Discovery of novel antagonists on β2-adrenoceptor from natural products using a label-free cell phenotypic assay

Label-free cell phenotypic assays were performed to establish a β2-adrenoceptor (β2-AR) target model in A431 cells and a β1-AR target model in transfected HEK293-β1 cells, using known β2-AR and β1-AR agonists and antagonists. A list of natural compounds was screened on the target models, among which seven new compounds were found to be antagonistically active against β2-AR. After receptor specificity evaluations on hydroxyl carboxylic acid receptor-2 (ΗΧΑ-2), histamine receptor (H1R), and β1-adrenoceptor (β1-AR), six out of the seven compounds, including nuciferine, epiberberine, harmaline, harmine, palmatine, and columbamine, exhibited specific antagonistic activity against β2-AR. Epiberberine and palmatine showed the strongest antagonistic activities against β2-AR with IC50 values of 2.3 ± 0.2 μM and 2.6 ± 0.3 μM, respectively. Docking palmatine to the crystal structure of human β2-AR (PDB 5X7D) suggested that the ligand forms a hydrogen bond with N312 and hydrophobic interaction with several amino acid residues in the binding pocket, such as D113 and V114. The kinetic binding profile of palmatine was further investigated using co-stimulation assays. Results suggested that palmatine was a competitive antagonist for β2-AR. The six novel β2-AR antagonists provide a promising chemical starting point for identification and optimization of drugs used for treating hypertension, glaucoma, and infantile hemangiomas. This study also lays the foundation for the in-depth investigation of biochemical mechanisms and pharmacological properties of natural compounds.