从而保护胆碱能回路，据信胆碱能回路介导记忆通路。更令人惊讶的是，这些药物对记忆没有实质性的影响，但有一个意想不到的、受欢迎的结果，将认知保存了一段略长的时间。本提案试图表征ChEIs的神经保护和保护机制，并对这一途径进行微调，以改善ChEIs的这一有益特性。我们的目标是测试新型乙酰胆碱酯酶抑制剂(AChEIs)或丁酰胆碱酯酶抑制剂(BChEIs)对AD病理生理学中的关键步骤的作用，以更好地了解这些药物的效果。我们建议研究ChEI药物在将APP加工为潜在毒性AB的淀粉样变通路上的机制。我们的研究最初集中在这些药物对AB产生的影响，最近已扩展到突触蛋白标记物和其他神经保护作用。该提案的结果是确定CHEIs阻断潜在毒性AB水平的机制，并利用这一特性开发新的治疗剂。

DESCRIPTION (provided by applicant): Alzheimer's disease (AD) is characterized by cholinergic dysfunction and depositions of the amyloid beta-peptide (AB) derived from AB precursor protein (APP). Four current FDA-approved drugs for AD belong to the category of cholinesterase inhibitor (ChEI). These drugs are useful for treatment of mild to moderate AD but limited use in advanced stages. Our goal is to test novel acetylcholinesterase inhibitors (AChEIs) or butyrylcholinesterase inhibitors (BChEIs) against pivotal steps in the pathophysiology of AD to better understand the drugs' effects. Our focus is to test new therapeutic strategies to both validated and novel disease targets. Our hypothesis is that certain ChEIs have neuroprotective activity independent of their cholinergic activity due to their AB-lowering property. Our proposal to study the mechanism of ChEI drugs on amyloidogenic pathways is based on our results that certain ChEIs significantly reduced AB levels in cultured cells and animals. The specific aims are: 1] To study the effect of novel AChEIs and BChEIs on APP pathway steps. We will examine i) the effect of novel ChEIs on APP pathway: AB and BACE levels, ii) the effect of substitution of functional groups, iii) APPmRNA 5'-UTR, and iv) synaptic proteins. 2] To test the effect of a novel group of ChEIs on AB deposition in vivo. We will study the effects of different doses of selective AChEIs and BChEIs on APP and AB in double transgenic APPSWE-Tau amyloid plaque plus tangle producing mice. 3] To examine the role of ChE enzyme in AChE knockout mice. We will study the role of the AChE enzyme on APP, AB peptides and synaptic protein markers in the brain tissue samples from novel AChE knockout mice. 4] To investigate the effect of ChEIs on molecular markers in human (archived) samples. To validate the animal studies, we will test the effect of treatment of selected ChEIs on APP and AB levels in human plasma and/or CSF samples. The primary end-points are quantitative and functional: Cell survival, enzyme assay and levels of APP pathway protein/peptides and synaptic protein markers. We will mechanistically select ChEIs that interact with the peripheral allosteric binding domain of ChE and with the esteratic and anionic binding domains and test them in cell culture and animal models. This work will indicate unique effect of ChEIs on AB and synaptic proteins, independent of their selectivity for the enzyme. This property will help maximize their beneficial effects on amyloid and synaptic proteins, which can be utilized to design better therapeutic agents for AD. PUBLIC HEALTH RELEVANCE: Alzheimer's disease (AD) is characterized by a reduction in the presynaptic markers of the cholinergic system, particularly in areas of the brain related to memory and learning, and by depositions of the amyloid beta peptide (AB), which is derived from the AB precursor protein (APP). Four of five current FDA-approved drugs for AD are cholinesterase inhibitors (ChEI), which increase the brain's supply of acetylcholine (ACh) by inhibiting cholinesterases (ChE) enzyme and thus preserve cholinergic circuits, which are believed to mediate memory pathways. Rather surprisingly, these drugs did not have a substantial effect on memory, but had an unexpected and welcome outcome of preserving cognition for a slightly extended period of time. The present proposal attempts to characterize the mechanisms of neuropreservation and protection by the ChEIs and fine tune this pathway to improve this beneficial property of ChEIs. Our goal is to test novel acetylcholinesterase inhibitors (AChEIs) or butyrylcholinesterase inhibitors (BChEIs) against pivotal steps in the pathophysiology of AD to better understand the effects of the drugs. We propose to study the mechanism of ChEI drugs on amyloidogenic pathways that process APP to potentially toxic AB. Our studies initially focused on the effects of these drugs on AB production and have recently been extended to synaptic protein markers and other neuroprotective effects. The outcome of the proposal is to identify mechanisms by which ChEIs block potentially toxic AB levels and to utilize this property in developing novel therapeutic agents.