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.