Optimizations of a novel fluorescence polarization-based high-throughput screening assay for β-catenin/LEF1 interaction inhibitors
Optimizations of a novel fluorescence polarization-based high-throughput screening assay for β-catenin/LEF1 interaction inhibitors
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β-连环蛋白/LEF1 相互作用抑制剂的新型基于荧光偏振的高通量筛选测定的优化
DOI：
10.1016/j.ab.2020.113966
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发表时间：
2021-01-01
影响因子：
2.9
通讯作者：
Liu, Xiaoping
中科院分区：
文献类型：
--
作者：
Chen, Yunyu;Fu, Zhenghao;Liu, Xiaoping
Wnt/β-catenin信号通路的异常激活在非小细胞肺癌(NSCLC)的发生、发展和转移中起重要作用。对这一途径的高效抑制为治疗非小细胞肺癌提供了新的途径。此外，β-catenin/LEF1相互作用调节β-catenin核转运以及Wnt/β-catenin信号通路中关键癌基因的转录。因此，阻断这种相互作用将是治疗非小细胞肺癌转移的一种有前景的治疗策略。到目前为止，还没有关于发现β-连环蛋白/LEF1相互作用抑制剂的经济和快速的高通量筛选(HTS)方法的报道。在这项研究中，我们建立了一种新的基于荧光偏振(FP)的HTS方法来鉴定β-连环蛋白/LEF1相互作用抑制剂。对FITC-LEF1的序列、孵育时间、温度和DMSO抗性进行了优化，获得了高达0.77的Z‘因子。通过建立的FP筛选试验对天然产物库进行中试筛选，确定血根碱类似物为潜在的β-连环蛋白/LEF1相互作用抑制剂。GST下拉实验和表面等离子体共振(SPR)分析表明，β-连环蛋白/LEF1相互作用是血根那林潜在的体外抗癌靶点。这种新开发的FP筛选方法对于快速发现针对β-连环蛋白/LEF1相互作用的新型Wnt抑制剂至关重要。
Aberrant activation of the Wnt/beta-catenin signaling pathway is prominent in the development and metastasis of non-small cell lung cancer (NSCLC). Highly effective inhibition of this pathway highlights a therapeutic avenue against NSCLC. Moreover, beta-catenin/LEF1 interaction regulates beta-catenin nuclear transport as well as the transcriptions of the key oncogenes in Wnt/beta-catenin signaling pathway. Therefore, interruption of this interaction would be a promising therapeutic strategy for NSCLC metastasis. To date, no economical and rapid high-throughput screening (HTS) assay has been reported for the discovery of beta-catenin/LEF1 interaction inhibitors. In this study, we developed a novel fluorescence polarization (FP)-based HTS assay to identify beta-catenin/LEF1 interaction inhibitors. The FITC-LEF1 sequence, incubation time, temperature, and DMSO resistance were optimized, and then a high Z' factor of 0.77 was achieved. A pilot screening of a natural product library via this established FP screening assay identified sanguinarine analogues as potential beta-catenin/LEF1 interaction inhibitors. GST pull-down and surface plasmon resonance (SPR) assay demonstrated that beta-catenin/LEF1 interaction is a potential anticancer target of sanguinarine in vitro. This newly developed FP screening assay will be vital for the rapid discovery of novel Wnt inhibitors targeting beta-catenin/LEF1 interaction.