Combining the rapid MTT formazan exocytosis assay and the MC65 protection assay led to the discovery of carbazole analogs as small molecule inhibitors of Aβ oligomer-induced cytotoxicity
Combining the rapid MTT formazan exocytosis assay and the MC65 protection assay led to the discovery of carbazole analogs as small molecule inhibitors of Aβ oligomer-induced cytotoxicity
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DOI：
10.1016/j.brainres.2006.10.093
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发表时间：
2007-01-26
期刊：
影响因子：
2.9
通讯作者：
Jin, Lee-Way
中科院分区：
文献类型：
--
作者：
Hong, Hyun-Seok;Maezawa, Izumi;Jin, Lee-Way
淀粉样蛋白- β (A β)寡聚物诱导的细胞毒性小分子抑制剂的发现，无论是应用于细胞外还是积累在神经内，都是阿尔茨海默病（AD）药物开发的重要目标，但由于缺乏有效的筛选方法而受到限制。在这里，我们使用两种基于细胞的方法来描述我们的方法。第一种方法利用细胞外应用的A β低聚物的独特能力，快速诱导由3-（4,5-二甲基噻唑-2 -基）-2,5-二苯基溴化四氮唑（MTT）形成的甲醛的胞吐。我们采用了一个简短的方案来量化这种毒性，并迅速从两个化合物文库中鉴定出两种新的抑制剂，代号为CP2和AS。使用我们之前发表的MC65保护试验对相同的文库进行了第二次独立筛选，该试验确定了与细胞内A β低聚物相关的毒性抑制剂，也选择了相同的两个先导，这表明两种测定方法都选择了这些化合物所显示的相同的抗A β低聚物特性。我们进一步证明，AS可减弱现有A - β寡聚物的渐进性聚集，降低细胞内A -寡聚物的水平，并阻止A - β寡聚物诱导的初级皮质神经元死亡，其作用与CP2所证明的相似。我们的研究结果表明，当结合使用时，这两种方法将产生更少的错误结果，并且很有可能识别出在改善神经元内和细胞外部位a β寡聚物诱导的毒性方面有希望的线索。这两种检测方法简单，适用于大量药物文库的快速筛选，并且易于自动化。(c) 2006 Elsevier B.V.版权所有
The discovery of small molecule inhibitors of cytotoxicity induced by amyloid-beta (A beta) oligomers, either applied extracellularly or accumulated intraneuronally, is an important goal of drug development for Alzheimer's disease (AD), but has been limited by the lack of efficient screening methods. Here we describe our approach using two cell-based methods. The first method takes advantage of the unique ability of extracellularly applied A beta oligomers to rapidly induce the exocytosis of formazan formed by 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT). We employed a short protocol to quantify this toxicity, and quickly identified two novel inhibitors, code-named CP2 and AS, from two compound libraries. A second independent screen of the same libraries using our previously published MC65 protection assay, which identifies inhibitors of toxicity related to intracellular A beta oligomers, also selected the same two leads, suggesting that both assays select for the same anti-A beta oligomer properties displayed by these compounds. we further demonstrated that AS attenuated the progressive aggregation of existing A beta oligomers, reduced the level of intracellular A oligomers, and prevented the A beta oligomer-induced death of primary cortical neurons, effects similar to those demonstrated by CP2. Our results suggest that, when combined, the two methods would generate fewer false results and give a high likelihood of identifying leads that show promises in ameliorating A beta oligomer-induced toxicities within both intraneuronal and extracellular sites. Both assays are simple, suitable for rapid screening of a large number of medicinal libraries, and amenable for automation. (c) 2006 Elsevier B.V. All rights reserved.