A Sos-derived peptidimer blocks the Ras signaling pathway by binding both Grb2 SH3 domains and displays antiproliferative activity.
With the aim of interrupting the growth factor-stimulated Ras signaling pathway at the level of the Grb2-Sos interaction, a peptidimer, made of two identical proline-rich sequences from Sos linked by a lysine spacer, was designed using structural data from Grb2 and a proline-rich peptide complexed with its SH3 domains. The peptidimer affinity for Grb2 is 40 nM whereas that of the monomer is 16 microM, supporting the dual recognition of both Grb2 SH3 domains by the dimer. At 50 nM, the peptidimer blocks selectively Grb2-Sos complexation in ER 22 (CCL 39 fibroblasts overexpressing epidermal growth factor receptor) cellular extracts. The peptidimer specifically recognizes Grb2 and does not interact with PI3K or Nck, two SH3 domain-containing adaptors. The peptidimer was modified to enter cells by coupling to a fragment of Antennapedia homeodomain. At 10 microM, the conjugate inhibits the Grb2-Sos interaction (100%) and MAP kinase (ERK1 and ERK2) phosphorylation (60%) without modifying cellular growth of ER 22 cells. At the same concentration, the conjugate also inhibits both MAP kinase activation induced by nerve growth factor or epidermal growth factor in PC12 cells, and differentiation triggered by nerve growth factor. Finally, when tested for its antiproliferative activity, the conjugate was an efficient inhibitor of the colony formation of transformed NIH3T3/HER2 cells grown in soft agar, with an IC50 of around 1 microM. Thus, the designed peptidimers appear to be interesting leads to investigate signaling and intracellular processes and for designing selective inhibitors of tumorigenic Ras-dependent processes.
Cussac
D
D
Département de Pharmacochimie Moléculaire et Structurale, INSERM U266-CNRS UMR 8600, UFR des Sciences Pharmaceutiques et Biologiques, 75270 Paris Cedex 06, France.
Vidal
M
M
Leprince
C
C
Liu
W Q
WQ
Cornille
F
F
Tiraboschi
G
G
Roques
B P
BP
Garbay
C
C
D016475:3T3 Cells; D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002455:Cell Division; D002457:Cell Extracts; D019281:Dimerization; D004789:Enzyme Activation; D066246:ErbB Receptors / Q000378:metabolism*; D051380:GRB2 Adaptor Protein; D020662:Guanine Nucleotide Exchange Factors; D051379:Mice; D008969:Molecular Sequence Data; D016716:PC12 Cells; D011506:Proteins / Q000378:metabolism*; D016283:Proto-Oncogene Proteins p21(ras) / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D014162:Transfection; D020663:ras Guanine Nucleotide Exchange Factors; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002457:Cell Extracts; D066246:ErbB Receptors; D051380:GRB2 Adaptor Protein; D020662:Guanine Nucleotide Exchange Factors; D011506:Proteins; D016283:Proto-Oncogene Proteins p21(ras); D018719:Erb-b2 Receptor Tyrosine Kinases; D020663:ras Guanine Nucleotide Exchange Factors; C495949:Grb2 protein, mouse; C495950:Grb2 protein, rat