Pharmacological Characterisation of Novel Adenosine Receptor A 3 R Antagonists
Pharmacological Characterisation of Novel Adenosine Receptor A 3 R Antagonists
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新型腺苷受体 A 3 R 拮抗剂的药理学表征
DOI：
10.1101/693796
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发表时间：
2019
期刊：
影响因子：
--
通讯作者：
Barkan K
中科院分区：
文献类型：
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作者：
Barkan K
腺苷a3受体（A3R）属于一个由四种腺苷受体（AR）亚型组成的家族，它们在全身发挥着不同的作用。A3R拮抗剂已被描述为包括哮喘在内的许多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，我们筛选了39种潜在的A3R拮抗剂，使用激动剂诱导的cAMP抑制。通过cAMP积累试验评估阳性命中的AR亚型选择性。通过Schild分析（pa2值）和荧光配体结合来确定拮抗剂的亲和力。构效关系研究表明，3-（二氯苯基）-异恶唑基部分或α-位置含氮的芳香氮杂环对碳胺基碳的损失显著减弱了K18的拮抗效力。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在A3R正位位点上对结合重要的残基。我们证明了K18是一种特异性的A3R（< 1µM）竞争性拮抗剂，它含有一个3-（二氯苯基）-异恶唑基团，通过羰基氧基carboyloxyximidamide片段与1,3-噻唑环连接。最后，我们介绍了一个模型，可以从实时荧光配体结合研究中估计快速解缔合化合物的平衡结合亲和力。这些结果证明了选择性竞争A3R拮抗剂的药理学特征及其正畸结合模式的描述。我们的发现可能为药物发现提供新的见解。
The adenosine A3receptor (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 (pA2values) 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.