Development of a functional HTS assay for the APJ receptor

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.