Small Molecule Screen for Apolipoprotein-E/Alzheimer's Disease

阿尔茨海默病（AD）直接困扰着500万美国人，并间接影响着估计1300万护理人员。仅次于年龄，AD的第二大风险因素是APOE（载脂蛋白E）基因型，其中APOE 4携带者患AD的风险显着高于非APOE 4（主要是APOE 3）携带者。我们已经发现了载脂蛋白-E的模拟物，其是衍生自全蛋白（apoE 133 - 149）的残基133 - 149的小肽家族。这些apoE-肽显示出神经恢复特征，包括抗炎和神经保护特性，以及刺激髓鞘重建的活性（Li et al. 2006，Lynch et al. 2004，Hoane et al. 2007，Laskowitz et al. 2007，Tukhovskaya et al. 2008，Li et al.提交，Christensen et al.提交，Saransteva et al.修订提交）。 在更完整的阿尔茨海默病小鼠模型中，出现阿尔茨海默病样病理，神经元丢失和行为缺陷，（Colton等2006，Wilcock等2008，Colton等未发表），这些apoE-肽的给药减少了淀粉样斑块样结构和神经胶质缠结样结构;在学习和记忆任务中显著改善行为表现（请参见初步结果）。我们最近发现apoE肽在细胞内通过一种新的机制激活蛋白磷酸酶2A（PP 2A）。该机制涉及apoE-肽与I2 PP 2A（PP 2A的抑制剂#2）结合以形成apoE/I2 PP 2A复合物，该复合物显然不能抑制PP 2A磷酸酶活性。由于阿尔茨海默氏症是一种多组分疾病，涉及PP 2A活性丧失、I2 PP 2A增加、炎症增加和磷酸化tau水平增加，我们认为apoE-肽介导的PP 2A活化至更健康的水平可能在减缓、停止和/或逆转疾病进程方面具有显著和多方面的优势。 该提案的目标是发现和开发口服活性的小分子，其功能类似于apoE-肽，以抑制I2 PP 2A蛋白，从而增加PP 2A磷酸酶活性。因此，我们建议开发一个高通量筛选的基础上的小分子与apoE-肽/I2 PP 2A复合物形成的竞争。该竞争测定将用于筛选100，000种不同小分子的文库。假设命中率为1%，我们预测在该竞争测定中将鉴定出e100个小分子，其抑制apoE-肽和I2 PP 2A之间的复合物形成。然后在基于细胞的测定中测定这100个“命中”，以评估用脂多糖（LPS）和每种化合物处理细胞后它们的抗炎活性。然后测试每种确认具有抗炎活性的化合物在全细胞中激活PP 2A磷酸酶活性的能力。然后测试5种最有效的化合物在全动物中以剂量依赖性方式抑制LPS刺激的细胞因子释放的能力。使用我们公布的方法（Lynch et al. 2003），我们将间接测定这5种化合物的血脑屏障渗透和脑活性。在未来的提议中，抑制血液和大脑中炎症的化合物将用药物化学方法进行化学修饰，并在阿尔茨海默病的动物模型中进一步评估药物样质量。 阿尔茨海默病（AD）直接影响500万美国人，间接影响估计1300万护理人员。在年龄之后，APOE（载脂蛋白-E）基因型是第二大风险因素：APOE 4携带者患AD的风险显著高于非APOE 4（主要是APOE 3）携带者。我们已经发现了一个家族的小肽，作为apoE模拟物的功能。这些肽来源于载脂蛋白-E（apoE）全蛋白的残基133-149，显示出神经恢复特征，包括抗炎和神经保护特性，以及刺激髓鞘重建的活性（Li等人，2006年; Lynch等人，2004年; Hoane等人，2007年; Laskowitz等人，2007年; Tukhovskaya等人，2008年; Li等人提交，Chirstensen等人提交，Saransteva等人提交修订）。 在我们的初步研究中，这些apoE模拟肽减少了淀粉样蛋白斑块、神经元缠结的存在，并改善了我们更完整的阿尔茨海默病转基因小鼠模型的学习和记忆能力。我们现在希望发现和开发与我们的apoE模拟肽以相同方式起作用的小分子。这将需要几个层次的筛选，以找到少数化合物，可以口服给药，并显示在实验动物的大脑保护活性。

DESCRIPTION (provided by applicant): Alzheimer's disease (AD) afflicts 5 million Americans directly, and indirectly affects an estimated 13 million caregivers. Behind age, the second largest risk factor for AD is APOE (apolipoprotein-E) genotype where APOE4 carriers are at significantly higher risk for AD than their non-APOE4 (mainly APOE3) counterparts. We have discovered mimetics of apolipoprotein-E which are a family of small peptides derived from residues 133-149 of the holoprotein (apoE 133-149). These apoE-peptides display neurorestorative features that include anti-inflammatory and neuroprotective properties, and activities that stimulate the rebuilding of myelin (Li et al. 2006, Lynch et al. 2004, Hoane et al. 2007, Laskowitz et al. 2007, Tukhovskaya et al. 2008, Li et al. submitted, Christensen et al. submitted, Saransteva et al. revision submitted). In more complete mouse models of Alzheimer's disease that develop Alzheimer-like pathology, neuronal loss and behavioral deficits (Colton et al. 2006, Wilcock et al. 2008, Colton et al. unpublished), administration of these apoE-peptides reduced both amyloid plaque-like structures and neurofibrillary tangle- like structures; and significantly improved behavioral performance in a learning and memory task (please see Preliminary Results). We recently discovered that apoE-peptides function inside the cell to activate Protein Phosphatase 2A (PP2A) by a novel mechanism. This mechanism involves the binding of apoE-peptides to I2PP2A (Inhibitor #2 of PP2A) to