Small Molecule Structure Correctors Abolish Detrimental Effects of Apolipoprotein E4 in Cultured Neurons
Small Molecule Structure Correctors Abolish Detrimental Effects of Apolipoprotein E4 in Cultured Neurons
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DOI：
10.1074/jbc.m111.276162
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发表时间：
2012-02-17
影响因子：
4.8
通讯作者：
Mahley, Robert W.
中科院分区：
文献类型：
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作者：
Chen, Hung-Kai;Liu, Zhaoping;Mahley, Robert W.
载脂蛋白E4（apoE 4）是晚发性阿尔茨海默病的主要遗传危险因子，具有分子内相互作用的病理构象。ApoE 4结构域相互作用介导apoE 4的有害作用，包括降低线粒体细胞色素c氧化酶亚基1水平，降低线粒体运动性和减少体外神经突生长。突变型apoE 4（apoE 4-R61 T）缺乏结构域相互作用，行为类似于apoE 3，并且不引起有害作用。为了鉴定抑制结构域相互作用（即结构校正剂）和逆转apoE 4有害作用的小分子，我们建立了一种高通量的基于细胞的FRET初级测定法，其测定apoE 4结构域相互作用和基于细胞和功能的二级测定法。用FRET测定筛选ChemBridge文库鉴定了CB 9032258（二氮杂萘酮衍生物），其抑制神经元细胞中的结构域相互作用。在第二次功能试验中，CB 9032258恢复线粒体细胞色素c氧化酶亚基1水平，并挽救了apoE 4表达神经元细胞中线粒体运动和轴突生长的损伤。这些益处具有apoE 4特异性和剂量依赖性。修饰CB 9032258产生了明确的结构-活性关系和更多活性化合物，在FRET测定中具有增强的效力（IC 50分别为23和116 nM）。这些化合物在二级测定中有效地恢复了apoE 4表达细胞的功能活性。EPR结合实验表明，apoE 4的结构校正是由二氮杂萘酮的直接相互作用引起的。利用这些数据，构建了一个六特征药效团模型，为未来的药物设计。我们的研究结果作为一个概念的证明，apoE 4结构校正剂的药理学干预否定apoE 4在神经元细胞中的有害作用，并可以进一步开发为阿尔茨海默病治疗。
Apolipoprotein E4 (apoE4), the major genetic risk factor for late onset Alzheimer disease, assumes a pathological conformation, intramolecular domain interaction. ApoE4 domain interaction mediates the detrimental effects of apoE4, including decreased mitochondrial cytochrome c oxidase subunit 1 levels, reduced mitochondrial motility, and reduced neurite outgrowth in vitro. Mutant apoE4 (apoE4-R61T) lacks domain interaction, behaves like apoE3, and does not cause detrimental effects. To identify small molecules that inhibit domain interaction (i.e. structure correctors) and reverse the apoE4 detrimental effects, we established a high throughput cell-based FRET primary assay that determines apoE4 domain interaction and secondary cell- and function-based assays. Screening a ChemBridge library with the FRET assay identified CB9032258 (a phthalazinone derivative), which inhibits domain interaction in neuronal cells. In secondary functional assays, CB9032258 restored mitochondrial cytochrome c oxidase subunit 1 levels and rescued impairments of mitochondrial motility and neurite outgrowth in apoE4-expressing neuronal cells. These benefits were apoE4-specific and dose-dependent. Modifying CB9032258 yielded well defined structure-activity relationships and more active compounds with enhanced potencies in the FRET assay (IC50 of 23 and 116 nM, respectively). These compounds efficiently restored functional activities of apoE4-expressing cells in secondary assays. An EPR binding assay showed that the apoE4 structure correction resulted from direct interaction of a phthalazinone. With these data, a six-feature pharmacophore model was constructed for future drug design. Our results serve as a proof of concept that pharmacological intervention with apoE4 structure correctors negates apoE4 detrimental effects in neuronal cells and could be further developed as an Alzheimer disease therapeutic.