The affinities of several unlabeled ligands determined by HTRF on membrane preparations or on intact cells were similar to those determined by usual radioligand binding methods
A series of fluorescent ligands designed for vasopressin and oxytocin G protein-coupled receptors was synthesized and characterized to develop fluorescence polarization or homogeneous time-resolved fluorescence (HTRF) binding assays. These ligands, labeled with europium pyridine-bis-bipyridine cryptate or with Alexa 488,546,647 selectively bound to the vasopressin V1a and oxytocin receptors with high affinities and exhibited antagonistic properties. The affinities of several unlabeled ligands determined by our homogeneous assays on membrane preparations or on intact cells into 96- and 384-well plate formats were similar to those determined by usual radioligand binding methods. Compared to other binding assays, the polarization and HTRF binding assays are nonradiaoactive, therefore safer to perform, yet very sensitive and homogeneous, therefore easier and faster to automate. These methods are thus suitable for efficient drug high-throughput screening procedures and can easily be applied to other G protein-coupled receptor models.
J Med Chem. 2007;50(20):4976-85.