Deciphering the Agonist Binding Mechanism to the Adenosine A1 Receptor

Abstract

Despite being amongst the most characterized G protein-coupled receptors (GPCRs), adenosine receptors (ARs) have always been a difficult target in drug design. To date, no agonist other than the natural effector and the diagnostic regadenoson has been approved for human use. Recently, the structure of the adenosine A1 receptor (A1R) was determined in the active, Gi protein complexed state; this has important repercussions for structure-based drug design. Here, we employed supervised molecular dynamics simulations and mutagenesis experiments to extend the structural knowledge of the binding of selective agonists to A1R. Our results identify new residues involved in the association and dissociation pathway, suggest the binding mode of N6-cyclopentyladenosine (CPA) related ligands, and highlight the dramatic effect that chemical modifications can have on the overall binding mechanism.

Discovery and Structure–Activity Relationship Studies of Novel Adenosine A1 Receptor-Selective Agonists

“…The reason for this could be the divergent amino acid composition of the extracellular loops between hA 1 R and rA 1 R, which would favor different binding paths to the orthosteric site. 24 Overall, the compounds displayed a very similar binding profile across the human and rat A 1 R, suggesting that further studies in rats would be highly relevant for the potential use of the compounds in humans. The adenosine and NECA-derived analog pairs also display very similar affinities for both human and rat A 1 R, suggesting that it is the R 2 substituent on the phenoxy or benzyloxy ring that is key in determining the compound affinity for A 1 R. At the hA 1 R, the compounds with the highest affinity are 27−29, 49, 51, 53, and 54.…”

“…BnOCPA has previously been identified as a high-potency A 1 R-selective full agonist. 13,22 Using insights from BnOCPA MD simulations 22,24 and with the aim of further improving the A 1 R selectivity and potency, we designed extended BnOCPA derivatives 15−18. Their binding and activity at human A 1 R (hA 1 R) were then explored using both a NanoBRET binding assay and a cAMP accumulation assay, respectively (Table 1).…”

“…We also included the mutant T257 6.58 A since we have previously shown that this residue is a good discriminator between different A 1 R agonists. 24 We did not consider mutations of N254 6.55 or F171 ELC2 since these are known to prevent ligand binding to the A 1 R (including CA200645) and therefore cannot be studied. 24,37 Furthermore, we did not consider mutating T270 7.35 since, when we compared the sequences of the hA 1 R and the rA 1 R, we observed that, in the rA 1 R, the equivalent residue at position T270 7.35 is an Ile.…”

“…22 Molecular dynamics (MD) simulations using the cryo-EM structure of the active adenosine-bound A 1 R-heterotrimeric G i2 protein complex (PDB code 6D9H 23 ) proposed four binding modes of BnOCPA 22 due to the high flexibility of the N 6 appended benzyloxy group. 24 Based on these molecular modeling studies, we have designed a series of adenosine-and NECA-based compounds with extended N 6 -benzyloxy-and N 6 -phenoxycyclopentyl substituents (Chart 1) with the aim of improving the potency at A 1 R while maintaining or improving the subtype selectivity. To test the potency, selectivity, and affinity of the designed compounds at the adenosine receptors, we have employed the cAMP accumulation and NanoBRET binding assays previously validated and employed at ARs.…”

Selective activation of Gαob by an adenosine A1 receptor agonist elicits analgesia without cardiorespiratory depression

“…To better understand the unusual signalling properties of BnOCPA and the highly specific Gα coupling to Gob, we carried out dynamic docking simulations to study the basic orthosteric binding mode of BnOCPA in an explicit, fully flexible environment using the active cryo-EM structure of the A 1 R (PDB code 6D9H; Supplementary Movie 1). We previously reported that modifications at position N 6 of the adenine scaffold modulated the agonist binding path to A 1 R 69 . More precisely, N 6 -cyclopentyl analogues (CPA and HOCPA) markedly interact with the extracellular loop 2 (ECL2) compared to adenosine, while BnOCPA (which bears the N 6 -cyclopentyl-2-benzyloxy group) is most prone to engage residues of the A 1 R located at the top of transmembrane helix 1 (TM1) and TM7.…”