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 pat hway at the level of the Grb2-Sos interaction, a peptidimer, made of two id entical proline-rich sequences from Sos linked by a lysine spacer, was desi gned using structural data from Grb2 and a proline-rich peptide complexed w ith its SH3 domains. The peptidimer affinity for Grb2 is 40 nM whereas that of the monomer is 16 mu M, supporting the dual recognition of both Grb2 SH 3 domains by the dimer. At 50 nM, the peptidimer blocks selectively Grb2-So s 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 ada ptors. The peptidimer was modified to enter cells by coupling to a fragment of Antennapedia homeodomain. At 10 mu M, the conjugate inhibits the Grb2-S os 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 g rowth factor or epidermal growth factor in PC12 cells, and differentiation triggered by nerve growth factor. Finally,when tested for its antiprolifera tive activity, the conjugate was an efficient inhibitor of the colony forma tion of transformed NIH3T3/HER2 cells grown in soft agar, with an IC50 of a round 1 yM. Thus, the designed peptidimers appear to be interesting leads t o investigate signaling and intracellular processes and for designing selec tive inhibitors of tumorigenic Ras-dependent processes.