Potentiating effect of distant sites in non-phosphorylated cyclic peptide antagonists of the Grb2-SH2 domain.

Without the presence of a phosphotyrosyl group, a phage library derived non-phosphorylated cyclic peptide ligand of Grb2-SH2 domain attributed its high affinity and specificity to well-defined and highly favored interactions of its structural elements with the binding pocket of the protein. We have disclosed a significant compensatory role of the Glu(2-) sidechain for the absence of the phosphate functionality on Tyr(0) in the peptide ligand, cyclo(CH(2)CO-Glu(2-)-Leu-Tyr(0)-Glu-Asn-Val-Gly-Met(5+)-Tyr-Cys)-amide (termed G1TE). In this study, we report the importance of hydrophobic residue at the Tyr+5 site in G1TE. Both acidic and basic amino acid substitutes are disfavored at this position, and replacement of Met with beta-tert-butyl-Ala was found to improve the antagonist properties. Besides, the polarity of the cyclization linkage was implicated as important in stabilizing the favored binding conformation. Oxidation of the thioether linkage into sulfoxide facilitated the binding to Grb2-SH2 markedly. Simultaneous modification of the three distant sites within G1TE provided the best agent with an IC(50) of 220 nM, which is among the most potent non-phosphorous- and non-phosphotyrosine-mimic containing Grb2-SH2 domain inhibitors yet reported. This potent peptidomimetic provides a novel template for the development of chemotherapeutic agents for the treatment of erbB2-related cancer. Biological assays on G1TE(Gla(2-)) in which the original residue of Glu(2-) was substituted by gamma-carboxyglutamic acid (Gla) indicated that it could inhibit the interaction between activated GF receptor and Grb2 protein in cell homogenates of MDA-MB-453 breast cancer cells at the 2 microM level. More significantly, both G1TE(Gla(2-)) alone and the conjugate of G1TE(Gla(2-)) with a peptide carrier can effectively inhibit intracellular association of erbB2 and Grb2 in the same cell lines with IC(50) of 50 and 2 microM, respectively.

[1]  P. Furet,et al.  Structure-based design and synthesis of high affinity tripeptide ligands of the Grb2-SH2 domain. , 1998, Journal of medicinal chemistry.

[2]  G. Milne,et al.  Potent inhibition of Grb2 SH2 domain binding by non-phosphate-containing ligands. , 1999, Journal of medicinal chemistry.

[3]  P. Roller,et al.  Functional preference of the constituent amino acid residues in a phage-library-based nonphosphorylated inhibitor of the Grb2-SH2 domain. , 2001, The journal of peptide research : official journal of the American Peptide Society.

[4]  T. Burke,et al.  Inhibition of Grb2 SH2 domain binding by non-phosphate-containing ligands. 2. 4-(2-Malonyl)phenylalanine as a potent phosphotyrosyl mimetic. , 2000, Journal of medicinal chemistry.

[5]  P. Roller,et al.  Significant compensatory role of position Y-2 conferring high affinity to non-phosphorylated inhibitors of Grb2-SH2 domain. , 1999, Bioorganic & medicinal chemistry letters.

[6]  M. Kraus,et al.  Overexpression of the EGF receptor‐related proto‐oncogene erbB‐2 in human mammary tumor cell lines by different molecular mechanisms. , 1987, The EMBO journal.

[7]  C R King,et al.  Multiple Grb2‐protein complexes in human cancer cells , 1997, International journal of cancer.

[8]  D. Cussac,et al.  The Grb2 adaptor , 1995, FEBS letters.

[9]  D. Cowburn.  Src homology adaptor proteins: more than the sum of the parts? , 1995, Structure.

[10]  T Pawson,et al.  SH2 and SH3 domains , 1993, Current Biology.

[11]  B. Schaffhausen.  SH2 domain structure and function. , 1995, Biochimica et biophysica acta.

[12]  P. Roller,et al.  Global optimization of conformational constraint on non-phosphorylated cyclic peptide antagonists of the Grb2-SH2 domain. , 2003, Bioorganic & medicinal chemistry.

[13]  W C Shakespeare,et al.  SH2 domain inhibition: a problem solved? , 2001, Current opinion in chemical biology.

[14]  Dajun Yang,et al.  Potent Grb2-SH2 domain antagonists not relying on phosphotyrosine mimics. , 2003, Bioorganic & medicinal chemistry letters.

[15]  D. Krag,et al.  Nonphosphorylated Peptide Ligands for the Grb2 Src Homology 2 Domain* , 1997, The Journal of Biological Chemistry.

[16]  T. Schumacher,et al.  Specificity and affinity motifs for Grb2 SH2-ligand interactions , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[17]  P. Roller,et al.  Structural requirements for Tyr in the consensus sequence Y-E-N of a novel nonphosphorylated inhibitor to the Grb2-SH2 domain. , 1999, Biochemical and biophysical research communications.

[18]  Y. Lin,et al.  Conformational and topological requirements of cell-permeable peptide function. , 2009, The journal of peptide research : official journal of the American Peptide Society.

[19]  Gabriel Lopez-Berestein,et al.  Growth inhibition of breast cancer cells by Grb2 downregulation is correlated with inactivation of mitogen-activated protein kinase in EGFR, but not in ErbB2, cells , 1999, Oncogene.

[20]  Rf Rekker,et al.  THE HYDROPHOBIC FRAGMENTAL CONSTANT; AN EXTENSION TO A 1000 DATA POINT SET , 1979 .

[21]  T Pawson,et al.  SH2 and SH3 domains: elements that control interactions of cytoplasmic signaling proteins. , 1991, Science.

[22]  P. Furet,et al.  Structure-based design of compounds inhibiting Grb2-SH2 mediated protein-protein interactions in signal transduction pathways. , 2000, Current pharmaceutical design.