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

描述（由申请人提供）：阿尔茨海默病（AD）直接折磨着500万美国人，并间接影响着大约1300万护理人员。在年龄之后，AD的第二大危险因素是APOE（载脂蛋白e）基因型，APOE4携带者比非APOE4（主要是APOE3）携带者患AD的风险明显更高。我们已经发现了载脂蛋白e的模拟物，这是一个从全蛋白（apoE 133-149）残基133-149衍生的小肽家族。这些载子e肽显示神经恢复功能，包括抗炎和神经保护特性，以及刺激髓磷脂重建的活性（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，Christensen等人提交，Saransteva等人提交修订）。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、神经元丧失和行为缺陷（Colton et al. 2006, Wilcock et al. 2008, Colton et al.未发表），给药这些载脂蛋白肽减少了淀粉样斑块样结构和神经原纤维缠结样结构；并且在学习和记忆任务中的行为表现显著改善（请参见初步结果）。我们最近发现apoe肽在细胞内通过一种新机制激活蛋白磷酸酶2A （PP2A）。该机制涉及apoE-肽与I2PP2A （PP2A的抑制剂#2）结合，形成apoE/I2PP2A复合物，该复合物显然无法抑制PP2A磷酸酶活性。由于阿尔茨海默氏症是一种涉及PP2A活性丧失、I2PP2A增加、炎症增加和磷酸化tau水平增加的多组分疾病，我们认为apoE肽介导的PP2A激活到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的多维优势。本提案的目标是寻找和开发具有口服活性的小分子，其功能类似于apoE-肽，可以抑制I2PP2A蛋白，从而增加PP2A磷酸酶的活性。因此，我们建议开发一种基于apoE-peptide/I2PP2A复合物形成的小分子竞争的高通量筛选方法。该竞赛分析将用于筛选包含100,000种不同小分子的文库。假设命中率为1%，我们预测在这个竞争分析中将鉴定出100个抑制apoe肽和I2PP2A之间复合物形成的小分子。这100个“命中”将在以细胞为基础的实验中进行分析，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。每一种被证实具有抗炎活性的化合物都将被测试其在全细胞中激活PP2A磷酸酶活性的能力，然后在全动物身上测试5种最有效的化合物以剂量依赖的方式抑制LPS刺激的细胞因子释放的能力。使用我们发表的方法（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动。在未来的建议中，抑制血液和大脑炎症的化合物将通过药物化学方法进行化学修饰，并进一步评估阿尔茨海默病动物模型的药物样质量。

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 unpublished), administration of these apoE-peptides reduced both amyloid plaque-like structures and neurofibrillary tangle- like structures; and significantly improved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.