Cussac D, Vidal M, Leprince C, Liu W Q, Cornille F, Tiraboschi G, Roques B P, Garbay C
Département de Pharmacochimie Moléculaire et Structurale, INSERM U266-CNRS UMR 8600, UFR des Sciences Pharmaceutiques et Biologiques, 75270 Paris Cedex 06, France.
FASEB J. 1999 Jan;13(1):31-8. doi: 10.1096/fasebj.13.1.31.
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.
为了在Grb2 - Sos相互作用水平上中断生长因子刺激的Ras信号通路，利用来自Grb2和与其SH3结构域复合的富含脯氨酸肽的结构数据，设计了一种肽二聚体，它由两个来自Sos的相同富含脯氨酸序列通过赖氨酸间隔连接而成。肽二聚体对Grb2的亲和力为40 nM，而单体的亲和力为16 μM，这支持了二聚体对Grb2两个SH3结构域的双重识别。在50 nM时，肽二聚体在ER 22（过表达表皮生长因子受体的CCL 39成纤维细胞）细胞提取物中选择性地阻断Grb2 - Sos复合。该肽二聚体特异性识别Grb2，不与PI3K或Nck（两种含SH3结构域的衔接蛋白）相互作用。通过与触角足同源结构域的一个片段偶联，对肽二聚体进行修饰使其能够进入细胞。在10 μM时，该偶联物抑制Grb2 - Sos相互作用（100%）和MAP激酶（ERK1和ERK2）磷酸化（60%），而不改变ER 22细胞的细胞生长。在相同浓度下，该偶联物还抑制PC12细胞中神经生长因子或表皮生长因子诱导的MAP激酶激活以及神经生长因子触发的分化。最后，当测试其抗增殖活性时，该偶联物是软琼脂中生长的转化NIH3T3/HER2细胞集落形成的有效抑制剂，IC50约为1 μM。因此，所设计的肽二聚体似乎是研究信号传导和细胞内过程以及设计致癌性Ras依赖性过程的选择性抑制剂的有趣先导物。
