Probing structure-activity relationship in β-arrestin2 recruitment of diversely substituted adenosine derivatives.

In the adenosine receptor (AR) subfamily of G protein-coupled receptors (GPCRs), biased agonism has been described for the human AAR, AAR and AAR. While diverse AAR agonists have been evaluated for receptor binding and G-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 hAAR-NanoBit-βarr2 HEK293T cell line. Their activity profiles were compared with a cAMP accumulation assay in stable hAAR CHO cells. Structural features linked to βarr2 activation were further investigated by the evaluation of an additional ten AAR ligands. The AAR-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 AAR-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 AAR-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 AAR ligands with respect to G-protein and βarr2 signalling.