The 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 (adenosine receptors) orthosteric binding sites, obtaining highly selective antagonists is a challenging but critical task. Here we screen 39 potential A₃R, antagonists 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. Structure–activity relationship investigations revealed that loss of the 3-(dichlorophenyl)-isoxazolyl moiety or the aromatic nitrogen heterocycle with nitrogen at α-position to the carbon of carboximidamide group significantly attenuated K18 antagonistic potency. Mutagenic studies supported by molecular dynamic simulations combined with Molecular Mechanics—Poisson Boltzmann Surface Area calculations identified the residues important for binding in the A₃R orthosteric site. We 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. Finally, we introduce a model that enables estimates of the equilibrium binding affinity for rapidly disassociating compounds from real-time fluorescent ligand-binding studies. These results demonstrate the pharmacological characterisation of a selective competitive A₃R antagonist and the description of its orthosteric binding mode. Our findings may provide new insights for drug discovery.

腺苷 A ₃受体 (A ₃ R) 属于四种腺苷受体 (AR) 亚型家族，这些亚型在体内都发挥着不同的作用。 A ₃ R拮抗剂已被描述为包括哮喘在内的多种疾病的潜在治疗方法。鉴于（腺苷受体）正位结合位点之间的相似性，获得高选择性拮抗剂是一项具有挑战性但至关重要的任务。在这里，我们利用激动剂诱导的 cAMP 抑制筛选 39 种潜在的 A ₃ R 拮抗剂。通过 cAMP 积累测定评估阳性命中的 AR 亚型选择性。使用Schild分析（pA ₂值）和荧光配体结合来确定拮抗剂亲和力。结构-活性关系研究表明，3-(二氯苯基)-异恶唑基部分或在甲脒基团的碳的α位上带有氮的芳香氮杂环的丢失显着减弱了K18拮抗效力。分子动力学模拟结合分子力学-泊松玻尔兹曼表面积计算支持的诱变研究确定了对 A ₃ R 正构位点结合重要的残基。我们证明，K18 含有通过羰基羧酰亚胺片段与 1,3-噻唑环连接的 3-(二氯苯基)-异恶唑基团，是一种特异性 A ₃ R (< 1 µM) 竞争性拮抗剂。最后，我们引入了一个模型，可以估计实时荧光配体结合研究中快速解离化合物的平衡结合亲和力。 这些结果证明了选择性竞争性A ₃ R 拮抗剂的药理学特征及其正位结合模式的描述。我们的研究结果可能为药物发现提供新的见解。