Macrocyclic peptide-based inhibition and imaging of hepatocyte growth factor

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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

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.

References

  1. 1.

    Naka, D. et al. Activation of hepatocyte growth factor by proteolytic conversion of a single chain form to a heterodimer. J. Biol. Chem. 141, 20114–20119 (1992).

    Google Scholar 

  2. 2.

    Kataoka, H. et al. Activation of hepatocyte growth factor/scatter factor in colorectal carcinoma. Cancer Res. 60, 6148–6159 (2000).

    CAS  PubMed  Google Scholar 

  3. 3.

    Kawaguchi, M. & Kataoka, H. Mechanisms of hepatocyte growth factor activation in cancer tissues. Cancers 6, 1890–1904 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  4. 4.

    K Trusolino, L., Bertotti, A. & Comoglio, P. M. MET signalling: principles and functions in development, organ regeneration and cancer. Nat. Rev. Mol. Cell. Biol. 11, 834–848 (2010).

    Article  Google Scholar 

  5. 5.

    Gherardi, E., Birchmeier, W., Birchmeier, C. & Woude, G. V. Targeting MET in cancer: rationale and progress. Nat. Rev. Cancer 12, 89–103 (2012).

    CAS  PubMed  Article  Google Scholar 

  6. 6.

    Sakai, K., Aoki, S. & Matsumoto, K. Hepatocyte growth factor and MET in drug discovery. J. Biochem. 157, 271–284 (2015).

    CAS  PubMed  Article  Google Scholar 

  7. 7.

    Burggraaf, J. et al. Detection of colorectal polyps in humans using an intravenously administered fluorescent peptide targeted against c-MET. Nat. Med. 21, 955–961 (2015).

    CAS  PubMed  Article  Google Scholar 

  8. 8.

    Han, Z. et al. Analysis of progress and challenges for various patterns of c-MET-targeted molecular imaging: a systematic review. EJNMMI Res. 7, 41 (2017).

    PubMed  PubMed Central  Article  Google Scholar 

  9. 9.

    Engelman, J. A. et al. MET amplification leads to gefitinib resistance in lung cancer by activating ERBB3 signaling. Science 16, 1039–1043 (2007).

    Article  Google Scholar 

  10. 10.

    Yano, S. et al. Hepatocyte growth factor induces gefitinib resistance of lung adenocarcinoma with epidermal growth factor receptor-activating mutations. Cancer Res. 68, 9479–9487 (2008).

    CAS  PubMed  Article  Google Scholar 

  11. 11.

    Straussman, R. et al. Tumour micro-environment elicits innate resistance to RAF inhibitors through HGF secretion. Nature 487, 500–504 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  12. 12.

    Corso, S. & Giordano, S. Cell-autonomous and non-cell-autonomous mechanisms of HGF/MET-driven resistance to targeted therapies: from basic research to a clinical perspective. Cancer Discov. 3, 978–992 (2013).

    CAS  PubMed  Article  Google Scholar 

  13. 13.

    Cecchi, F., Rabe, D. C. & Bottaro, D. P. Targeting the HGF/MET signaling pathway in cancer therapy. Expert Opin. Ther. Targets 16, 553–572 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  14. 14.

    Furlan, A. et al. Thirty years of research on met receptor to move a biomarker from bench to bedside. Cancer Res. 74, 6737–6744 (2014).

    CAS  PubMed  Article  Google Scholar 

  15. 15.

    Peinado, H. et al. Melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through MET. Nat. Med. 18, 883–891 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  16. 16.

    Bendinelli, P., Maroni, P., Matteucci, E. & Desiderio, M. A. Epigenetic regulation of HGF/MET receptor axis is critical for the outgrowth of bone metastasis from breast carcinoma. Cell Death Dis. 8, e2578 (2017).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  17. 17.

    Matsumoto, K. et al. Hepatocyte growth factor/MET in cancer progression and biomarker discovery. Cancer Sci. 108, 296–307 (2017).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  18. 18.

    Grootjans, W. et al. PET in the management of locally advanced and metastatic NSCLC. Nat. Rev. Clin. Oncol. 12, 395–407 (2015).

    PubMed  Article  Google Scholar 

  19. 19.

    Stamos, J. et al. Crystal structure of the HGF beta-chain in complex with the sema domain of the met receptor. EMBO J. 23, 2325–2335 (2004).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  20. 20.

    Kirchhofer, D. et al. Structural and functional basis of the serine protease-like hepatocyte growth factor beta-chain in met binding and signaling. J. Biol. Chem. 279, 39915–39924 (2004).

