Article

Small-molecule inhibition of TLR8 through stabilization of its resting state

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Abstract

Endosomal Toll-like receptors (TLR3, TLR7, TLR8, and TLR9) are highly analogous sensors for various viral or bacterial RNA and DNA molecular patterns. Nonetheless, few small molecules can selectively modulate these TLRs. In this manuscript, we identified the first human TLR8-specific small-molecule antagonists via a novel inhibition mechanism. Crystal structures of two distinct TLR8–ligand complexes validated a unique binding site on the protein–protein interface of the TLR8 homodimer. Upon binding to this new site, the small-molecule ligands stabilize the preformed TLR8 dimer in its resting state, preventing activation. As a proof of concept of their therapeutic potential, we have demonstrated that these drug-like inhibitors are able to suppress TLR8-mediated proinflammatory signaling in various cell lines, human primary cells, and patient specimens. These results not only suggest a novel strategy for TLR inhibitor design, but also shed critical mechanistic insight into these clinically important immune receptors.

  • Compound

    7-(3-(trifluoromethyl)phenyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

  • Compound

    (Z)-N'-((3-(trifluoromethyl)benzoyl)oxy)-7-(3-(trifluoromethyl)phenyl)pyrazolo[1,5-a]pyrimidine-2-carboximidamide

  • Compound

    4-(4-Hydroxy-3,5-dimethylphenyl)-2-methylphthalazin-1(2H)-one

  • Compound

    2,6-Dimethyl-4-(3-methyl-4-oxo-3,4-dihydrophthalazin-1-yl)phenyl (4-(trifluoromethoxy)phenyl)carbamate

  • Compound

    7-(m-Tolyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

  • Compound

    7-(3-(trifluoromethyl)phenyl)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid

  • Compound

    2-Methyl-4-(7-methoxyl-4-quinolinyl)-phenol

  • Compound

    2-Methyl-4-(7-hydroxyl-4-quinolinyl)-phenol

  • Compound

    Ethyl 7-(3-(trifluoromethyl)phenyl)pyrazolo [1,5-a]pyrimidine-3-carboxylate

  • Compound

    Ethyl 7-m-tolylpyrazolo[1,5-a]pyrimidine-3-carboxylate

  • Compound

    7-(m-Tolyl)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid

  • Compound

    7-(3-(trifluoromethyl)phenyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

  • Compound

    7-(2-(Trifluoromethyl)phenyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

  • Compound

    7-(4-(Trifluoromethyl)phenyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

  • Compound

    7-Phenylpyrazolo[1,5-a]pyrimidine-3-carboxamide

  • Compound

    7-(3-Nitrophenyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

  • Compound

    7-(3-Fluorophenyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

  • Compound

    7-(3-Chlorophenyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

  • Compound

    7-(3,5-Bis(trifluoromethyl)phenyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

  • Compound

    7-(2-Methoxyphenyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

  • Compound

    7-(3-Methoxyphenyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

  • Compound

    7-(Pyridin-3-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

  • Compound

    7-(3-Ethylphenyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

  • Compound

    N-Methyl-7-(3-(trifluoromethyl)phenyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

  • Compound

    N, N-Diethyl-7-(3-(trifluoromethyl)phenyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

  • Compound

    2-Methyl-4-(2-methyl-4-quinolinyl)-phenol

  • Compound

    2-Methyl-4-(naphthalen-1-yl)phenol

  • Compound

    4-Phenyl-1(2H)-phthalazinone

  • Compound

    4-(4-Hydroxy-3-methylphenyl)-1(2H)-phthalazinone

  • Compound

    (E)-3-(Dimethylamino)-(3-(trifluoromethyl)phenyl)prop-2-en-1-one

  • Compound

    4-Chloro-7-methoxy-quinoline

  • Compound

    2-Methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-phenol

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Acknowledgements

This work was funded by the National Institute of Health (NIH R01GM101279 to H.Y.), National Natural Science Foundation of China (No. 21572114 to H.Y. and No. 81401333 to J.L.), the University Key Scientific Research Program Foundation of Henan Province (No. 5201039140120 to S.Z.) and Grant-in-Aid from the Japanese Ministry of Education, Culture, Sports, Science, and Technology (H.T., U.O., and T.S.), CREST JST (T.S.), the Takeda Science Foundation (U.O. and T.S.), the Mochida Memorial Foundation for Medical and Pharmaceutical Research (U.O.), the Naito Foundation (U.O.), and the Daiichi Sankyo Foundation of Life Science (U.O.). We thank X. Wang and W. Wang for their assistance with high-throughput screening and data analysis. We thank J. Dragavon for his assistance with confocal microscopy. We thank Y. Yamada and A. Shinoda for automated data collection at Photon Factory.

Author information

Author notes

    • Shuting Zhang
    • , Zhenyi Hu
    •  & Hiromi Tanji

    These authors contributed equally to this work.

Affiliations

  1. School of Pharmaceutical Sciences, Center of Basic Molecular Science, Key Laboratory of Bioorganic Phosphorus Chemistry and Chemical Biology (Ministry of Education), Department of Chemistry, Tsinghua University, Beijing, China.

    • Shuting Zhang
    • , Shuangshuang Jiang
    •  & Hang Yin
  2. Department of Chemistry and Biochemistry and BioFrontiers Institute, University of Colorado Boulder, Boulder, Colorado, USA.

    • Shuting Zhang
    • , Zhenyi Hu
    • , Nabanita Das
    •  & Hang Yin
  3. School of Chemistry and Chemical Engineering, Henan Normal University, Xinxiang, China.

    • Shuting Zhang
  4. Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan.

    • Hiromi Tanji
    • , Kentaro Sakaniwa
    • , Umeharu Ohto
    •  & Toshiyuki Shimizu
  5. Department of Rheumatology and Clinical Immunology, Peking Union Medical College Hospital and Chinese Academy of Medical Sciences, Key Laboratory of Rheumatology and Clinical Immunology (Ministry of Education), Beijing, China.

    • Jing Li
  6. Department of Orthopedics, Peking Union Medical College Hospital, Beijing, China.

    • Jin Jin
    •  & Yanyan Bian

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Contributions

H.Y. designed the project and supervised data analysis. S.Z. designed the experiments in consultation with H.Y. S.Z. performed the cell line establishment and high throughput screening. S.Z. and Z.H. performed chemical synthesis of compounds, cell culture and cellular inhibition studies. S.Z., Z.H., S.J., and N.D. performed immunoblotting experiments. H.T., K.S., U.O., and T.S. expressed protein, solved the crystal structure, performed isothermal titration calorimetry and gel-filtration experiments. J.L., J.J., and Y.B. contributed to the patient PBMC and synovial tissues extraction. S.J. performed PBMC and synovial cells extraction. S.J. and S.Z. performed primary cell and patient specimen studies. H.Y. and S.Z. wrote the manuscript with input from Z.H., S.J., and H.T.

Competing interests

H.Y., S.Z., and Z.H. have filed a patent application based on the technology reported in this manuscript.

Corresponding authors

Correspondence to Toshiyuki Shimizu or Hang Yin.

Supplementary information

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  1. 1.

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    Supplementary Results, Supplementary Tables 1–4, Supplementary Figures 1–17

  2. 2.

    Life Sciences Reporting Summary

  3. 3.

    Supplementary Note 1

    Chemical synthesis and characterizations