Subtype-Selective Fluorescent Ligands as Pharmacological Research Tools for the Human Adenosine A2A 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 realtime 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.