靶向β-catenin/TCF4相互作用小分子抑制剂酶联免疫吸附法高通量筛选模型的优化与应用

Optimizations of an ELISA-like high-throughput screening assay for the discovery of β-catenin/TCF4 interaction antagonists

摘要:

在经典的Wnt/β-catenin信号通路中，β-catenin/TCF4 (T-cell factor 4) 相互作用在非小细胞肺癌 (Non-small cell lung cancer，NSCLC) 的生长分化、化疗耐药、转移复发等过程中发挥着重要的促进作用，已成为新型靶向性抗NSCLC转移药物开发的理想靶标之一。为了高效地发现抑制β-catenin/TCF4相互作用的苗头化合物，本研究在应用酶联免疫吸附实验 (Enzyme-linked immunosorbent assay，ELISA) 原理的基础上，通过优化GST-TCF4 βBD包被浓度和β-catenin反应浓度，建立ELISA高通量筛选模型并成功应用于苗头化合物的筛选。ELISA筛选模型优化实验结果表明，选用2 μg/mL GST-TCF4 βBD和0.5 μg/mL β-catenin建立ELISA高通量筛选模型，其Z′因子值为0.83，并成功筛选到具有良好抑制活性的白花丹素 (Plumbagin)。肿瘤细胞增殖实验结果表明，白花丹素对A549、H1299、MCF7和SW480细胞具有明显的细胞毒性。TOPFlash实验结果证实，白花丹素对转染的HEK293细胞内β-catenin介导的转录活性具有显著的抑制作用，β-catenin/TCF4相互作用可能是白花丹素抗癌活性的潜在分子靶标之一。文中以β-catenin/TCF4相互作用为靶标，通过系统的实验优化方案，成功地建立了适用于药物高通量筛选的ELISA筛选模型，为高效筛选靶向β-catenin/TCF4相互作用的小分子抑制剂奠定了实验基础。

Abstract:

In canonical Wnt/β-catenin signaling pathway, β-catenin/TCF4 (T-cell factor 4) interaction plays an important role in the pathogenesis and development of non-small cell lung cancer (NSCLC), and it is tightly associated with the proliferation, chemoresistance, recurrence and metastasis of NSCLC. Therefore, suppressing β-catenin/TCF4 interaction in Wnt/β-catenin signaling pathway would be a new therapeutic avenue against NSCLC metastasis. In this study, considering the principle of enzyme-linked immunosorbent assay (ELISA), an optimized high-throughput screening (HTS) assay was developed for the discovery of β-catenin/TCF4 interaction antagonists. Subsequently, this ELISA-like screening assay was performed using 2 μg/mL GST-TCF4 βBD and 0.5 μg/mL β-catenin, then a high Z′ factor of 0.83 was achieved. A pilot screening of a natural product library using this ELISA-like screening assay identified plumbagin as a potential β-catenin/TCF4 interaction antagonist. Plumbagin remarkably inhibited the proliferation of A549, H1299, MCF7 and SW480 cell lines. More importantly, plumbagin significantly suppressed the β-catenin-responsive transcription in TOPFlash assay. In short, this newly developed ELISA-like screening assay will be vital for the rapid screening of novel Wnt inhibitors targeting β-catenin/TCF4 interaction, and this interaction is a potential anticancer target of plumbagin in vitro.