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

载脂蛋白-E/阿尔茨海默病的小分子筛查

基本信息

批准号：
7947726
负责人：
MICHAEL PETER VITEK
金额：
$ 43.31万
依托单位：
COGNOSCI, INC.
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-09-15 至 2013-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7947726
关键词：
Address Affect Aftercare Age Alzheimer like pathology Alzheimer&apos s Disease American Animal Model Animals Anti-Inflammatory Agents Anti-inflammatory Apolipoprotein E Ascorbic Acid Back Behavioral Binding Biochemical Biological Assay Blood Blood - brain barrier anatomy Brain Businesses Caregivers Cells Complex Detection Disease Doctor of Philosophy Dose Enzyme Inhibitor Drugs Enzyme Inhibitors Family Flowcharts Fluorescence Fluorescence Resonance Energy Transfer Food Funding Mechanisms Future Genes Genotype Goals Grant Health Human Immunoprecipitation Individual Inflammation Inflammatory Inflammatory Response Ingestion Institution Intravenous Learning Libraries Lipopolysaccharides Measures Mediating Memory Messenger RNA Methods Mus Myelin NIH Program Announcements Neurofibrillary Tangles Pathology Patients Penetration Peptides Performance Peripheral Pharmaceutical Chemistry Pharmaceutical Preparations Phosphoric Monoester Hydrolases Pilot Projects Principal Investigator Property Protein Dephosphorylation Protein Phosphatase 2A Regulatory Subunit PR53 Protein phosphatase Proteins Publishing Qualifying Reporting Risk Risk Factors Sailor Screening procedure Scurvy Senile Plaques Signal Transduction Structure Testing Therapeutic Transgenic Mice Transgenic Organisms United States National Institutes of Health Wild Type Mouse apolipoprotein E-3 apolipoprotein E-4 base cytokine functional mimics functional outcomes high risk high throughput screening human disease improved inhibitor/antagonist intraperitoneal mimetics miniaturize mouse model neuron loss neuroprotection neurorestoration novel phosphatase inhibitor prevent public health relevance response small molecule tau Proteins tau aggregation tau-1

项目摘要

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.

描述（由申请人提供）：阿尔茨海默病 (AD) 直接影响 500 万美国人，并间接影响估计 1300 万护理人员。除了年龄之外，AD 的第二大风险因素是 APOE（载脂蛋白-E）基因型，其中 APOE4 携带者患 AD 的风险明显高于非 APOE4（主要是 APOE3）携带者。我们发现了载脂蛋白-E 的模拟物，它是源自全蛋白 (apoE 133-149) 残基 133-149 的小肽家族。这些 apoE 肽显示出神经恢复功能，包括抗炎和神经保护特性以及刺激髓磷脂重建的活性（Li et al. 2006、Lynch et al. 2004、Hoane et al. 2007、Laskowitz et al. 2007、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，这些模型出现了类似阿尔茨海默病的病理、神经元损失和行为缺陷（Colton et al. 2006，Wilcock et al. 2008，Colton et al. unpublished），施用这些 apoE 肽可减少淀粉样斑块-类结构和神经原纤维缠结样结构；并显着提高了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 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。由于阿尔茨海默病是一种多成分疾病，涉及 PP2A 活性丧失、I2PP2A 增加、炎症增加和磷酸 tau 水平增加，因此我们认为 apoE 肽介导的 PP2A 激活至更健康的水平可能具有显着且多方面的作用。 -在减缓、停止和/或逆转病程方面的维度优势。该提案的目标是寻找并开发具有口服活性的小分子，其功能类似于 apoE 肽，可抑制 I2PP2A 蛋白，从而增加 PP2A 磷酸酶活性。因此，我们建议开发一种基于小分子与 apoE-肽/I2PP2A 复合物形成竞争的高通量筛选。该竞争测定将用于筛选 100,000 个不同小分子的文库。假设命中率为 1%，我们预测在该竞争测定中将鉴定出 e 100 个小分子，它们抑制 apoE-肽和 I2PP2A 之间的复合物形成。然后，这 100 个“命中”将在基于细胞的测定中进行测定，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。然后将测试每种被确认具有抗炎活性的化合物在整个细胞中激活 PP2A 磷酸酶活性的能力。然后将在整个动物中测试 5 种最有效的化合物在一定剂量下抑制 LPS 刺激的细胞因子释放的能力依赖时尚。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性和大脑活性。在未来的提案中，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，并在阿尔茨海默病动物模型中进一步评估其药物性质。公共卫生相关性：阿尔茨海默病 (AD) 直接影响 500 万美国人，并间接影响估计 1300 万护理人员。除了年龄之外，APOE（载脂蛋白-E）基因型是第二大风险因素：APOE4 携带者患 AD 的风险明显高于非 APOE4（主要是 APOE3）携带者。我们发现了一系列具有 apoE 模拟物功能的小肽。这些肽源自载脂蛋白-E (apoE) 全蛋白的残基 133-149，具有神经恢复功能，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，Chirstensen 等人提交，Saransteva 等人提交修订版）。在我们的初步研究中，这些 apoE 模拟肽减少了淀粉样斑块、神经原纤维缠结的存在，并提高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 apoE 模拟肽具有相同作用的小分子。这将需要进行多个级别的筛选，以找到少数可以口服并在实验动物大脑中显示出保护活性的化合物。