Reversible and site-specific immobilization of β2-adrenergic receptor by aptamer-directed method for receptor-drug interaction analysis
Reversible and site-specific immobilization of β2-adrenergic receptor by aptamer-directed method for receptor-drug interaction analysis
复制标题
通过适体定向方法对 β(2)-肾上腺素能受体进行可逆和位点特异性固定，用于受体-药物相互作用分析
DOI：
10.1016/j.chroma.2020.461091
复制
发表时间：
2020-07-05
影响因子：
4.1
通讯作者：
Zhao，Xinfeng
中科院分区：
文献类型：
--
作者：
Gao，Juan;Chang，Zhongman;Zhao，Xinfeng
固定化蛋白质对药物发现、诊断和体内生物学相互作用分析方法的发展产生了深远的影响。由于G蛋白偶联受体功能的丧失，传统的方法受到了极大的挑战。我们将β2-肾上腺素能受体（β2-AR）适体引入受体的固定化中。这通过将受体缀合的硅胶与含有受体的细胞裂解物混合来实现。我们发现，适体导向的方法使固定化的β2-AR在7天内具有良好的稳定性，并在亚型受体水平上具有较高的配体识别特异性。采用非线性色谱法、峰衰减分析法和注射依赖性方法对固定化β2-AR用于药物-受体相互作用分析的可行性进行了评价。沙丁胺醇、甲氧那明、盐酸麻黄碱、氯丙那林、妥洛特罗、班布特罗、普萘洛尔和ICI 118551通过一种类型的结合位点与受体结合。结合常数在三种方法中呈现出良好的一致性，但与放射性配体结合试验的数据存在明显差异。关于这些结果，我们的结论是，适体导向的方法可能会成为一种替代的可逆和位点特异性固定GPCR直接从复杂的矩阵;固定的受体是定性的药物-受体相互作用分析。
Immobilized protein makes a profound impact on the development of assays for drug discovery, diagnosis andin vivobiological interaction analysis. Traditional methods are enormously challenged by the G-protein coupled receptor ascribed to the loss of receptor functions. We introduced a β2-adrenergic receptor (β2-AR) aptamer into the immobilization of the receptor. This was achieved by mixing the receptor conjugated silica gel with cell lysates containing the receptor. We found that the aptamer-directed method makes immobilized β2-AR good stability in seven days and high specificity of ligand recognition at the subtype receptor level. Feasibility of the immobilized β2-AR in drug-receptor interaction analysis was evaluated by injection amount-dependent method, nonlinear chromatography, and peak decay analysis. Salbutamol, methoxyphenamine, ephedrine hydrochloride, clorprenaline, tulobuterol, bambuterol, propranolol and ICI 118551 bound to the receptor through one type of binding sites. The association constants presented good agreement within the three methods but exhibited clear differences from the data by radio-ligand binding assay. Regarding these results, we concluded that the aptamer-directed method will probably become an alternative for reversible and site-specific immobilization of GPCRs directly from complex matrices; the immobilized receptor is qualitative for drug-receptor interaction analysis.