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

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Abstract

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.

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Funding

This work was funded by “Project supported by the State Key Program of National Natural Science of China (Grant No.U1508221)”. This work is also supported by “Projects of National Science Foundation of China (81473436, 81403100, 21405155 and 81402916).”

Author information

Author notes

  1. Pengyu Zhang and Jixia Wang contributed equally to this work.

Authors and Affiliations

  1. Liaoning Provincial Core Lab of Medical Molecular Biology, Dalian Medical University, Dalian, 116044, China

    Pengyu Zhang & Ying Zhao

  2. Key Lab of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, 116023, China

    Jixia Wang, Xiuli Zhang, Lala Qu, Chaoran Wang, Jiatao Feng, Weijia Zhou, Yanfang Liu, Tao Hou, Han Zhou & Xinmiao Liang

  3. DICP-CMC Innovation Institute of Medicine, Taizhou, 225300, China

    Jixia Wang, Chaoran Wang, Jiatao Feng, Yanfang Liu, Zhiwei Wang & Xinmiao Liang

  4. Co-innovation Center of Neuroregeneration, Nantong University, Nantong, 226019, China

    Xiuli Zhang & Xinmiao Liang

  5. State Key Laboratory of Fine Chemicals, School of Chemistry, Dalian University of Technology, Dalian, 116023, China

    Anhui Wang

  6. Laboratory of Molecular Modeling and Design, State Key Laboratory of Molecular Reaction Dynamics, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, 116023, China

    Anhui Wang

Authors
  1. Pengyu Zhang
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  2. Jixia Wang
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  3. Ying Zhao
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  4. Xiuli Zhang
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  5. Lala Qu
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  6. Chaoran Wang
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  7. Jiatao Feng
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  8. Anhui Wang
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  9. Weijia Zhou
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  10. Yanfang Liu
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  11. Tao Hou
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  12. Han Zhou
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  13. Zhiwei Wang
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  14. Xinmiao Liang
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Contributions

Pengyu Zhang and Jixia Wang designed and performed most of the experiments, interpreted data, and wrote the paper. Anhui Wang designed and performed docking studies. Weijia Zhou, Yanfang Liu, Chaoran Wang, and Jiatao Feng provided and identified natural compounds. Lala Qu, Tao Hou, Han Zhou, and Zhiwei Wang analyzed the data and revised the manuscript. Ying Zhao, Xiuli Zhang, and Xinmiao Liang conceived the idea, designed experiment plan, and revised the manuscript.

Corresponding authors

Correspondence to Ying Zhao or Xiuli Zhang.

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The authors declare that they have no conflict of interest.

Electronic supplementary material

Supplemental Information includes information on 191 natural compounds used in this study.

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Zhang, P., Wang, J., Zhao, Y. et al. Discovery of novel antagonists on β2-adrenoceptor from natural products using a label-free cell phenotypic assay. Naunyn-Schmiedeberg's Arch Pharmacol 391, 1411–1420 (2018). https://doi.org/10.1007/s00210-018-1555-8

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  • DOI: https://doi.org/10.1007/s00210-018-1555-8

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