Subtype-Selective Fluorescent Ligands as Pharmacological Research Tools for the Human Adenosine A 2A Receptor.

Among class A G protein-coupled receptors (GPCR), the human adenosine A _2A receptor (hA _2A AR) remains an attractive drug target. However, translation of A _2A AR ligands into the clinic has proved challenging and an improved understanding of A _2A AR pharmacology could promote development of more efficacious therapies. Subtype-selective fluorescent probes would allow detailed real-time pharmacological investigations both in vitro and in vivo. In the present study, two families of fluorescent probes were designed around the known hA _2A AR selective antagonist preladenant (SCH 420814). Both families of fluorescent antagonists retained affinity at the hA _2A AR, selectivity over all other adenosine receptor subtypes and allowed clear visualization of specific receptor localization through confocal imaging. Furthermore, the Alexa Fluor 647-labeled conjugate allowed measurement of ligand binding affinities of unlabeled hA _2A AR antagonists using a bioluminescence resonance energy transfer (NanoBRET) assay. The fluorescent ligands developed here can therefore be applied to a range of fluorescence-based techniques to further interrogate hA _2A AR pharmacology and signaling.