    CAS  PubMed  Article  Google Scholar 

  21. 21.

    Kirchhofer, D. et al. Utilizing the activation mechanism of serine proteases to engineer hepatocyte growth factor into a met antagonist. Proc. Natl Acad. Sci. USA 104, 5306–5311 (2007).

    CAS  PubMed  Article  Google Scholar 

  22. 22.

    Landgraf, K. E. et al. An allosteric switch for pro-HGF/MET signaling using zymogen activator peptides. Nat. Chem. Biol. 10, 567–573 (2014).

    CAS  PubMed  Article  Google Scholar 

  23. 23.

    Gherardi, E. et al. Structural basis of hepatocyte growth factor/scatter factor and MET signalling. Proc. Natl Acad. Sci. USA 103, 4046–4051 (2006).

    CAS  PubMed  Article  Google Scholar 

  24. 24.

    Winter, A. et al. Developing antagonists for the MET-HGF/SF protein–protein interaction using a fragment-based approach. Mol. Cancer Ther. 15, 3–14 (2016).

    CAS  PubMed  Article  Google Scholar 

  25. 25.

    Tam, E. M. et al. Noncompetitive inhibition of hepatocyte growth factor-dependent MET signaling by a phage-derived peptide. J. Mol. Biol. 385, 79–90 (2009).

    CAS  PubMed  Article  Google Scholar 

  26. 26.

    Valeur, E. et al. New modalities for challenging targets in drug discovery. Angew. Chem. Int. Ed. Engl. 56, 10294–10323 (2017).

    CAS  PubMed  Article  Google Scholar 

  27. 27.

    Dougherty, P. G., Qian, Z. & Pei, D. Macrocycles as protein-protein interaction inhibitors. Biochem. J. 474, 1109–1125 (2017).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  28. 28.

    Villar, E. A. et al. How proteins bind macrocycles. Nat. Chem. Biol. 10, 723–731 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  29. 29.

    Millward, S. W., Fiacco, S., Austin, R. J. & Roberts, R. W. Design of cyclic peptides that bind protein surfaces with antibody-like affinity. ACS Chem. Biol. 2, 625–634 (2007).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  30. 30.

    Heinis, C., Rutherford, T., Freund, S. & Winter, G. Phage-encoded combinatorial chemical libraries based on bicyclic peptides. Nat. Chem. Biol. 5, 502–507 (2009).

    CAS  PubMed  Article  Google Scholar 

  31. 31.

    Shi, Y., Yang, X., Garg, N. & Van Der Donk, W. A. Production of lantipeptides in Escherichia coli. J. Am. Chem. Soc. 133, 2338–2341 (2011).

    CAS  PubMed  Article  Google Scholar 

  32. 32.

    Schlippe, Y. V., Hartman, M. C., Josephson, K. & Szostak, J. W. In vitro selection of highly modified cyclic peptides that act as tight binding inhibitors. J. Am. Chem. Soc. 134, 10469–10477 (2012).

    PubMed  Article  Google Scholar 

  33. 33.

    Li, Y. et al. Versatile protein recognition by the encoded display of multiple chemical elements on a constant macrocyclic scaffold. Nat. Chem. 10, 441–448 (2018).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  34. 34.

    Kale, S. S. et al. Cyclization of peptides with two chemical bridges affords large scaffold diversities. Nat. Chem. 10, 715–723 (2018).

    CAS  PubMed  Article  Google Scholar 

  35. 35.

    Passioura, T. & Suga, H. Flexizyme-mediated genetic reprogramming as a tool for noncanonical peptide synthesis and drug discovery. Chemistry. 19, 6530–6536 (2013).

    CAS  PubMed  Article  Google Scholar 

  36. 36.

    Josephson, K., Ricardo, A. & Szostak, J. W. mRNA display: from basic principles to macrocycle drug discovery. Drug Discov. Today 19, 388–399 (2014).

    CAS  PubMed  Article  Google Scholar 

  37. 37.

    Goto, Y., Katoh, T. & Suga, H. Flexizymes for genetic code reprogramming. Nat. Protoc. 6, 779–790 (2011).

    CAS  PubMed  Article  Google Scholar 

  38. 38.

    Ito, K. et al. Artificial human met agonists based on macrocycle scaffolds. Nat. Commun. 6, 6372 (2015).

    Article  Google Scholar 

  39. 39.

    Veronese, F. M. & Pasut, G. PEGylation, successful approach to drug delivery. Drug Discov. Today 10, 1451–1458 (2005).

    CAS  PubMed  Article  Google Scholar 

  40. 40.

