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An allosteric switch for pro-HGF/Met signaling using zymogen activator peptides

Nature Chemical Biology volume 10, pages 567–573 (2014)Cite this article

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Abstract

Stimulation of hepatocyte growth factor (HGF) signaling through the Met receptor is an attractive approach for promoting tissue repair and preventing fibrosis. Using structure-guided peptide phage display combined with an activity-based sorting strategy, we engineered allosteric activators of zymogen-like pro-HGF to bypass proteolytic activation and reversibly stimulate pro-HGF signaling through Met. Biochemical, structural and biological data showed that zymogen activator peptides (ZAPtides) potently and selectively bind the activation pocket within the serine protease–like β-chain of pro-HGF and display titratable activation of pro-HGF–dependent Met signaling, leading to cell survival and migration. To further demonstrate the versatility of our ZAPtide platform, we identified allosteric activators for pro–macrophage stimulating protein and a zymogen serine protease, Protein C, which also provides evidence for target selectivity. These studies reveal that ZAPtides use molecular mimicry of the trypsin-like N-terminal insertion mechanism and establish a new paradigm for selective pharmacological activation of plasminogen-related growth factors and zymogen serine proteases.

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Figure 1: Using the ZAPtide phage library to target the HGF β activation pocket.
Figure 2: Optimizing ZAP.14 using activity-based phage sorting.
Figure 3: ZAP2.3 is a high-affinity cyclic peptide activator of zHGF β.
Figure 4: ZAP2.3 retains the ZAP.14 structure within the disulfide-locked scaffold.
Figure 5: ZAPtides allosterically activate scHGF and control cell survival and migration.
Figure 6: ZAPtides activate the serine protease–like domain of pro-MSP and the zymogen protease Protein C.

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Acknowledgements

We thank gCell, Oligonucleotide Synthesis, DNA Sequencing and the Protein Expression facilities at Genentech for their support. We thank T. Lipari and D. Kirchhofer for providing full-length HGF proteins and helpful discussions. We also thank M. Sagolla and the Center for Advanced Light Microscopy at Genentech for help with migration assays. We acknowledge the use of synchrotron X-ray sources at Advanced Photon Source, Argonne National Laboratory and Stanford Synchrotron Radiation Lightsource, supported by the Department of Energy Office of Science.

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  1. Kyle E Landgraf & Micah Steffek

    Present address: Present addresses: AvidBiotics, Corp., San Francisco, California, USA (K.E.L.) and Novartis Vaccines and Diagnostics, Inc., Emeryville, California, USA (M.S.).,

Authors and Affiliations

  1. Department of Early Discovery Biochemistry, Genentech, Inc., San Francisco, California, USA

    Kyle E Landgraf, Clifford Quan, Jeffrey Tom, Lydia Santell, Henry R Maun & Robert A Lazarus

  2. Department of Structural Biology, Genentech, Inc., San Francisco, California, USA

    Micah Steffek, Christine Yu & Charles Eigenbrot

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Contributions

K.E.L. designed the research, performed the experiments, analyzed data and wrote the paper. M.S. and C.Y. performed the crystallography. C.Q. and J.T. carried out peptide synthesis, purification and characterization. H.R.M. and L.S. generated key HGF, Met, MSP or RON protein reagents. C.E. determined the crystal structures. R.A.L. guided the research, analyzed data and wrote the paper.

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Correspondence to Kyle E Landgraf or Robert A Lazarus.

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All authors are, or were while the research was conducted, employees of Genentech, Inc.

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Landgraf, K., Steffek, M., Quan, C. et al. An allosteric switch for pro-HGF/Met signaling using zymogen activator peptides. Nat Chem Biol 10, 567–573 (2014). https://doi.org/10.1038/nchembio.1533

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