Modeling the Adenosine Receptors:  Comparison of the Binding Domains of A2A Agonists and Antagonists

A three-dimensional model of the human A2A adenosine receptor (AR) and its docked ligands was built by homology to rhodopsin and validated with site-directed mutagenesis and the synthesis of chemically complementary agonists. Different binding modes of A2AAR antagonists and agonists were compared by using the FlexiDock automated docking procedure, with manual adjustment. Putative binding regions for the 9H-purine ring in agonist NECA 3 and the 1H-[1,2,4]triazolo[1,5-c]quinazoline ring in antagonist CGS15943 1 overlapped, and the exocyclic amino groups of each were H-bonded to the side chain of N6.55. For bound agonist, H-bonds formed between the ribose 3‘- and 5‘-substituents and the hydrophilic amino acids T3.36, S7.42, and H7.43, and the terminal methyl group of the 5‘-uronamide interacted with the hydrophobic side chain of F6.44. Formation of the agonist complex destabilized the ground-state structure of the A2AAR, which was stabilized through a network of H-bonding and hydrophobic interactions in the transmembrane helical domain (TM) regions, facilitating a conformational change upon activation. Both flexibility of the ribose moiety, required for the movement of TM6, and its H-bonding to the receptor were important for agonism. Two sets of interhelical H-bonds involved residues conserved among ARs but not in rhodopsin: (1) E131.39 and H2787.43 and (2) D522.50, with the highly conserved amino acids N2807.45 and S2817.46, and N2847.49 with S913.39. Most of the amino acid residues lining the putative binding site(s) were conserved among the four AR subtypes. The A2AAR/3 complex showed a preference for an intermediate conformation about the glycosidic bond, unlike in the A3AR/3 complex, which featured an anti-conformation. Hydrophilic amino acids of TMs 3 and 7 (ribose-binding region) were replaced with anionic residues for enhanced binding to amine-derivatized agonists. We identified new neoceptor (T88D)-neoligand pairs that were consistent with the model.