Noncompetitive inhibition of hepatocyte growth factor-dependent Met signaling by a phage-derived peptide |
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Authors: | Tam Eric M Runyon Steven T Santell Lydia Quan Clifford Yao Xiaoyi Kirchhofer Daniel Skelton Nicholas J Lazarus Robert A |
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Institution: | 1 Department of Protein Engineering, Genentech, Inc., South San Francisco, CA 94080, USA 2 Department of Small Molecule Drug Discovery, Genentech, Inc., South San Francisco, CA 94080, USA |
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Abstract: | Dysregulation of hepatocyte growth factor (HGF)-induced signaling via its receptor tyrosine kinase Met results in tumor progression and metastasis. To initiate signaling, pro-HGF must be proteolytically activated to reveal a secondary Met binding site within the serine protease-like β-chain of HGF. Although HGF/Met is a large complex, we sought to discover relatively small antagonists that might interfere with this critical Met binding region. Pools of disulfide-constrained random peptide libraries displayed on phage were selected for binding to HGF, ultimately resulting in a disulfide-constrained 15-mer peptide (VNWVCFRDVGCDWVL) termed HB10, which bound to the recombinant human HGF β-chain (HGF β) and competitively inhibited binding to Met with an IC50 of 450 nM. In MDA-MB435 cells, HB10 reduced HGF-dependent Met phosphorylation by 70%, and phosphorylation of downstream kinases AKT and ERK1/ERK2 by 74% and 69%, respectively. Addition of HB10 also inhibited HGF-dependent migration of these cells with an IC50 of ∼ 20 μM. The 2D 1H-NMR structure of HB10 revealed a β-hairpin loop stabilized by the disulfide bond and cross-strand pairing of Trp3 and Trp13. HGF β mutants deficient in Met binding also have reduced HB10 binding, suggesting an overlapping binding site. Notably HB10 did not inhibit full length HGF binding to Met. Thus steric hindrance of the interaction between HGF β domain binding to Met is sufficient for inhibiting full-length HGF-dependent Met signaling and cell migration that is consistent with a noncompetitive inhibitory mechanism of Met signal transduction. |
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Keywords: | HGF hepatocyte growth factor K1-K4 Kringle domains 1-4 pro-HGF single chain precursor form of HGF scHGF a noncleavable single chain form of HGF (R424A/R494E) HGF β serine protease-like domain of HGF (Val495-Ser728) scHGF β a noncleavable single chain form of HGF β (Asn479-Ser728 R494E) HRG β1 heregulin β1 PBS phosphate-buffered saline BSA bovine serum albumin ELISA enzyme-linked immunosorbent assay HRP horseradish peroxidase NOE nuclear Overhauser effect |
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