Subtype-selective Fluorescent Ligands as Pharmacological Research Tools for the Human Adenosine A2A Receptor.

Among the class A G protein-coupled receptors (GPCR), the human adenosine A2A receptor (hA2AAR) represents an attractive drug target and has been the subject of intensive medicinal chemistry research during the last forty years. However, translation of A2AAR ligands into the clinic has proved challenging. A better understanding of the molecular mechanisms underlying the A2AAR-specific pharmacology, under either physiological or pathological conditions, could promote the development of more efficacious therapies. This can be facilitated by exploiting subtype-selective fluorescent probes which allow detailed real-time pharmacological investigations in both in vitro and in vivo environments. In the present study, two families of fluorescent probes containing BODIPY-, Cy5- and AF647-based dyes were prepared, the design of which was based upon the known hA2AAR selective antagonist preladenant (SCH 420814). Structural modification of preladenant with an amino alkyl spacer yielded the corresponding amino functionalized congener amenable for conjugation with six commercially available fluorophores. Both families of fluorescent antagonists retained affinity at the hA2AAR and, most importantly, selectivity over all of the other adenosine receptor subtypes. The novel hA2AAR fluorescent conjugates allowed clear visualization of specific receptor localization through confocal imaging. Within the second family of hA2AAR fluorescent ligands, the AlexaFluor647-labelled conjugate allowed measurement of ligand binding affinities of unlabeled hA2AAR antagonists through the recently described bioluminescence resonance energy transfer (NanoBRET) approach. The novel hA2AAR fluorescent ligands developed here can be broadly applied to a range of fluorescence-based techniques to study the signaling and dynamics of A2AAR receptors in their native cellular environment providing a robust and specific platform for further pharmacological investigations.