form an apoE/I2PP2A complex that apparently is unable to inhibit PP2A phosphatase activity. Since Alzheimer's is a multi-component disorder involving loss of PP2A activity, an increase in I2PP2A, increased inflammation and increased levels of phospho-tau, we submit that apoE- peptide-mediated activation of PP2A to more healthy levels may have a significant and multi-dimensional advantage in slowing, halting and/or reversing the course of disease. The goal of this proposal is to find and develop orally active, small molecules that function like apoE- peptides to inhibit the I2PP2A protein, and thereby increase PP2A phosphatase activity. Thus, we propose to develop a high throughput screen based on competition of small molecules with apoE-peptide/I2PP2A complex formation. This competition assay will be used to screen a library of 100,000 diverse small molecules. Assuming a hit rate of 1%, we predict that e 100 small molecules will be identified in this competition assay that inhibit complex formation between apoE-peptide and I2PP2A. These 100 "hits" will then be assayed in a cell- based assay to assess their anti-inflammatory activity after treatment of cells with lipopolysaccharide (LPS) and each compound. Each compound that is confirmed to have anti-inflammatory activity will then be tested for its ability to activate PP2A phosphatase activity in whole cells The 5 most potent compounds will then be tested in whole animals for their ability to inhibit LPS stimulated cytokine release in a dose dependent fashion. Using our published method (Lynch et al. 2003), we will indirectly assay blood brain barrier penetration and brain activity of these 5 compounds. In future proposals, compounds that inhibit inflammation in the blood and in the brain, will be chemically modified with medicinal chemistry approaches and further evaluated for drug- like qualities in animal models of Alzheimer's disease. PUBLIC HEALTH RELEVANCE: Alzheimer's disease (AD) afflicts 5 million Americans directly, and indirectly affects an estimated 13 million caregivers. Behind age, APOE (apolipoprotein-E) genotype is the second largest risk factor: APOE4 carriers are at significantly higher risk for AD than their non-APOE4 (mainly APOE3) counterparts. We have discovered a family of small peptides that function as apoE mimetics. Derived from residues 133-149 of the apolipoprotein-E (apoE) holoprotein, these peptides display neurorestorative features that include anti- inflammatory and neuroprotective properties, and activities that stimulate the rebuilding of myelin (Li et al. 2006, Lynch et al. 2004, Hoane et al. 2007, Laskowitz et al. 2007, Tukhovskaya et al. 2008, Li et al. submitted, Chirstensen et al. submitted, Saransteva et al. revision submitted). These apoE mimetic peptides, in our pilot studies, reduced the presence of amyloid plaques, neurofibrillary tangles, and improved the learning and memory ability of our more complete transgenic mouse model of Alzheimer's disease. We now wish to find and develop small molecules that act in the same way as our apoE-mimetic peptides. This will require several levels of screening to find the few compounds that can be orally administered and show protective activity in the brains of experimental animals.