SummaryBackground and PurposeThe adenosine A3 receptor (A3R) belongs to a family of four adenosine receptor (AR) subtypes 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 antagonists is a challenging but critical task.Experimental approach39 potential A3R, antagonists were screened using agonist-induced inhibition of cAMP. Positive hits were assessed for AR subtype selectivity 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 dynamic (MD) simulations and consistent calculated binding free energy differences between K18 and congeners, using a homology model of A3R, combined with mutagenesis studies.Key ResultsWe demonstrate that K18, which contains a 3-(dichlorophenyl)-isoxazole group connected through carbonyloxycarboximidamide fragment with a 1,3-thiazole ring, is a specific A3R (<1 µM) competitive antagonist. Structure-activity relationship investigations revealed that loss of the 3-(dichlorophenyl)-isoxazole group significantly attenuated K18 antagonistic potency. Mutagenic studies supported by MD simulations identified the residues important for binding in the A3R orthosteric site. 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 ImplicationsThese results demonstrate the pharmacological characterisation of a selective competitive A3R antagonist and the description of its orthosteric binding mode. Our findings may provide new insight for drug discovery.What is already knownThe search for AR subtype specific compounds often leads to ones with multiple subtype bindingWhat this study addsThis study demonstrates the pharmacological characterisation of a selective competitive A3R antagonistMD simulations identified the residues important for binding in the A3R orthosteric siteClinical significanceThis study offers insight into A3R antagonists that may provide new opportunities for drug discovery