Wang Hongjie, Nefzi Adel, Fields Gregg B, Lakshmana Madepalli K, Minond Dmitriy
Torrey Pines Institute for Molecular Studies, 11350 SW Village Parkway, Port Saint Lucie, 34987 FL, USA.
Torrey Pines Institute for Molecular Studies, 11350 SW Village Parkway, Port Saint Lucie, 34987 FL, USA.
Anal Biochem. 2014 Aug 15;459:24-30. doi: 10.1016/j.ab.2014.05.007. Epub 2014 May 20.
Activation of nonamyloidogenic processing of amyloid precursor protein (APP) has been hypothesized to be a viable approach for Alzheimer's disease drug discovery. However, until recently, the lack of HTS-compatible assay technologies precluded large scale screening efforts to discover molecules that potentiate nonamyloidogenic pathways. We have developed an HTS-compatible assay based on AlphaLISA technology that quantitatively detects soluble APPα (sAPPα), a marker of nonamyloidogenic processing of APP, released from live cells in low volume, 384-well plates. The assay exhibited good QC parameters (Z'>0.5, S/B>2). A pilot screen of 801 compounds yielded a novel chemotype that increased the release of sAPPα 2-fold at 5μM. These results suggest that the AlphaLISA-based HTS assay is robust and sensitive and can be used to screen large compound collections to discover molecules that potentiate the release of sAPPα. Additionally, we demonstrated that increase of APP processing by nonamyloidogenic pathways will result in decrease of release of amyloidogenic Aβ40 fragments.
淀粉样前体蛋白（APP）的非淀粉样生成加工过程的激活被认为是阿尔茨海默病药物研发的一种可行方法。然而，直到最近，缺乏与高通量筛选（HTS）兼容的检测技术阻碍了大规模筛选以发现增强非淀粉样生成途径的分子。我们基于AlphaLISA技术开发了一种与HTS兼容的检测方法，该方法可在低体积的384孔板中定量检测从活细胞释放的可溶性APPα（sAPPα），这是APP非淀粉样生成加工过程的一个标志物。该检测方法表现出良好的质量控制参数（Z'>0.5，S/B>2）。对801种化合物的初步筛选产生了一种新型化学类型，在5μM时可使sAPPα的释放增加2倍。这些结果表明，基于AlphaLISA的HTS检测方法强大且灵敏，可用于筛选大量化合物库以发现增强sAPPα释放的分子。此外，我们证明通过非淀粉样生成途径增加APP加工将导致淀粉样生成的Aβ40片段释放减少。
