Development of a fluorescence polarization-based high-throughput screening assay to identify antagonists targeting β-catenin/TCF4 interaction
10.16438/j.0513-4870.2019-0998
- VernacularTitle: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
- Author:
Yun-yu CHEN
1
;
Ke HU
1
;
Zheng-hao FU
1
;
Xia-yi NIU
1
;
Jing ZHANG
2
;
Xiao-ping LIU
1
Author Information
1. Institute for Drug Screening and Evaluation, Wannan Medical College, Wuhu 241002, China
2. Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China
- Publication Type:Research Article
- Keywords:
Wnt inhibitor;
italic>β-catenin/TCF4 interaction;
fluorescence polarization;
high-throughput screening;
sanguinarine;
chelerythrine
- From:
Acta Pharmaceutica Sinica
2020;55(5):884-891
- CountryChina
- Language:Chinese
-
Abstract:
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 HisTrapTM column. 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-1 β-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.
