Ligand-Specific Binding and Activation of the Human Adenosine A_2B Receptor

Abstract

Adenosine A2B receptors, which play a role in inflammation and cancer, are of considerable interest as novel drug targets. To gain deeper insights into ligand binding and receptor activation, we exchanged amino acids predicted to be close to the binding pocket. The alanine mutants were stably expressed in CHO cells and characterized by radioligand binding and cAMP assays using three structural classes of ligands: xanthine (antagonist), adenosine, and aminopyridine derivatives (agonists). Asn2827.45 and His2807.43 were found to stabilize the binding site by intramolecular hydrogen bond formation as in the related A2A receptor subtype. Trp2476.48, Val2506.51, and particularly Ser2797.42 were shown to be important for binding of nucleosidic agonists. Leu813.28, Asn1865.42, and Val2506.51 were discovered to be crucial for binding of the xanthine-derived antagonist PSB-603. Leu813.28, which is not conserved among adenosine receptor subtypes, may be important for the high selectivity of PSB-603. The N1865.42A mutant resulted in an increased potency for agonists. The interactions of the non-nucleosidic agonist BAY60–6583 were different from those of the nucleosides: while BAY60–6583 appeared not to interact with Ser2797.42, its interactions with Trp2476.48 and Val2506.51 were significantly weaker compared to those of NECA. Moreover, our results discount the hypothesis of Trp2476.48 serving as a “toogle switch” because BAY60–6583 was able to activate the corresponding mutant. This study reveals distinct interactions of structurally diverse ligands with the human A2B receptor and differences between closely related receptor subtypes (A2B and A2A). It will contribute to the understanding of G protein-coupled receptor function and advance A2B receptor ligand design.

Simulation and comparative analysis of binding modes of nucleoside and non-nucleoside agonists at the A2B adenosine receptor

“…The obtained A 2B AR homology models were checked by using the Protein Geometry Monitor application within MOE (Environment), which provides a variety of stereochemical measurements for inspection of the structural quality in a given protein, such as backbone bond lengths, angles and dihedrals, Ramachandran φ-ψ dihedral plots, and quality of side chain rotamer and non-bonded contact. The final A 2B AR models contain a disulfide bridge given by two cysteine residues belonging to TM3 and extracellular loop (EL) 2 domains (Cys78 3.25 - where 3.25 indicates the residue position within helix (Ballesteros and Weinstein 1995) - and Cys171, respectively), in agreement with recent mutagenesis studies (Schiedel et al 2011) and as observed in the case of recently reported modelling analyses on this AR subtype (Sherbiny et al 2009; Thimm et al 2013; Inamdar et al 2013).

Figure 2 Sequence alignment of the four human AR subtypes.

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“…Considering the positioning of the different substituents within the binding cavity, the orientation of the pyridine agonists results somehow comparable to the one obtained and schematically described by Sherbiny and colleagues (Sherbiny et al 2009) but significantly different respect to the ones obtained in more recent docking studies at the same AR subtype (Thimm et al 2013) and at the A 2A AR (Lane et al 2012). In detail, the interaction of the scaffold with the A 2B AR binding site is given as H-bonding between the N1 atom and the 6-amino group of pyridines and the amine and carbonyl groups of Asn254 6.55 amide function, respectively.…”

“…Ile67 2.64 forms hydrophobic interaction with the 4-aromatic group of pyridines, while Ala64 2.61 forms on the one hand a stable H-bond interaction with the para -hydroxyl group of the 4-aromatic substituent of compound 8 (3QAK-based model), on the other hand a hydrophobic interaction with 4-aromatic group of 12. Comparing the results for this section with previously reported data, it can be underlined that Tyr10 1.35 showed to be critical for compound 12 activity as evidenced from mutagenesis results (Thimm et al 2013), while Ile67 2.64 and Ala64 2.61 were already reported to possibly interact with the 4-substituent of pyridine derivatives (Sherbiny et al 2009). The effect of Ser279 7.42 and His280 7.43 is evident for nucleoside agonists but not so well defined for non-nucleoside derivatives.…”

BAY60-6583 Acts as a Partial Agonist at Adenosine A2B Receptors

“…This was surprising given the fact that in functional assays, BAY60-6583 and NECA had been found to be almost equipotent (Peeters et al, 2011;Schiedel et al, 2011;Seibt et al, 2013;Thimm et al, 2013). To further examine the pharmacological profile of BAY60-6583, we performed sodium shift experiments.…”

The second extracellular loop of GPCRs determines subtype-selectivity and controls efficacy as evidenced by loop exchange study at A2 adenosine receptors

“…In Fig. 9, the X-ray structure of the wt A 2A AR [30], a homology model of the wt A 2B AR [42], and a homology model of the A 2B (EL2-A 2A )AR mutant based on the X-ray structure of the active A 2A AR conformation [17] are depicted. The amino acid residues that were mutated to alanine are highlighted in the A 2B AR model.…”