Adenosine A₃ receptor (A₃R) is a promising drug target cancer and for a number of other conditions like inflammatory diseases, including asthma and rheumatoid arthritis, glaucoma, chronic obstructive pulmonary disease, and ischemic injury. Currently, there is no experimentally determined structure of A₃R. We explored the binding profile of O4-{[3-(2,6-dichlorophenyl)-5-methylisoxazol-4-yl]carbonyl}-2-methyl-1,3-thiazole-4-carbohydroximamide (K18), which is a new specific and competitive antagonist at the orthosteric binding site of A₃R. MD simulations and MM-GBSA calculations of the WT A₃R in complex with K18 combined with in vitro mutagenic studies show that the most plausible binding conformation for the dichlorophenyl group of K18 is oriented toward trans-membrane helices (TM) 5, 6 and reveal important residues for binding. Further, MM-GBSA calculations distinguish mutations that reduce or maintain or increase antagonistic activity. Our studies show that selectivity of K18 toward A₃R is defined not only by direct interactions with residues within the orthosteric binding area but also by remote residues playing a significant role. Although V169^5.30 is considered to be a selectivity filter for A₃R binders, when it was mutated to glutamic acid, K18 maintained antagonistic potency, in agreement with our previous results obtained for agonists binding profile investigation. Mutation of the direct interacting residue L90^3.32 in the low region and the remote L264^7.35 in the middle/upper region to alanine increases antagonistic potency, suggesting an empty space in the orthosteric area available for increasing antagonist potency. These results approve the computational model for the description of K18 binding at A₃R, which we previously performed for agonists binding to A₃R, and the design of more effective antagonists based on K18.

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