Development of immobilized beta1-adrenoceptor chromatography for rapid discovery of ligands specifically binding to...

The discovery of beta1-adrenoceptor (β₁-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 β₁-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 β₁-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 β₁-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 β₁-AR with association constants of 1.07 × 10⁴, 6.54 × 10³, and 1.45 × 10⁴ M^-1. 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 β₁-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 β₁-AR ligands with high efficiency from complex matrices like herbal extract.