Abstract
Among class A G protein-coupled receptors (GPCR), the human adenosine A2A receptor (hA2AAR) remains an attractive drug target. However, translation of A2AAR ligands into the clinic has proved challenging and an improved understanding of A2AAR 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 hA2AAR selective antagonist preladenant (SCH 420814). Both families of fluorescent antagonists retained affinity at the hA2AAR, 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 hA2AAR 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 hA2AAR pharmacology and signaling.
Highlights
The adenosine receptors belong to class A of the G proteincoupled receptor (GPCR) superfamily with the latter accounting for about 30% of the molecular targets for currently marketed drugs.[1]
The adenosine A2A receptor (A2AAR) represents an attractive drug target which has been the subject of intensive medicinal chemistry research over the last 40 years.[3,4]
We report the design, synthesis, and pharmacological evaluation of a novel series of subtypeselective fluorescent ligands (12−17) for the human adenosine hA2AAR receptor based upon preladenant (a high affinity[29] and highly selective[29] hA2AAR antagonist), which are amenable to use in a variety of fluorescence-based techniques
Summary
The adenosine receptors belong to class A of the G proteincoupled receptor (GPCR) superfamily with the latter accounting for about 30% of the molecular targets for currently marketed drugs.[1]. Given the higher affinity displayed at the A2AAR, the BODIPY-labeled ligands were selected to assess whether the novel synthesized probes were able to retain the functional antagonistic ability of the parent compound preladenant This was performed using a previously described CRE-SPAP reporter gene assay[50] in CHO cells expressing the human adenosine hA2AAR receptors. We have reported the successful design, development, and pharmacological evaluation of a novel series of hA2AAR fluorescent antagonists, encompassing BODIPYbearing probes and water-soluble Cy5-labeled and AF647labeled probes, based on the high affinity and selective preladenant scaffold. Derivatization of the amino functionalized congener 11 with two families of fluorescent tags led to six hA2AAR fluorescent antagonists with retained affinity and, most importantly, selectivity at the A2AAR subtype Both families of compounds allowed visualization of A2AARs expressed in live cells as demonstrated by the high fluorescence intensity at the cell membranes. Membrane preparations of Terbium-labeled SNAP-HEK293-A2AAR cells: The following described steps were conducted at 4 °C to circumvent receptor degradation
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