Noncompetitive inhibition of hepatocyte growth factor-dependent Met signaling by a phage-derived peptide.
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 beta-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 beta-chain (HGF beta) and competitively inhibited binding to Met with an IC(50) 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 IC(50) of approximately 20 microM. The 2D (1)H-NMR structure of HB10 revealed a beta-hairpin loop stabilized by the disulfide bond and cross-strand pairing of Trp3 and Trp13. HGF beta 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 beta 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.
Tam
Eric M
EM
Department of Protein Engineering, Genentech, Inc., South San Francisco, CA 94080, USA.
Runyon
Steven T
ST
Santell
Lydia
L
Quan
Clifford
C
Yao
Xiaoyi
X
Kirchhofer
Daniel
D
Skelton
Nicholas J
NJ
Lazarus
Robert A
RA
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects; D017228:Hepatocyte Growth Factor / Q000378:metabolism*; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry / Q000494:pharmacology*; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects; D025941:Protein Interaction Mapping; D019859:Proto-Oncogene Proteins c-met / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D012996:Solutions; D013329:Structure-Activity Relationship
D019151:Peptide Library; D010455:Peptides; D011994:Recombinant Proteins; D012996:Solutions; D017228:Hepatocyte Growth Factor; D019859:Proto-Oncogene Proteins c-met