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Macrocyclic peptide-based inhibition and imaging of hepatocyte growth factor

Nature Chemical Biology volume 15, pages 598–606 (2019)Cite this article

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Abstract

Activation of hepatocyte growth factor (HGF) by proteolytic processing is triggered in cancer microenvironments, and subsequent signaling through the MET receptor is involved in cancer progression. However, the structure of HGF remains elusive, and few small/medium-sized molecules can modulate HGF. Here, we identified HiP-8, a macrocyclic peptide consisting of 12 amino acids, which selectively recognizes active HGF. Biochemical analysis and real-time single-molecule imaging by high-speed atomic force microscopy demonstrated that HiP-8 restricted the dynamic domains of HGF into static closed conformations, resulting in allosteric inhibition. Positron emission tomography using HiP-8 as a radiotracer enabled noninvasive visualization and simultaneous inhibition of HGF–MET activation status in tumors in a mouse model. Our results illustrate the conformational change in proteolytic activation of HGF and its detection and inhibition by a macrocyclic peptide, which may be useful for diagnosis and treatment of cancers.

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Fig. 1: A potent macrocyclic peptide inhibitor of HGF.
Fig. 2: HiP-8’s selectivity for other growth factors.
Fig. 3: HiP-8 binds at interfaces of multiple domains only present in tcHGF.
Fig. 4: HS-AFM observations of tcHGF, scHGF and tcHGF/HiP-8 complex.
Fig. 5: HiP-8-PEG11 detects tcHGF co-localized with MET activation in clinical tissue sections.
Fig. 6: Imaging and targeting of HGF–MET activation in tumors using HiP-8-PEG11 in a mouse model.

Data availability

The authors declare that all data supporting the findings of this study are available within the article and its Supplementary information or from the authors upon reasonable request.

Code availability

The authors declare that no custom code was used in this study. Software code or mathematical algorithm used in this study is available within the article and its Supplementary Information or from the authors upon reasonable request.

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Acknowledgements

This work was supported by World Premier International Research Center Initiative (WPI), MEXT, Japan. This work was supported in part by the A-STEP (Adaptable and Seamless Technology Transfer Program through Target-driven R&D) (grant no. AS262Z) from the Japan Science and Technology Agency (JST), the Medical Research Fund of Takeda Science Foundation, the Mitani Foundation for Research and Development, the Grant-in-Aid for JSPS Scientific Research (C) (no. 16K08544) to K.S., a Grant-in-Aid for JSPS Scientific Research (B) (no. 15K14473) to K.M., Project for Cancer Research and Therapeutic Evolution (P-CREATE) from the Japan Agency for Medical Research and development (AMED) to Y.W., H.M., K.M. and T.P., Basic Science and Platform Technology Program for Innovative Biological Medicine from AMED to H. Suga, a Grant-in-Aid for JSPS Research Activity Start-up (no. 16H06830) to H. Sato, a Grant-in-Aid for JSPS Fellows (no. 23-7727) to K.I., a Grant-in-Aid for JSPS Scientific Research (B) (no. 18K01836) to M.S. This work was performed under the Cooperative Research Program of the Institute for Protein Research, Osaka University (no. CR15-05) and an Extramural Collaborative Research Grant from the Cancer Research Institute (Kanazawa University). We thank T. Ando (Kanazawa University) for providing HS-AFM apparatus, T. Uchihashi (Nagoya University) for providing the analytical software of HS-AFM, Y. Kanayama and R. Zochi for their assistance in 64Cu production, Y. Wada and E. Hayashinaka for their assistance in reconstructing the PET images and Enago (www.enago.jp) for the English language review.

Author information

Author notes

  1. Toby Passioura

    Present address: Sydney Analytical, The University of Sydney, Sydney New, South Wales, Australia

  2. These authors contributed equally: Toby Passioura, Hiroki Sato.

Authors and Affiliations

  1. Division of Tumor Dynamics and Regulation, Cancer Research Institute, Kanazawa University, Kanazawa, Japan

    Katsuya Sakai, Hiroki Sato & Kunio Matsumoto

  2. WPI-Nano Life Science Institute (WPI-NanoLSI), Kanazawa University, Kanazawa, Japan

    Katsuya Sakai, Hiroki Sato, Seiji Yano, Mikihiro Shibata & Kunio Matsumoto

  3. Department of Chemistry, Graduate School of Science, The University of Tokyo, Tokyo, Japan

    Toby Passioura, Kenichiro Ito & Hiroaki Suga

  4. Mathematical and Physical Sciences, Graduate School of Natural Science & Technology, Kanazawa University, Kanazawa, Japan

    Hiroki Furuhashi

  5. Laboratory of Protein Synthesis and Expression, Institute for Protein Research, Osaka University, Osaka, Japan

    Masataka Umitsu & Junichi Takagi

  6. Medicine/New Industry Creation Hatchery Center, Tohoku University, Sendai, Japan

    Yukinari Kato

  7. Laboratory for Molecular Delivery and Imaging Technology, RIKEN Center for Biosystems Dynamics Research, Kobe, Japan

    Hidefumi Mukai, Shota Warashina & Maki Zouda

  8. Laboratory for Pathophysiological and Health Science, RIKEN Center for Biosystems Dynamics Research, Kobe, Japan

    Yasuyoshi Watanabe

  9. Division of Medical Oncology, Cancer Research Institute, Kanazawa University, Kanazawa, Japan

    Seiji Yano

  10. High-speed AFM for Biological Application Unit, Institute for Frontier Science Initiative, Kanazawa University, Kanazawa, Japan

