Pharmacological Characterisation of Novel Adenosine Receptor A3R Antagonists

Background and Purpose: The adenosine A3 receptor (A3R) belongs to a family of four adenosine receptor (AR) subtypes (A1R, A2AR, A2BR and A3R), which all play distinct roles throughout the body. A3R antagonists have been described as potential treatments for numerous diseases including asthma. Given the similarity between ARs orthosteric binding sites, obtaining highly selective receptor antagonists is a challenging but critical task. Experimental approach: 39 potential A3R, antagonists were functionally screened using agonist-induced inhibition of cAMP. Selectivity and specificity of potential antagonists was determined in cell lines expressing each of the AR subtypes, again through cAMP accumulation assays. The antagonist affinity was determined using Schild analysis (pA2 values) and fluorescent ligand binding. Further, a likely binding pose of the most potent antagonist (K18) was determined through molecular dynamics (MD) simulations and consistent calculated binding free energy differences between K18 and congeners, using a homology model of A3R, combined with mutagenesis studies. Key Results: We demonstrate that K18, which included a 3-(dichlorophenyl)-isoxazole group connected through carbonyloxycarboximidamide fragment with a thiazole ring, is a sub-micromolar specific A3R competitive antagonist. Mutagenic studies supported by MD simulations identified the residues important for binding in the orthosteric site of the A3R. Structure-activity relationship investigation revealed that the absence of a chloro substituent led to affinity loss. Finally, we introduce a model that enables estimates of the equilibrium binding affinity for rapidly disassociating compounds from real-time fluorescent ligand binding studies. Conclusions and Implications: These results demonstrate the pharmacological characterisation of a selective competitive A3R antagonist and the description of its orthosteric binding mode through a combined approach of mutagenesis studies and MD simulations.