Modeling the Adenosine Receptors: Comparison of the Binding Domains of A_(2A) Agonists and Antagonists

摘要

A three-dimensional model of the human A_(2A) 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 A_(2A)AR 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 I overlapped, and the exocyclic amino groups of each were H-bonded to the side chain of N~(6.55). For bound agonist, H-bonds formed between the ribose 3'- and 5'-substituents ad the hydrophilic amino acids T~(3.36), S~(7.42), and H~(7.43), and the terminal methyl group of the 5'-uronamide interacted with the hydrophobic side chain of F~(6.44). Formation of the agonist complex destabilized the ground-state structure of the A_(2A)AR, 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) E13~(7.46), and H278~(7.43) and (2) D52~(2.50), with the highly conserved amino acids N280~(7.45) and S281~(7.46), and N284~(7.49) with S91~(3.39). Most of the amino acid residues lining the putative binding site(s) were conserved among the four AR subtypes. The A_(2A)AR/3 complex showed a preference for an intermediate conformation about the glycosidic bond, unlike in the A_3AR/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.