    Mikihiro Shibata

  11. Tumor Microenvironment Research Unit, Institute for Frontier Science Initiative, Kanazawa University, Kanazawa, Japan

    Kunio Matsumoto

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Contributions

K.S., H. Suga and K.M. conceived and designed the study. K.S. expressed and purified HGF and HGF fragment proteins. M.U. and J.T. expressed and purified Xa-modified scHGF, tcHGF, K2–4–SP and K4–SP proteins. K.I., K.S. and T.P. performed RaPID and peptide synthesis. K.S., H. Sato and K.I performed cell-based assays. K.S. and K.I. performed biochemical analysis. H. Sato performed immunohistochemistry and the in vivo efficacy studies. M.S. and H.F. performed HS-AFM observations. H.M., H. Sato, S.W., M.Z. and Y.W. designed and performed PET studies. Y.K. developed t5A11. S.Y. developed HGF-expressing PC-9. All authors analyzed the experimental data, discussed the results and were involved in preparation of the manuscript.

Corresponding authors

Correspondence to Hiroaki Suga or Kunio Matsumoto.

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Supplementary information

Supplementary Information

Supplementary Figures 1–21

Reporting Summary

Supplementary Video 1 HS-AFM analysis of NK1.

NK1 consisting of two domains attached to the AP-mica surface. Pixel sizes: 120 × 72 pixels. 80 × 47 nm2. This experiment was repeated three times independently with similar results.

Supplementary Video 2 HS-AFM analysis of NK4.

NK4 attached to the AP-mica surface predominantly through NK1. NK4 had additional flexible domains corresponding to K2–K4, which did not interact with the AP-mica surface. Pixel sizes: 120 × 72 pixels. 80 × 47 nm2. This experiment was repeated three times independently with similar results.

Supplementary Video 3 HS-AFM analysis of SP.

SP sparingly bound to the AP-mica surface. Pixel sizes: 180 × 108 pixels. 200 × 120 nm2. This experiment was repeated three times independently with similar results.

Supplementary Video 4 HS-AFM analysis of scHGF.

scHGF attached to the AP-mica surface predominantly through NK1. scHGF had additional flexible domains corresponding to K2–SP, which did not interact with the AP-mica surface. The SP domain was bended toward N-terminus in scHGF. Pixel sizes: 120 × 72 pixels. 80 × 47 nm2. This experiment was repeated three times independently with similar results.

Supplementary Video 5 HS-AFM analysis of tcHGF.

tcHGF attached to the AP-mica surface predominantly through NK1. tcHGF had additional flexible domains corresponding to K2–SP, which did not interact with the AP-mica surface. The SP domain was more open toward N-terminus in tcHGF compared to scHGF. Pixel sizes: 120 × 72 pixels. 80 × 47 nm2. This experiment was repeated three times independently with similar results.

Supplementary Video 6 HS-AFM analysis of tcHGF/HiP-8 complex (Shape 1).

A representative tcHGF/HiP-8 complex on the AP-mica surface showing a static elongated shape (Shape 1). Pixel sizes: 120 × 72 pixels. 80 × 47 nm2. This experiment was repeated three times independently with similar results.

Supplementary Video 7 HS-AFM analysis of tcHGF/HiP-8 complex (Shape 2).

A representative tcHGF/HiP-8 complexe on the AP-mica surface showing a static closed circular shape (Shape 2). Pixel sizes: 120 × 72 pixels. 80 × 47 nm2. This experiment was repeated three times independently with similar results.

Supplementary Video 8 HS-AFM analysis of scHGF treated with HiP-8.

A representative scHGF treated with HiP-8 on the AP-mica surface showing an unchanged molecular shape and flexible conformation compared with free scHGF. Pixel sizes: 120 × 72 pixels. 80 × 47 nm2. This experiment was repeated twice independently with similar results.

Supplementary Video 9 HS-AFM analysis of tcHGF/t5A11 antibody complex.

Three representative tcHGF/t5A11 antibody complexes on the AP-mica surface. t5A11 bound to tcHGF between NK1 and SP domains. tcHGF molecules maintain their flexible conformation. Pixel sizes: 120 × 72 pixels. 80 × 47 nm2. This experiment was repeated twice independently with similar results.

Supplementary Video 10 Dynamic maximum intensity projection PET image of mice bearing PC-9 tumors intravenously administered with 64Cu-labeled HiP-8-PEG11.

This experiment was repeated three times independently with similar results.

Supplementary Video 11 Rotating maximum intensity projection image along with z axis of mice bearing PC-9 tumors intravenously administered with 64Cu-labeled HiP-8-PEG11 at 90 min.

This experiment was repeated three times independently with similar results.

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Sakai, K., Passioura, T., Sato, H. et al. Macrocyclic peptide-based inhibition and imaging of hepatocyte growth factor. Nat Chem Biol 15, 598–606 (2019). https://doi.org/10.1038/s41589-019-0285-7

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