Optimizations of a novel fluorescence polarization-based high-throughput screening assay for β-catenin/LEF1 interaction inhibitors

Aberrant activation of the Wnt/β-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, β-catenin/LEF1 interaction regulates β-catenin nuclear transport as well as the transcriptions of the key oncogenes in Wnt/β-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 β-catenin/LEF1 interaction inhibitors. In this study, we developed a novel fluorescence polarization (FP)-based HTS assay to identify β-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 β-catenin/LEF1 interaction inhibitors. GST pull-down and surface plasmon resonance (SPR) assay demonstrated that β-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 β-catenin/LEF1 interaction. [Display omitted] •Development of a fluorescence polarization (FP) screening assay for the discovery of β-catenin/LEF1 interaction inhibitors.•The FP screening assay identified sanguinarine analogues as promising β-catenin/LEF1 interaction inhibitors.•β-Catenin/LEF1 interaction is a potential anticancer target of sanguinarine in vitro.