2. Academic Validation
  3. A piggyBac-based TANGO GFP assay for high throughput screening of GPCR ligands in live cells

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

  • Cell Commun Signal. 2019 May 23;17(1):49. doi: 10.1186/s12964-019-0359-x.
Fei Li 1 2,  Xi Jiang 2,  Ling-Ling Luo 1,  Yue-Ming Xu 3,  Xing-Xu Huang 1,  Cheng Huang 4,  Yu Zhang 5
Affiliations

Affiliations

  • 1 School of Life Science and Technology, ShanghaiTech University, Shanghai, China.
  • 2 School of Pharmacy, Shanghai University of Traditional Chinese Medicine, Shanghai, China.
  • 3 iHuman Institute, ShanghaiTech University, Shanghai, China.
  • 4 School of Pharmacy, Shanghai University of Traditional Chinese Medicine, Shanghai, China. [email protected].
  • 5 School of Life Science and Technology, ShanghaiTech University, Shanghai, China. [email protected].
PMID: 31122241 DOI: 10.1186/s12964-019-0359-x
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.

Keywords

GPCRs; High signal-to-background ratio; Ligand piggyBac-TANGO; Live cells; Stable readout.

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