Limits...
Luciferase reporter gene assay on human 5-HT receptor: which response element should be chosen?

Chen Y, Xu Z, Wu D, Li J, Song C, Lu W, Huang J - Sci Rep (2015)

Bottom Line: However, 5-HT receptors couple to multiple G-proteins regulate respective downstream signalling pathways and are usually detected using different response elements.The potencies and efficacies of the reported ligands (agonists and antagonists) were determined and compared.Our results indicate that CRE-luciferase reporter gene is sensitive and reliable to detect the activities of G protein-coupled 5-HT receptors.

View Article: PubMed Central - PubMed

Affiliation: Shanghai Key Laboratory of new drug design, School of Pharmacy, East China University of Science and Technology, Shanghai 200237, China.

ABSTRACT
Serotonin (5-HT) receptors are valuable molecular targets for antipsychotic drug discovery. Current reported methods for detecting 5-HT receptors, such as cAMP accumulation and calcium influx assay, are often demanding specialized instruments and inconvenient. The luciferase reporter gene assay, based on the responsible-element-regulated expression of luciferase, has been widely applied in the high-throughput functional assay for many targets because of its high sensitivity and reliability. However, 5-HT receptors couple to multiple G-proteins regulate respective downstream signalling pathways and are usually detected using different response elements. Hence, finding a suitable response element to fulfil the detection of different 5-HT receptors and make the results of luciferase reporter gene assays generalizable is very useful for active compounds screening. Here, we conducted three luciferase reporter assays using CRE, NFAT, and SRE response elements attached to 5-HT to detect the activation of different 5-HT receptors in CHO-K1 cells. The potencies and efficacies of the reported ligands (agonists and antagonists) were determined and compared. Our results indicate that CRE-luciferase reporter gene is sensitive and reliable to detect the activities of G protein-coupled 5-HT receptors.

No MeSH data available.


Related in: MedlinePlus

Screening of a small compound library.Graphical presentation for the screening results of 133 compounds at 50 μM in the CRE-based luciferase assay. Each dot represents one compound. (A, B) Compounds above the red line showing more than 2.0 fold increasing of relative luciferase activity were defined as active agonists. (C, D) Compounds above the red line showing more than 50% inhibition were defined as active antagonist.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4306921&req=5

f4: Screening of a small compound library.Graphical presentation for the screening results of 133 compounds at 50 μM in the CRE-based luciferase assay. Each dot represents one compound. (A, B) Compounds above the red line showing more than 2.0 fold increasing of relative luciferase activity were defined as active agonists. (C, D) Compounds above the red line showing more than 50% inhibition were defined as active antagonist.

Mentions: In order to verify the ability of the CRE-luc assay, we performed a screening using a small compound library consisting of 133 compounds at 50 μM. Active compounds were defined as those inhibiting at least 50% of the luciferase expression and relative luciferase activity increases to at least 2.0 times. Applying this high threshold, we identified 14 active compounds as agonists and 3 active compounds as antagonist for 5-HT7A receptor; 4 active compounds as agonists and 7 active compounds as antagonist for 5-HT2A receptor. These results demonstrated that the screening assay is available for library screening (Fig. 4).


Luciferase reporter gene assay on human 5-HT receptor: which response element should be chosen?

Chen Y, Xu Z, Wu D, Li J, Song C, Lu W, Huang J - Sci Rep (2015)

Screening of a small compound library.Graphical presentation for the screening results of 133 compounds at 50 μM in the CRE-based luciferase assay. Each dot represents one compound. (A, B) Compounds above the red line showing more than 2.0 fold increasing of relative luciferase activity were defined as active agonists. (C, D) Compounds above the red line showing more than 50% inhibition were defined as active antagonist.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4306921&req=5

f4: Screening of a small compound library.Graphical presentation for the screening results of 133 compounds at 50 μM in the CRE-based luciferase assay. Each dot represents one compound. (A, B) Compounds above the red line showing more than 2.0 fold increasing of relative luciferase activity were defined as active agonists. (C, D) Compounds above the red line showing more than 50% inhibition were defined as active antagonist.
Mentions: In order to verify the ability of the CRE-luc assay, we performed a screening using a small compound library consisting of 133 compounds at 50 μM. Active compounds were defined as those inhibiting at least 50% of the luciferase expression and relative luciferase activity increases to at least 2.0 times. Applying this high threshold, we identified 14 active compounds as agonists and 3 active compounds as antagonist for 5-HT7A receptor; 4 active compounds as agonists and 7 active compounds as antagonist for 5-HT2A receptor. These results demonstrated that the screening assay is available for library screening (Fig. 4).

Bottom Line: However, 5-HT receptors couple to multiple G-proteins regulate respective downstream signalling pathways and are usually detected using different response elements.The potencies and efficacies of the reported ligands (agonists and antagonists) were determined and compared.Our results indicate that CRE-luciferase reporter gene is sensitive and reliable to detect the activities of G protein-coupled 5-HT receptors.

View Article: PubMed Central - PubMed

Affiliation: Shanghai Key Laboratory of new drug design, School of Pharmacy, East China University of Science and Technology, Shanghai 200237, China.

ABSTRACT
Serotonin (5-HT) receptors are valuable molecular targets for antipsychotic drug discovery. Current reported methods for detecting 5-HT receptors, such as cAMP accumulation and calcium influx assay, are often demanding specialized instruments and inconvenient. The luciferase reporter gene assay, based on the responsible-element-regulated expression of luciferase, has been widely applied in the high-throughput functional assay for many targets because of its high sensitivity and reliability. However, 5-HT receptors couple to multiple G-proteins regulate respective downstream signalling pathways and are usually detected using different response elements. Hence, finding a suitable response element to fulfil the detection of different 5-HT receptors and make the results of luciferase reporter gene assays generalizable is very useful for active compounds screening. Here, we conducted three luciferase reporter assays using CRE, NFAT, and SRE response elements attached to 5-HT to detect the activation of different 5-HT receptors in CHO-K1 cells. The potencies and efficacies of the reported ligands (agonists and antagonists) were determined and compared. Our results indicate that CRE-luciferase reporter gene is sensitive and reliable to detect the activities of G protein-coupled 5-HT receptors.

No MeSH data available.


Related in: MedlinePlus