College of Life Sciences, Northwest University, Xi'an 710069, China.
College of Chemistry and Materials Science, Northwest University, Xi'an 710069, China.
J Chromatogr A. 2022 Aug 16;1677:463298. doi: 10.1016/j.chroma.2022.463298. Epub 2022 Jul 2.
The discovery of beta1-adrenoceptor (β-AR) ligands is viewed as an enormous demand for fighting ailments mediated by the receptor including cardiovascular diseases. Such pursuit is gravely challenged due to the lack of lead screening methods with high efficiency. This work developed a chromatographic method for pursuing β-AR ligand from the herbal extract by fusing epidermal growth factor receptor (EGFR) as a tag at its C-terminus to stably express the fusion receptor in E. coli, immobilizing the expressed EGFR-tagged β-AR onto ibrutinib-derivatized amino microspheres, and applying the immobilized receptor in the analysis of ligand-receptor interaction and herbal extract. Comprehensive characterizations like X-ray photoelectron spectroscopy and retention behaviors of canonical drugs demonstrated high specificity and good stability of the immobilized β-AR prepared through the covalent reaction between the EGFR and ibrutinib decorated on the microsphere surface. Frontal analysis of atenolol, metoprolol, and esmolol confirmed their bindings to β-AR with association constants of 1.07 × 10, 6.54 × 10, and 1.45 × 10 M. The thermodynamic analysis provided proof of electrostatic interaction, hydrogen bonds, and van der Waals force driving those interactions. Pulegone was recognized as a bioactive compound that specifically binding to β-AR from the extract of Ziziphora clinopodioides Lam by analyzing the retention peak through reverse-phase high performance liquid chromatography coupled with tandem mass spectrometry. These results, taken together, indicated that the current method is possible to provide an alternative for discovering β-AR ligands with high efficiency from complex matrices like herbal extract.
β1-肾上腺素受体（β-AR）配体的发现被认为是对抗包括心血管疾病在内的受受体介导的疾病的巨大需求。由于缺乏高效的先导筛选方法，这种追求受到了严重的挑战。本工作通过在其 C 末端融合表皮生长因子受体（EGFR）作为标签，在大肠杆菌中稳定表达融合受体，将表达的 EGFR 标记的β-AR 固定在伊布替尼衍生的氨基微球上，将固定化受体应用于配体-受体相互作用和草药提取物的分析，开发了一种从草药提取物中寻找β-AR 配体的色谱方法。X 射线光电子能谱和典型药物的保留行为等综合表征表明，通过 EGFR 与微球表面修饰的伊布替尼之间的共价反应制备的固定化β-AR 具有高特异性和良好的稳定性。阿替洛尔、美托洛尔和艾司洛尔的前沿分析证实了它们与β-AR 的结合，其结合常数分别为 1.07×10、6.54×10 和 1.45×10 M。热力学分析证明了静电相互作用、氢键和范德华力驱动这些相互作用。通过反相高效液相色谱-串联质谱分析，从迷迭香属植物提取物中鉴定出香芹酮是一种特异性结合β-AR 的生物活性化合物。这些结果表明，该方法有可能为从草药等复杂基质中高效发现β-AR 配体提供一种替代方法。
