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

Among class A G protein-coupled receptors (GPCR), the human adenosine A receptor (hAAR) remains an attractive drug target. However, translation of AAR ligands into the clinic has proved challenging and an improved understanding of AAR 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 hAAR selective antagonist preladenant (SCH 420814). Both families of fluorescent antagonists retained affinity at the hAAR, 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 hAAR 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 hAAR pharmacology and signaling.