ApoE4 Structure Correctors as a Therapeutic Approach for Alzheimers Disease

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。尽管努力开发AD疗法，但FDA批准的那些疗法并不能有效地减缓神经变性或疾病进展。我们研究的重点是发现靶向载脂蛋白（apo）E4毒性作用的新型药物，以阻断AD的神经退行性变。ApoE 4是AD的主要遗传危险因子，并且apoE 4携带者占所有AD病例的65-80%。ApoE 4增加了AD的发病率，降低了AD的发病年龄，大量证据表明它在AD神经退行性变中起着基础性作用。我们建议确定小分子探针，可以“纠正”apoE 4的病理构象（结构校正剂），消除其神经毒性作用，并有可能作为新的药物导致治疗或预防AD进展。apoE的两种主要亚型apoE 3和apoE 4在脑中表达。它们的区别在于一个氨基酸，并且单个取代（半胱氨酸至精氨酸）将apoE 3从支持神经元维持的分子转化，促进神经突生长和神经修复，并保护神经元至神经病理形式（apoE 4）。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。ApoE 4在其氨基末端和羧基末端结构域之间显示分子内结构域相互作用，导致紧凑的结构。破坏apoE 4结构域与小分子结构校正剂的相互作用，将apoE 4转化为apoE 3样构象，并逆转apoE 4特异性对神经元的有害作用。在初步筛选中，我们发现小分子结构校正剂可以破坏apoE 4结构域的相互作用，防止有毒apoE 4片段的形成，并保护线粒体和神经元免于退化。本提案的目的是鉴定新型化学系列的apoE 4结构校正剂，其可以被开发成具有改善的效力和药学性质的化学探针，这将潜在地导致新一代治疗AD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，以筛选NIH分子库小分子库（MLSMR），以确定apoE 4的小分子结构校正剂。初步测定将测量绿色荧光蛋白（GFP）-apoE 4水平。将使用基于GFP-apoE 4-eDHFR荧光共振能量转移的二级测定来选择性地确认命中。此外，基于三级细胞的测定将测量apoE 4对复合物IV的细胞色素c氧化酶亚基1的毒性作用，并测量神经突生长。这些最终试验将进一步验证候选化合物在保护线粒体功能和预防神经元变性方面的作用特异性和生物相关性。从这些研究中鉴定的探针可用于进一步研究apoE 4在AD中的作用，并将为开发治疗AD的新药奠定基础。

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.