ApoE4 Structure Correctors as a Therapeutic Approach for Alzheimers Disease

ApoE4 结构校正剂作为阿尔茨海默病的治疗方法

• 批准号: 8328029
• 负责人: ROBERT W. MAHLEY
• 金额: $ 4.69万
• 依托单位: J. DAVID GLADSTONE INSTITUTES
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-05-01 至 2014-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8328029
• 关键词: Accounting; Affect; Age of Onset; AlamarBlue; Alzheimer' s Disease; Alzheimer' s disease model; American; Amino Acids; Amyloid; Apolipoprotein E; Arginine; Biological; Biological Assay; Biological Testing; Brain; Cells; Chemicals; Clinical; Cysteine; Development; Disease; Disease Progression; Drug Delivery Systems; Enzymes; Evaluation; FDA approved; Fluorescence Resonance Energy Transfer; Foundations; Generations; Goals; Green Fluorescent Proteins; Lead; Life; Maintenance; Measures; Mitochondria; Molecular Bank; Molecular Conformation; Monitor; Nerve Degeneration; Neurites; Neurodegenerative Disorders; Neurons; Neuropathogenesis; PTGS1 gene; Pathogenesis; Pharmaceutical Chemistry; Pharmaceutical Preparations; Pharmacologic Substance; Property; Protein Isoforms; Proteins; Publishing; Respiratory Chain; Role; Screening procedure; Series; Specificity; Structure; Tertiary Protein Structure; Testing; Therapeutic; Toxic Actions; Toxic effect; United States National Institutes of Health; apolipoprotein E-3; apolipoprotein E-4; base; complex IV; cytochrome c oxidase; cytotoxic; effective therapy; efficacy testing; genetic risk factor; high throughput screening; improved; neurotoxic; novel; prevent; protein structure function; relating to nervous system; repaired; repository; response to injury; scaffold; small molecule; therapeutic development

DESCRIPTION (provided by applicant): Alzheimer's disease (AD) is the most common neurodegenerative disorder, affecting over 5 million Americans. Despite efforts to develop AD therapies, those that are FDA approved do not effectively slow neurodegeneration nor disease progression. The focus of our studies is to discover novel drugs targeting the toxic actions of apolipoprotein (apo) E4 to block neurodegeneration in AD. ApoE4 is the major genetic risk factor for AD, and apoE4 carriers account for 65-80% of all cases of AD. ApoE4 increases the occurrence and lowers the age of onset of AD, and considerable evidence suggests that it has a fundamental role in AD neurodegeneration. We propose to identify small-molecule probes that can "correct" the pathological conformation of apoE4 (structure correctors), abolish its neurotoxic effects, and potentially serve as new drug leads to treat or prevent AD progression. Two major isoforms of apoE, apoE3 and apoE4, are expressed in the brain. They differ by one amino acid, and that single substitution (cysteine to arginine) converts apoE3 from a molecule that supports neuronal maintenance, promotes neurite outgrowth and neural repair, and protects neurons to a neuropathological form (apoE4). This amino acid difference gives rise to profound differences in the tertiary protein structure and function. ApoE4 displays an intramolecular domain interaction between its amino- and carboxyl-terminal domains, leading to a compact structure. Disrupting apoE4 domain interaction with small-molecule structure correctors converts apoE4 into an apoE3-like conformation and reverses the apoE4-specific detrimental effects on neurons. In pilot screens, we found small-molecule structure correctors that disrupt apoE4 domain interaction, prevent the formation of toxic apoE4 fragments, and protect mitochondria and neurons from degenerating. The objective of the current proposal is to identify novel chemical series of apoE4 structure correctors that can be developed into chemical probe(s) with improved potency and pharmaceutical properties that will potentially lead to a new generation of drugs to treat AD. To achieve this goal, we developed cell-based high-throughput screening assays to screen the NIH Molecular Libraries Small Molecule Repository (MLSMR) to identify small-molecule structure correctors of apoE4. A primary assay will measure green fluorescent protein (GFP)-apoE4 levels. A secondary assay based on GFP- apoE4-eDHFR fluorescence resonance energy transfer will be used to confirm hits selectively. In addition, tertiary cell-based assays will measure the toxic effects of apoE4 on cytochrome c oxidase subunit 1 of complex IV and measure neurite outgrowth. These final assays will further validate the specificity of action and biological relevance of the candidate compounds in protecting mitochondrial function and preventing neuronal degeneration. Probes identified from these studies can be used to further study the role of apoE4 in AD and will serve as a foundation for the development of novel drugs to treat AD.

描述（由申请人提供）：阿尔茨海默病（AD）是最常见的神经退行性疾病，影响超过500万美国人。尽管人们努力开发阿尔茨海默病的治疗方法，但那些获得FDA批准的药物并不能有效减缓神经退行性变或疾病进展。我们的研究重点是发现新的药物靶向载脂蛋白（apo） E4的毒性作用，以阻止阿尔茨海默病的神经退行性变。ApoE4是AD的主要遗传危险因素，ApoE4携带者占所有AD病例的65-80%。ApoE4增加阿尔茨海默病的发生并降低发病年龄，大量证据表明它在阿尔茨海默病神经退行性变中起着重要作用。我们建议鉴定能够“纠正”apoE4病理构象（结构校正器）的小分子探针，消除其神经毒性作用，并可能作为治疗或预防AD进展的新药物导向。apoE的两种主要亚型，apoE3和apoE4，在大脑中表达。它们只差一个氨基酸，而单次取代（半胱氨酸到精氨酸）将apoE3从支持神经元维持的分子转化为促进神经突生长和神经修复的分子，并将神经元保护为神经病理形态（apoE4）。这种氨基酸的差异导致了三级蛋白质结构和功能的深刻差异。ApoE4在其氨基末端和羧基末端之间表现出分子内结构域相互作用，导致其结构紧凑。破坏apoE4结构域与小分子结构校正器的相互作用，将apoE4转化为apoe3样构象，并逆转apoE4对神经元的特异性有害影响。在初步筛选中，我们发现了小分子结构纠正剂，它可以破坏apoE4结构域的相互作用，防止有毒apoE4片段的形成，并保护线粒体和神经元免受退化。当前提案的目的是确定新的apoE4结构校正的化学系列，这些化学系列可以开发成具有更高效力和药物特性的化学探针，这可能会导致新一代治疗阿尔茨海默病的药物。为了实现这一目标，我们开发了基于细胞的高通量筛选试验来筛选NIH分子文库小分子库（MLSMR），以鉴定apoE4的小分子结构校正器。初级试验将测量绿色荧光蛋白(GFP)-apoE4水平。基于GFP- apoE4-eDHFR荧光共振能量转移的二次分析将用于选择性地确认命中。此外，基于三级细胞的分析将测量apoE4对复合体IV的细胞色素c氧化酶亚基1的毒性作用，并测量神经突起的生长。这些最终的分析将进一步验证候选化合物在保护线粒体功能和防止神经元变性方面的作用特异性和生物学相关性。从这些研究中确定的探针可用于进一步研究apoE4在AD中的作用，并将为开发治疗AD的新型药物奠定基础。