Dataset Information

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

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.

SUBMITTER: Li F

PROVIDER: S-EPMC6533772 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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Publications

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

Li Fei F Jiang Xi X Luo Ling-Ling LL Xu Yue-Ming YM Huang Xing-Xu XX Huang Cheng C Zhang Yu Y

Cell communication and signaling : CCS 20190523 1

<h4>Background</h4>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.<h4>Methods</h4>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 ...[more]

PMID: 31122241

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Dataset Information

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

Background

Methods

Results

Conclusion

Publications

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

Similar Datasets

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