International Journal of High Throughput Screening
Open access peer-reviewed scientific and medical journals.
Dove Medical Press is now a member of the Open Access Initiative
An Author's Guide
A guide to help authors get their paper published.
Support Open Access and Dove Press
Promotional Article Monitoring - further details
Favored Author Program
Real benefits for authors, including fast-track processing of papers.
Development of a functional HTS assay for the APJ receptor
(3736) Total Article Views
Authors: Angela Giddings, Scott Runyon, James Thomas, et al
Published Date April 2010
Volume 2010:1 Pages 39 - 47
Angela Giddings1, Scott Runyon2, James Thomas2, Julianne Tajuba1, Katherine Bortoff1, Rangan Maitra1
1Center for Pharmacology and Toxicology, 2Center for Organic and Medicinal Chemistry, Division of Discovery Sciences, RTI International, Research Triangle Park, NC, USA
Abstract: The apelin receptor (APJ) is rapidly emerging as an attractive therapeutic target for various diseases. To date, only one nonpeptide APJ agonist has been reported and new chemotypes having improved pharmacological properties are needed to expedite maturation of this receptor as a viable target. Therefore, the development of a high-throughput screening (HTS) assay for APJ should facilitate the discovery of novel ligands as in vivo probes of APJ function. In this report, an HTS compatible assay for the APJ is demonstrated. This assay takes advantage of functional coupling of APJ with the promiscuous Gαq16 protein and consequent mobilization of intracellular calcium, which can be measured in a microplate format using a fluorescent calcium probe. The assay was developed and validated using several known peptides derived from preproapelin. Most of the apelin peptides tested activated APJ at subnanomolar concentrations in accordance with their reported median effective concentration values. The assay was deemed robust and reproducible with a Z-factor of ∼0.6 and tolerance to ethanol, dimethyl sulfoxide or a mixture of these two commonly used solvents to a final concentration of 1%. The assay was further validated by screening a small library of compounds for APJ activity. A hit rate of ∼0.5% was achieved and confirmed by counter-screening against parental Gαq16 cells lacking APJ. In conclusion, an HTS assay has been established to rapidly identify new ligands for the APJ. Novel APJ ligands having suitable drug-like properties are greatly needed to probe the in vivo pharmacology of this emerging therapeutic target.
Keywords: APJ, apelin, Gαq16, GPCR, HTS, assay
Cannotea Citeulike Del.icio.us Facebook LinkedIn Twitter
Readers of this article also read:
"You do a tremendous job!!" Ruben Restrepo, University of Texas Health Science Center, San Antonio.
- Does wine prevent dementia?
- Wine consumers’ environmental knowledge and attitudes: Influence on willingness to purchase
- China’s role in global competition in the wine industry: A new contestant and future trends
- Mycoflora and production of wine from fruits of soursop (Annona Muricata L.)