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

描述(申请人提供)：阿尔茨海默病(AD)直接影响500万美国人，间接影响约1300万照顾者。仅次于年龄，AD的第二大危险因素是APOE(载脂蛋白-E)基因，其中APOE4携带者患AD的风险显著高于非APOE4(主要是APOE3)携带者。我们已经发现了载脂蛋白-E的模拟物，这是一个由完整蛋白(apoE 133-149)的133-149残基衍生的小肽家族。这些apoE-肽显示出神经修复功能，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，Christensen等人。提交，Saransteva等人。已提交修订)。在更完整的阿尔茨海默病小鼠模型中，发展为阿尔茨海默病样病理、神经元丢失和行为缺陷(Colton等人。2006年，Wilcock等人。2008年，科尔顿等人。(未发表)，服用这些apoE-肽可以减少淀粉样斑块状结构和神经纤维缠结状结构；并显著改善学习和记忆任务中的行为表现(请参阅初步结果)。我们最近发现，apoE-肽通过一种新的机制在细胞内激活蛋白磷酸酶2A(PP2A)。这一机制涉及apoE-肽与I2PP2A(PP2A的抑制剂#2)结合形成apoE/I2PP2A复合体，该复合体显然不能抑制PP2A磷酸酶活性。由于阿尔茨海默病是一种多组分的疾病，涉及PP2A活性丧失、I2PP2A增加、炎症增加和磷酸化tau水平增加，我们认为apoE肽介导的PP2A激活到更健康的水平可能在减缓、阻止和/或逆转病程方面具有显著的多方面优势。这项建议的目标是找到并开发具有口服活性的小分子，其功能类似于apoE-肽，以抑制I2PP2A蛋白，从而提高PP2A磷酸酶活性。因此，我们建议开发一种基于apoE-肽/I2PP2A复合体形成的小分子竞争的高通量筛选方法。这种竞争分析将被用来筛选10万个不同的小分子文库。假设命中率为1%，我们预测在这个竞争实验中将发现100个小分子，它们抑制apoE-肽和I2PP2A之间的复合体的形成。然后，在用脂多糖(LPS)和每种化合物处理细胞后，将在基于细胞的测试中对这100种“命中”进行检测，以评估它们的抗炎活性。每一种被证实具有抗炎活性的化合物都将被测试其激活整个细胞中PP2A磷酸酶活性的能力，然后将在整个动物中测试它们以剂量依赖的方式抑制脂多糖刺激的细胞因子释放的能力。使用我们发表的方法(Lynch等人2003)，我们将间接测定这5种化合物的血脑屏障渗透率和脑活性。在未来的提案中，将使用药物化学方法对抑制血液和大脑中炎症的化合物进行化学修饰，并在阿尔茨海默病的动物模型中进一步评估药物的性质。 公共卫生相关性：阿尔茨海默病(AD)直接困扰着500万美国人，间接影响着大约1300万照顾者。ApoE(载脂蛋白-E)基因是仅次于年龄的第二大危险因素：ApoE4携带者患AD的风险显著高于非APOE4(主要是APOE3)携带者。我们已经发现了一系列小肽，它们具有载脂蛋白E模拟物的功能。这些多肽来源于载脂蛋白-E(ApoE)全蛋白的133-149残基，具有神经修复功能，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。已提交修订)。在我们的试点研究中，这些apoE模拟肽减少了淀粉样斑块和神经纤维缠结的存在，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。我们现在希望找到并开发与我们的载脂蛋白E模拟多肽具有相同作用的小分子。这将需要几个级别的筛选，以找到少数几种可以口服并在实验动物的大脑中显示保护活性的化合物。

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.