Identifying potential ligands specifically binding to beta1-adrenoceptor from Radix Aconiti Lateralis Praeparata...

As expressed predominantly in cardiac tissue, beta1-adrenoceptor (β₁-AR) is broadly accepted as one of the main targets for drugs against cardiovascular ailments. However, the discovery of β₁-AR ligand is gravely challenged due to the lack of efficient screening method. This work developed a general strategy for pursuing β₁-AR ligands from the herbal extract by immobilizing haloalkane dehalogenase (Halo)-tagged β₁-AR onto microspheres coated with 6-chlorohexanoic acid, and applying the immobilized β₁-AR in the analysis of ligand-receptor interaction. The morphology was characterized by scanning electron microscope (SEM) and X-ray photoelectron spectroscopy (XPS). The chromatographic specificity of the immobilized receptor column was evaluated by determining the association constants of atenolol, esmolol and metoprolol using stepwise frontal analysis plus injection amount-dependent method. The potential ligands binding to β₁-AR was screened by collecting the peak with retention time longer than the void time, and identified the collection by reverse phase liquid chromatography coupled with tandem mass spectrometry. The association constants of the three drugs to β₁-AR were (3.33 ± 0.29)× 10⁶ M^-1, (2.33 ± 0.23)× 10⁶ M^-1 and (2.06 ± 0.03)× 10⁶ M^-1, indicating a desired specificity of the immobilized receptor for recognizing its ligands. Molecular docking showed that van der Waals, hydrogen bonds, and hydrophobic interactions were the principal interaction forces for the receptor-drug complexes. Benzoylmesaconine was screened as the potential ligand of β₁-AR in Radix Aconiti Lateralis Praeparata extract. The association constant of the ligand was (1.06 ± 0.02)× 10⁵ M^-1, hinting structural modification may be required before clinical application. The immobilized β₁-AR is possible to provide a rapid method for screening potential ligands in herbal extract.