G-protein coupled receptor (GPCR) activation regulates cell signaling via several second messengers, including cAMP, inositol phospholipids and calcium. Upon activation of a Gq-type receptor, inositol-triphosphate (IP3) is formed that is used by the DiscoverX HitHunter assay to identify Gq-acting drugs.
The fluorescence polarization assay uses an IP3-binding protein saturated with fluorophore-coupled IP3. The protein-IP3-fluorophore complex exhibits low depolarization of fluorescence emission light due to its high molecular weight and small rotational movement. However, in the presence of native IP3, the fluorescent IP3 tracer competes with the native molecule and is released from the IP3-binding protein. The free tracer has a much lower molecular weight and, therefore, a higher rotational movement which finally results in depolarization of emission light.
A standard curve of IP3 was measured in 384 well format to validate the assay on the PHERAstar® microplate reader. With increasing analyte concentration, the fluorescence polarization value decreased and an EC50 of 13 nM was determined.