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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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.
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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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.
Supplementary Figures 1–21
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.
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.
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.
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.
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.
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.
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.
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.
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.