Targeted disruption of the hepatocyte growth factor β-chain/Met interface inhibits HGF-dependent Met signaling
Authors: Eric Tam, Steven Runyon, Lydia Santell, Michael Lipari, Clifford Quan, Xiaoyi Yao, Nicholas Skelton, Daniel Kirchhofer, Robert Lazarus
Journal: Cancer Research (2007)

AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA

2524 

Hepatocyte growth factor (HGF) binds the receptor tyrosine kinase Met to trigger a complex set of intracellular signaling pathways. Together, HGF and Met regulate cell proliferation, motility and morphogenesis, key processes that contribute to invasive tumor growth and angiogenesis. Previously, we have shown that upon proteolytic activation, there is a conformational rearrangement of the serine protease-like HGF β-chain (HGF β) that reveals a critical Met binding site required for signaling (1,2). Thus, we hypothesized that by targeting HGF at the β-chain/Met interface, we could inhibit signal transduction in the HGF/Met pathway. To screen for inhibitors, linear and disulfide constrained naive peptide libraries displayed on phage were sorted against HGF and HGF β. Following multiple rounds of enrichment, we identified a disulfide-constrained 15-mer peptide termed P10, originating from the library X4CX5CX4 (where X denotes all 20 amino acids). Peptide P10 binds to the HGF β-chain and blocks solution phase binding of HGF β to Met-IgG with an IC50 of 450 nM. In MDA-MB435 breast carcinoma cells, P10 (100 μM) reduced HGF-dependent Met phosphorylation by ~ 70% compared to HGF alone. Similarly, P10 also reduced downstream AKT and p44/42 phosphorylation by 74% and 69%, respectively. In a transwell assay, P10 was able to inhibit the migration of MDA-MB435 cells in response to HGF (1 nM) with an IC50 of ~ 20 μM. P10-S, an analog of P10 where the two cysteines were replaced with serines, was negative in all binding and activity assays, demonstrating the importance of the disulfide constraints on the tertiary structure for activity. The solution structure of P10 was solved by 2D 1H-NMR and revealed a β-hairpin loop, which is stabilized by the disulfide bond and hydrophobic stacking of two critical tryptophans. The N- and C-termini are in relatively close proximity and contain the majority of positions important for both binding and inhibition, determined by analysis of alanine scan analogs of P10. Notably, P10 does not bind murine HGF β, which shares ~ 90% identity to the human protein. Using various murine/human HGF β chimeras, we were able to map the binding epitope of P10 to surface residues of HGF β located near the Met interface. Currently, we are confirming this by generating crystals of P10 in complex with HGF β for X-ray analysis.

References 

1. D Kirchhofer, X Yao, M Peek, C Eigenbrot, MT Lipari, KL Billeci, HR Maun, P Moran, L Santell, C Wiesmann, and RA Lazarus. Structural and functional basis of the serine protease-like hepatocyte growth factor β-chain in Met binding and signaling. J Biol Chem (2004) 279: 39915 - 39924.

2. J Stamos, RA Lazarus, X Yao, D Kirchhofer, C Wiesmann. Crystal structure of the HGF β-chain in complex with the Sema domain of the Met receptor. EMBO J (2004) 23: 2325 - 2335.