靶向β-catenin/TCF4相互作用小分子抑制剂荧光偏振高通量筛选模型的建立与应用  

Development of a fluorescence polarization-based high-throughput screening assay to identify antagonists targeting β-catenin/TCF4 interaction

作　　者：陈云雨 胡克[1] 付正豪 牛夏忆 张晶[2] 刘晓平[1] CHEN Yun-yu;HU Ke;FU Zheng-hao;NIU Xia-yi;ZHANG Jing;LIU Xiao-ping(Institute for Drug Screening and Evaluation,Wannan Medical College,Wuhu 241002,China;Institute of Medicinal Biotechnology,Chinese Academy of Medical Sciences and Peking Union Medical College,Beijing 100050,China)

机构地区：[1]皖南医学院药物筛选与评价研究所,安徽芜湖241002 [2]中国医学科学院北京协和医学院医药生物技术研究所,北京100050

出　　处：《药学学报》2020年第5期884-891,共8页Acta Pharmaceutica Sinica

基　　金：国家自然科学基金资助项目(81703546);安徽省自然科学基金资助项目(1808085QH265);安徽省高校自然科学研究重大项目(KJ2019ZD30);吉林省科技发展计划项目(20160520045JH);中国医学科学院医学与健康科技创新工程(2017-I2M-1-012);安徽省大学生创新创业训练计划项目(S201910368083)。

摘　　要：基于荧光偏振(fluorescence polarization, FP)原理,建立并应用以β-catenin/TCF4 (T-cell factor 4)相互作用为靶标的小分子抑制剂荧光偏振高通量筛选模型获得苗头化合物。利用大肠杆菌原核表达系统,原核表达和分离纯化重组人β-catenin,以酶联免疫吸附实验(enzyme-linked immunosorbent assay, ELISA)进行生物学活性鉴定。以异硫氰酸荧光素(fluorescence isothiocyanate, FITC)标记的TCF4多肽为荧光探针,通过优化FITC-TCF4与β-catenin反应浓度,建立并应用靶向β-catenin/TCF4相互作用小分子抑制剂荧光偏振高通量筛选模型进行苗头化合物筛选。利用大肠杆菌原核表达系统成功进行了重组人β-catenin原核表达与分离纯化。ELISA实验证实了纯化的重组人β-catenin具有良好的生物学活性。选用20 nmol·L^-1FITC-TCF4和100 nmol·L^-1β-catenin,成功建立了Z’因子为0.88的荧光偏振高通量筛选模型。应用本筛选模型进行高通量筛选,成功筛选到了血根碱(sanguinarine)、白屈菜红碱(chelerythrine)和化合物S720具有良好的抑制活性。本研究成功建立了适用于靶向β-catenin/TCF4相互作用小分子抑制剂筛选的荧光偏振高通量筛选模型,为新型Wnt抑制剂的高效化和理性化发现奠定了基础。To develop a fluorescence polarization(FP)-based high-throughput screening(HTS) assay to identify novel small-molecule antagonists targeting β-catenin/TCF4(T-cell factor 4) interaction, recombinant human β-catenin was expressed in Escherichia coli Rosetta(DE3) cells and purified by HisTrapTMcolumn. The bioactivity of purified β-catenin was further analyzed by enzyme-linked immunosorbent assay(ELISA). According to FP principle, the β-catenin/TCF4 binding model was performed, and fluorescence isothiocyanate(FITC) labeled TCF4 peptide(FITC-TCF4) served as the molecular probe of adaptor for binding to β-catenin. The FITC-TCF4 and β-catenin working concentration were optimized, and the binding conditions(complex stability and dimethylsulfoxide(DMSO) tolerance) have been investigated yet for further hits screening. The results showed that recombinant human β-catenin was successfully expressed and purified β-catenin exhibited favorable bioactivity in ELISA binding assay. Subsequently, the FP-based HTS assay was performed using 20 nmol·L^-1 FITC-TCF4 and 100 nmol·L^-1β-catenin. Under these optimized conditions, a high Z′factor of 0.88 was achieved in a 384-well format and this FP-based HTS assay was very stable with regard to DMSO. Through screening of a natural-based product library(NBPL) using the established FP-based HTS assay, three hits(sanguinarine, chelerythrine, and compound S720)were identified as potential β-catenin/TCF4 interaction antagonists. Taken together, we have successfully developed a simple, robust and reliable FP-based HTS assay for screening of novel antagonists targeting β-catenin/TCF4 interaction.

关 键 词：Wnt抑制剂 β-catenin/TCF4相互作用 荧光偏振 高通量筛选 血根碱 白屈菜红碱 

分 类 号：R965.1[医药卫生—药理学]