An AlphaScreen-based high-throughput screen to identify inhibitors of Hsp90-cochaperone interaction.
An AlphaScreen-based high-throughput screen to identify inhibitors of Hsp90-cochaperone interaction.
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DOI：
10.1177/1087057108330114
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发表时间：
2009-03
影响因子：
--
通讯作者：
Regan L
中科院分区：
文献类型：
--
作者：
Yi F;Zhu P;Southall N;Inglese J;Austin CP;Zheng W;Regan L
Hsp90在促进许多致癌蛋白的折叠和成熟中发挥重要作用，已成为重要的抗癌药物靶点。在这里，我们描述了基于AlphaScreen™技术的第一个高通量筛选的发展，以识别一种新型的Hsp90抑制剂，这种抑制剂可以中断其与伴侣蛋白HOP的相互作用。该检测使用Hsp90的20-mer c -末端肽和HOP的TPR2A结构域。通过使用合成肽测量不同的相互作用，证明了检测的特异性，测量的ic50与报道的值很好地一致。该试验在12小时内保持稳定，可耐受高达5%的DMSO。我们首先通过筛选384孔格式的20,000种化合物来验证该分析。在进一步优化为1536孔格式后，与NCGC库中76,134个化合物进行筛选，信噪比（S/B）为78，Z因子为0.77。本实验可用于发现新的小分子Hsp90抑制剂，并可作为化学探针来研究cochaperones在Hsp90功能中的作用。这些分子有可能被开发成新的抗癌药物，可以单独使用，也可以与其他热休克蛋白90抑制剂联合使用。
Hsp90 has emerged as an important anti-cancer drug target because of its essential role in promoting the folding and maturation of many oncogenic proteins. Here we describe the development of the first high throughput screen, based on AlphaScreen™ technology, to identify a novel type of Hsp90 inhibitors that interrupt its interaction with the cochaperone HOP. The assay uses the 20-mer C-terminal peptide of Hsp90 and the TPR2A domain of HOP. Assay specificity was demonstrated by measuring different interactions using synthetic peptides, with measured IC50s in good agreement with reported values. The assay is stable over 12 hours and tolerates DMSO up to 5%. We first validated the assay by screening against 20,000 compounds in 384-well format. After further optimization into a 1536-well format, it was screened against a NCGC library of 76,134 compounds, with a signal-to-background (S/B) ratio of 78 and Z’ factor of 0.77. The present assay can be used for discovery of novel small molecule Hsp90 inhibitors that can be used as chemical probes to investigate the role of cochaperones in Hsp90 function. Such molecules have the potential to be developed into novel anti-cancer drugs, for use alone or in combination with other Hsp90 inhibitors.
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