A piggyBac-based TANGO GFP assay for high throughput screening of GPCR ligands in live cells

Cell Communication and Signaling

Fei Li¹  Ling-Ling Luo¹  Xing-Xu Huang¹  Yu Zhang¹  Cheng Huang²  Xi Jiang²  Yue-Ming Xu³ 

[1] School of Life Science and Technology, ShanghaiTech University;School of Pharmacy, Shanghai University of Traditional Chinese Medicine;iHuman Institute, ShanghaiTech University;

关键词: GPCRs;    Ligand piggyBac-TANGO;    High signal-to-background ratio;    Stable readout;    Live cells;   

DOI  :  10.1186/s12964-019-0359-x

来源: DOAJ

【 摘 要 】

Abstract Background GPCRs are considered essential for various physiological processes and have been the most productive drug targets. Therefore, development of the methods of GPCR ligands screening is a high priority for pharmaceutical industries and research institutions. Methods We developed a potential method (piggyBac-TANGO) based on the TANGO and PRESTO-TANGO assays. The system was optimized with a piggyBac transposon as a transgene vehicle, and eGFP was used as a reporter instead of luciferase. The assay was validated in the HEK 293T and U87-MG cell lines and antagonist activities of the compounds were assessed. The transgene copy number and long-term stability were evaluated by qPCR. Then, we performed a DRD2-targeted screening for natural products using the piggyBac-TANGO assay. Results The validation assay showed that using the piggyBac transposon as a transgene vehicle produced high signal-to-background ratio and stable readout confirmed by investigation of the transgene copy number and long-term stability. Use of eGFP instead of luciferase as a reporter enabled to create a high throughput system suitable for live cells. Moreover, the piggyBac-TANGO assay permitted versatile detection of antagonist activity of compounds and was not limited to a particular cell type. With the use of the piggyBac-TANGO assay, we have successfully identified a novel agonist of DRD2. Conclusion Thus, the results indicate that the piggyBac-TANGO method is a user-friendly, robust and imaging-based assay that provides a novel approach to high throughput GPCR-targeted ligand screening and drug development.

【 授权许可】

Unknown