In the adenosine receptor (AR) subfamily of G protein-coupled receptors
(GPCRs), biased agonism has been described for the human A1AR,
A2BAR and A3AR. While diverse A3AR agonists
have been evaluated for receptor binding and Gi-mediated cAMP signalling, the
β-arrestin2 (βarr2) pathway has been left largely unexplored. We
screened nineteen diverse adenosine derivatives for βarr2 recruitment
using a stable hA3AR-NanoBit®-βarr2 HEK293T
cell line. Their activity profiles were compared with a cAMP accumulation assay
in stable hA3AR CHO cells. Structural features linked to βarr2
activation were further investigated by the evaluation of an additional ten
A3AR ligands. The A3AR-selective reference agonist
2-Cl-IB-MECA, which is a full agonist in terms of cAMP inhibition, only showed
partial agonist behaviour in βarr2 recruitment. Highly
A3AR-selective (N)-methanocarba 5’-uronamide adenosine
derivatives displayed higher potency in both cAMP signalling and βarr2
recruitment than reference agonists NECA and 2-Cl-IB-MECA. Their
A3AR-preferred conformation tolerates C2-position substitutions, for
increased βarr2 efficacy, better than the flexible scaffolds of ribose
derivatives. The different amino functionalities in the adenosine scaffold of
these derivatives each seem to be important for signalling as well. In
conclusion, we have provided insights into ligand features that can help to
guide the future therapeutic development of biased A3AR ligands with
respect to G-protein and βarr2 signalling.