利用AlphaScreen技术开发一种用于发现ASK1信号体抑制剂的与高通量筛选兼容的检测方法。
Development of an HTS-compatible assay for the discovery of ASK1 signalosome inhibitors using alphascreen technology.
作者信息
Sturchler Emmanuel, Chen Weimin, Spicer Timothy, Hodder Peter, McDonald Patricia, Duckett Derek
机构信息
Department of Molecular Therapeutics, The Scripps Translational Science Institute , Jupiter, Florida.
出版信息
Assay Drug Dev Technol. 2014 May;12(4):229-37. doi: 10.1089/adt.2013.558.
Genetic target validation studies have demonstrated that the apoptosis signal-regulating kinase 1 (ASK1) represents an important target for the treatment of rheumatoid arthritis, cardiac diseases, and several neurodegenerative disorders. To identify small-molecule inhibitors of ASK1, we have developed a high-throughput screening-compatible, homogenous, biochemical assay using AlphaScreen technology. This novel assay design utilizes purified stress-activated ASK1 signalosome complex, and it monitors phosphorylation of its full-length native substrate, MKK6. The assay has been optimized in a 384-well format and validated by screening the Sigma LOPAC library. The results presented here demonstrate that the assay is sensitive and robust with a Z' factor value of 0.88±0.04 and a signal-to-background ratio of 11, indicating that this assay can be used to screen large chemical libraries to discover novel inhibitors of ASK1.
基因靶点验证研究表明，凋亡信号调节激酶1（ASK1）是治疗类风湿性关节炎、心脏病和几种神经退行性疾病的重要靶点。为了鉴定ASK1的小分子抑制剂，我们利用AlphaScreen技术开发了一种高通量筛选兼容的、均一的生化检测方法。这种新颖的检测设计使用纯化的应激激活ASK1信号体复合物，并监测其全长天然底物MKK6的磷酸化。该检测方法已在384孔板中进行了优化，并通过筛选Sigma LOPAC文库进行了验证。此处给出的结果表明，该检测方法灵敏且稳健，Z'因子值为0.88±0.04，信号背景比为11，这表明该检测方法可用于筛选大型化学文库以发现ASK1的新型抑制剂。
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