Enhancing the Bioactivity of Bicyclic Peptides Targeted to Grb7-SH2 by Restoring Cell Permeability

Abstract

The development of peptide inhibitors against intracellular targets depends upon the dual challenge of achieving a high affinity and specificity for the target and maintaining cellular permeability for biological activity. Previous efforts to develop bicyclic peptides targeted to the Grb7 signalling protein implicated in HER2+ve cancer progression have resulted in improved affinity. However, these same peptides demonstrated a lowered activity due to their decreased ability to penetrate cell membranes. Here, we report the testing of a new series of bicyclic G7 peptides designed to possess improved bioactivity. We discovered that the incorporation of two amino acids (Phe-Pro, Phe-Trp or Phe-Arg) within the bicyclic peptide framework maintains an enhanced binding affinity for the Grb7-SH2 domain compared to that of the first-generation monocyclic peptide G7-18NATE. Structure determination using X-ray crystallography revealed that the mode of binding by the expanded bicyclic G7 peptide is analogous to that of G7-18NATE. Interestingly, while the bicyclic peptide containing Phe-Trp did not display the highest affinity for Grb7-SH2 in the series, it was the most potent inhibitor of HER2+ve SKBR3 breast cancer cell migration when coupled to Penetratin. Together, this demonstrates that peptide flexibility as well as the amino acid tryptophan can play important roles in the uptake of peptides into the cell.

Up-regulated GRB7 protein in gastric cancer cells correlates with clinical properties and increases proliferation and stem cell properties

“…The PH domain of GRB7 has been shown to bind Filamin-A, an actin-binding protein involved in the regulation and control of membrane fold formation ( 23 ). The SH2 domain of GRB7 has been shown to bind to various phosphotyrosine motifs of its binding partners, such as EGFR, ERBB2, Ret, platelet-derived growth factor receptor (PDGFR), FAK, etc ( 24 – 26 ). In addition, the interaction between the SH2 domain of GRB7 and the phosphotyrosine motif of the signal protein leads to different cellular outcomes or tumorigenic functions ( 27 ).…”

Peptide therapeutics in the management of metastatic cancers

“…Interaction leads to phosphorylation and activation of Grb7 followed by mediation of cascades enhancing cell migration, invasion, growth and proliferation. 120 The SH2 domain of this protein has been targeted by the 11 residue peptide G7-18NATE. This cyclic peptide although highly active was unable to appropriately penetrate the cell and thus was co-delivered by fusion with the CPP, penetratin.…”

“… 123 Recently, further modifications have been made to allow better permeability with increased affinity and specificity. 120 It has now been recognised that many proteins promoting cell-survival and metastasis may contain cryptic segments that serve as growth inhibiting domains. These domains may be naturally exposed due to the presence of high concentrations of specific ligands in the microenvironment or due to other factors.…”