Site-Specific Immobilization of β2-AR Using O6-Benzylguanine Derivative-Functionalized Supporter for High-Throughput Receptor-Targeting Lead Discovery
Site-Specific Immobilization of β2-AR Using O6-Benzylguanine Derivative-Functionalized Supporter for High-Throughput Receptor-Targeting Lead Discovery
复制标题
使用 O-6-苄基鸟嘌呤衍生物功能化支持物对 β(2)-AR 进行位点特异性固定，以实现高通量受体靶向先导化合物发现
DOI：
10.1021/acs.analchem.9b01268
复制
发表时间：
2019-06-04
影响因子：
7.4
通讯作者：
Zheng, Xiaohui
中科院分区：
文献类型：
--
作者：
Wang, Jing;Wang, Yuxin;Zheng, Xiaohui
在过去的十年里，基于表面固定化GPCR的配体发现策略已经有了巨大的前景。本文介绍了一种固定化GPCR的制备方法。主要特点包括高特异性的共价固定和在药物-受体相互作用分析和配体筛选中的稳健应用。在我们使用β（2）-肾上腺素能受体（β（2）-AR）的示例性测定中，在大肠杆菌中表达的人DNA修复蛋白O-6-烷基鸟嘌呤-DNA烷基转移酶（hAGT）融合受体被直接捕获到聚乙二醇聚丙烯酰胺（PEGA）树脂上。我们观察到β（2）-AR在树脂上的均匀分布和生理功能。以β（2）-AR为固定相，快速测定了4种药物与β（2）-AR的结合。我们的结论是，O-6-苄基鸟嘌呤衍生物功能化的载体是有前途的特异性固定hAGT标记的蛋白质;固定化受体色谱在筛选受体结合的线索，从草药植物或传统的药物配方具有很大的潜力。
The past decade has witnessed the great promise of strategies for ligand discovery based on surface-immobilized GPCRs. We present here a method for preparation of immobilized GPCRs. Key features include covalent immobilization with high specificity and robust application in drug-receptor interaction analysis and ligand screening. In our example assay using beta(2)-adrenergic receptor (beta(2)-AR), the human DNA repair protein O-6-alkylguanine-DNA alkyltransferase (hAGT) fusion receptor expressed in Escherichia coli was directly captured onto polyethylene glycol polyacrylamide (PEGA) resin. We observed even distribution and physiological functions of beta(2)-AR on the resin. The immobilized beta(2)-AR as a stationary phase enabled us to rapidly determine the binding of four drugs to beta(2)-AR By coupling this assay to mass spectrometry, we screened rosmarinic acid as a bioactive compound targeting beta(2)-AR in Fructus Perillae. We concluded that O-6-benzylguanine derivative-functionalized supporter is promising for specific immobilization of hAGT-tagged proteins; immobilized receptor chromatography has great potential in screening receptor-binding leads from herbal plants or traditional medicine recipes.