Discovery of thioether-bridged cyclic pentapeptides binding to Grb2-SH2 domain with high affinity.

Blocking the interaction between phosphotyrosine (pTyr)-containing activated receptors and the Src homology 2 (SH2) domain of the growth factor receptor-bound protein 2 (Grb 2) is considered to be an effective and non-cytotoxic strategy to develop new anti-proliferate agents due to its potential to shut down the Ras activation pathway. In this study, a series of phosphotyrosine containing cyclic pentapeptides were designed and synthesized based upon the phage library derived cyclopeptide, G1TE. A comprehensive SAR study was also carried out to develop potent Grb2-SH2 domain antagonists based upon this novel template. With both the peptidomimetic optimization of the amino acid side-chains and the constraint of the backbone conformation guided by molecular modeling, we developed several potent antagonists with low micromolar range binding affinity, such as cyclic peptide 15 with an K(d)=0.359microM, which is providing a novel template for the development of Grb2-SH2 domain antagonists as potential therapeutics for certain cancers.

Structure-based design of potent Grb2-SH2 domain antagonists not relying on phosphotyrosine mimics.

Development of Grb2-SH2 domain antagonists is considered to be an effective and non-cytotoxic strategy to develop new antiproliferative agents because of their potential to shut down the Ras signaling pathway. We developed a concise route for the efficient synthesis of G1TE analogs on solid phase. Using this route, a series of cyclic peptides that do not rely on phosphotyrosine or its mimics were designed and synthesized based upon the phage library-derived cyclopeptide, G1TE. Considering that Gly7 plays prominent roles for G1TE binding to the Grb2-SH2 domain, we introduced different amino acids in the 7th position. The D-Ala7-containing peptide 3 demonstrates improved binding affinity by adopting favorable conformation for protein binding. This can be rationalized by molecular modeling. The optimization at the Leu2 position was also studied, and the resulting cyclopeptides exhibited remarkably improved binding affinity. Based upon these global modifications, a highly potent peptide ligand 9 was discovered with a Kd = 17 nM, evaluated by Biacore binding assay. This new analog is one of the most potent non-phosphorus-containing Grb2-SH2 antagonists reported to date. This potent peptidomimetic provides a new template for the development of non-pTyr containing Grb2-SH2 domain antagonists and acts as a chemotherapeutic lead for the treatment of erbB2-related cancer.