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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Project description:Quantitative high throughput screening (qHTS) assays use cells or tissues to screen thousands of compounds in a short period of time. Data generated from qHTS assays are then evaluated using nonlinear regression models, such as the Hill model, and decisions regarding toxicity are made using the estimates of the parameters of the model. For any given compound, the variability in the observed response may either be constant across dose groups (homoscedasticity) or vary with dose (heteroscedasticity). Since thousands of compounds are simultaneously evaluated in a qHTS assay, it is not practically feasible for an investigator to perform residual analysis to determine the variance structure before performing statistical inferences on each compound. Since it is well-known that the variance structure plays an important role in the analysis of linear and nonlinear regression models it is therefore important to have practically useful and easy to interpret methodology which is robust to the variance structure. Furthermore, given the number of chemicals that are investigated in the qHTS assay, outliers and influential observations are not uncommon. In this article we describe preliminary test estimation (PTE) based methodology which is robust to the variance structure as well as any potential outliers and influential observations. Performance of the proposed methodology is evaluated in terms of false discovery rate (FDR) and power using a simulation study mimicking a real qHTS data. Of the two methods currently in use, our simulations studies suggest that one is extremely conservative with very small power in comparison to the proposed PTE based method whereas the other method is very liberal. In contrast, the proposed PTE based methodology achieves a better control of FDR while maintaining good power. The proposed methodology is illustrated using a data set obtained from the National Toxicology Program (NTP). Additional information, simulation results, data and computer code are available online as supplementary materials.

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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