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

Abstract

In the adenosine receptor (AR) subfamily of G protein-coupled receptors (GPCRs), biased agonism has been described for the human A₁AR, A_2BAR and A₃AR. While diverse A₃AR agonists have been evaluated for receptor binding and G_i-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.

Keywords: A(3) adenosine receptor; Biased signaling; G protein-coupled receptor; Structure-activity relationship; β-arrestin2.