<h4>Summary</h4> <h4>Background and Purpose</h4> The adenosine A 3 receptor (A 3 R) belongs to a family of four adenosine receptor (AR) subtypes which all play distinct roles throughout the body. A 3 R 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. <h4>Experimental approach</h4> 39 potential A 3 R, 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 (pA 2 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 A 3 R, combined with mutagenesis studies. <h4>Key Results</h4> We demonstrate that K18, which contains a 3-(dichlorophenyl)-isoxazole group connected through carbonyloxycarboximidamide fragment with a 1,3-thiazole ring, is a specific A 3 R (<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 A 3 R 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. <h4>Conclusions and Implications</h4> These results demonstrate the pharmacological characterisation of a selective competitive A 3 R antagonist and the description of its orthosteric binding mode. Our findings may provide new insight for drug discovery. <h4>What is already known</h4> The search for AR subtype specific compounds often leads to ones with multiple subtype binding <h4>What this study adds</h4> This study demonstrates the pharmacological characterisation of a selective competitive A 3 R antagonist MD simulations identified the residues important for binding in the A 3 R orthosteric site <h4>Clinical significance</h4> This study offers insight into A 3 R antagonists that may provide new opportunities for drug discovery
