A BINDING-SITE MODEL AND STRUCTURE-ACTIVITY-RELATIONSHIPS FOR THE RATA(3)-ADENOSINE RECEPTOR

A novel adenosine receptor, the A(3) receptor, has recently been clone d. We have systematically investigated the hitherto largely unexplored structure-activity relationships (SARs) for binding at A(3) receptors , using (125)l-N-6-2-(4-aminophenyl)ethyladenosine as a radioligand an d membranes from Chinese hamster ovary cells stably transfected with t he rat A(3)-cDNA. As is the case for A(1) and A(2A) receptors, substit utions at the N-6 and 5' positions of adenosine, the prototypic agonis t ligand, may yield fairly potent compounds. However, the highest affi nity and A(3) selectivity is found for N-6,5'-disubstituted compounds, in contrast to A(1) and A(2a) receptors. Thus, N-6-benzyladenosine-5' -N-ethylcarboxamide is highly potent (K-I, 6.8 nM) and moderately sele ctive (13- and 14-fold versus A(1) and A(2a)) The N-6 region of the A( 3) receptor also appears to tolerate hydrophilic substitutions, in sha rp contrast to the other subtypes. Potencies of N-6,5'-disubstituted c ompounds in inhibition of adenylate cyclase via A(3) receptors paralle l their high affinity in the binding assay. None of the typical xanthi ne or nonxanthine (A(1)/A(2)) antagonists tested show any appreciable affinity for rat A(3) receptors. 1,3-Dialkylxanthines did not antagoni ze the A(3) agonist-induced inhibition of adenylate cyclase. A His res idue in helix 6 that is absent in A(3) receptors but present in A(1)/A (2) receptors may be causal in this respect. In a molecular model for the rat A(3) receptor, this mutation, together with an increased bulki ness of residues surrounding the ligand, make antagonist binding unfav orable when compared with a previously developed A(1) receptor model. Second, this A(3) receptor model predicted similarities with A(1) and A(2) receptors in the binding requirements for the ribose moiety and t hat xanthine-7-ribosides would bind to rat A(3) receptors. This hypoth esis was supported experimentally by the moderate affinity (K-l 6 mu M ) of 7-riboside of 1,3-dibutylxanthine, which appears to be a partial agonist at rat A(3) receptors. The model presented here, which is cons istent with the detailed SAR found in this study, may serve to suggest future chemical modification, site-directed mutagenesis, and SAR stud ies to further define essential characteristics of the ligand-receptor interaction and to develop even more potent and selective A(3) recept or ligands.