    Holmes, O. et al. Insights into the structure/function of hepatocyte growth factor/scatter factor from studies with individual domains. J. Mol. Biol. 367, 395–408 (2007).

    CAS  PubMed  Article  Google Scholar 

  41. 41.

    Lokker, N. A. et al. Structure-function analysis of hepatocyte growth-factor-identification of variants that lack mitogenic activity yet retain high-affinity receptor-binding. EMBO J. 11, 2503–2510 (1992).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  42. 42.

    Matsumoto, K., Kataoka, H., Date, K. & Nakamura, T. Cooperative interaction between α- and β-chains of hepatocyte growth factor on c-MET receptor confers ligand-induced receptor tyrosine phosphorylation and multiple biological responses. J. Biol. Chem. 273, 22913–22920 (1998).

    CAS  PubMed  Article  Google Scholar 

  43. 43.

    Umitsu, M. et al. Probing conformational and functional states of human hepatocyte growth factor by a panel of monoclonal antibodies. Sci. Rep. 6, 33149 (2016).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  44. 44.

    Shibata, M. et al. High-speed atomic force microscopy shows dynamic molecular processes in photoactivated bacteriorhodopsin. Nat. Nanotech. 5, 208–212 (2010).

    CAS  Article  Google Scholar 

  45. 45.

    Uchihashi, T., Iino, R., Ando, T. & Noji, H. High-speed atomic force microscopy reveals rotary catalysis of rotorless F1-ATPase. Science 333, 755–758 (2011).

    CAS  PubMed  Article  Google Scholar 

  46. 46.

    Ando, T., Uchihashi, T. & Scheuring, S. Filming biomolecular processes by high-speed atomic force microscopy. Chem. Rev. 114, 3120–3188 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  47. 47.

    Shibata, M. et al. Real-space and real-time dynamics of CRISPR-Cas9 visualized by high-speed atomic force microscopy. Nat. Commun. 8, 1430 (2017).

    PubMed  PubMed Central  Article  Google Scholar 

  48. 48.

    Chirgadze, D. Y. et al. Crystal structure of the NK1 fragment of HGF/SF suggests a novel mode for growth factor dimerization and receptor binding. Nat. Struct. Biol. 6, 72–79 (1999).

    CAS  PubMed  Article  Google Scholar 

  49. 49.

    Yu, H. et al. Macrocycle peptides delineate locked-open inhibition mechanism for microorganism phosphoglycerate mutases. Nat. Commun. 8, 14932 (2017).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  50. 50.

    Wu, A. M. & Olafsen, T. Antibodies for molecular imaging of cancer. Cancer J. 14, 191–197 (2008).

    CAS  PubMed  Article  Google Scholar 

  51. 51.

    Fukuta, K., Matsumoto, K. & Nakamura, T. Multiple biological responses are induced by glycosylation-deficient hepatocyte growth factor. Biochem. J. 388, 555–562 (2005).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  52. 52.

    Suzuki, Y. et al. Inhibition of MET/HGF receptor and angiogenesis by NK4 leads to suppression of tumor growth and migration in malignant pleural mesothelioma. Int. J. Cancer 127, 1948–1957 (2010).

    CAS  PubMed  Article  Google Scholar 

  53. 53.

    Isozaki, H. et al. Non-small cell lung cancer cells acquire resistance to the ALK inhibitor alectinib by activating alternative receptor tyrosine kinases. Cancer Res. 76, 1506–1516 (2016).

    CAS  PubMed  Article  Google Scholar 

  54. 54.

    Mukai, H., Wada, Y. & Watanabe, Y. The synthesis of 64Cu-chelated porphyrin photosensitizers and their tumor-targeting peptide conjugates for the evaluation of target cell uptake and PET image-based pharmacokinetics of targeted photodynamic therapy agents. Ann. Nucl. Med. 27, 625–639 (2013).

    CAS  PubMed  Article  Google Scholar 

  55. 55.

    Zeng, D. et al. New cross-bridged cyclam derivative CB-TE1K1P, an improved bifunctional chelator for copper radionuclides. Chem. Commun. 50, 43–45 (2014).

    Article  Google Scholar 

  56. 56.

    Mukai, H. et al. Quantitative evaluation of the improvement in the pharmacokinetics of a nucleic acid drug delivery system by dynamic PET imaging with (18)F-incorporated oligodeoxynucleotides. J. Control. Release 180, 92–99 (2014).

    CAS  PubMed  Article  Google Scholar 

Download references

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

Affiliations

Authors

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.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

Further reading

Search

Sign up for the Nature Briefing newsletter for a daily update on COVID-19 science.
Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing