Peptide inhibitors of src SH3-SH2-phosphoprotein interactions

Activated pp60c-src has been implicated in a number of human malignancies including colon carcinoma and breast adenocarcinoma. Association of the src SH2 domain with tyrosine-phosphorylated proteins plays a role in src-mediated signal transduction. Inhibitors of src SH2 domain-phosphoprotein interactions are, thus, of great interest in defining the role(s) of src in signal transduction pathways. To facilitate such studies, an enzyme-linked immunosorbent assay (ELISA) was developed to detect inhibitors of src SH2-phosphoprotein interactions. This assay measures inhibition of binding of a fusion construct (glutathione S-transferase src SH3-SH2) with autophosphorylated epidermal growth factor receptor tyrosine kinase domain. Activities of phosphopeptide segments derived from potential src SH2 cognate phosphoprotein partners were determined, with the focal adhesion kinase-derived segment VSETDDY*AEIIDE yielding the highest inhibitory activity. Structure activity studies starting from acetyl (Ac)-Y*EEIE have identified Ac-Y*Y*Y*IE as the most active compound screened in the ELISA. This compound is at least 20-fold more active than the parent peptide Ac-Y*EEIE. A high resolution (2 A) crystal structure of human src SH2 complexed with Ac-Y*EEIE was obtained and provided a useful framework for understanding the structure-activity relationships. Additionally, Ac-Y*EEIE was able to block interactions between src and its cellular phosphoprotein partners in vanadate-treated cell lysates from MDA-MB-468 breast carcinoma cells. However, it is unable to abrogate proliferation of MDA-MB-468 cells in culture, presumably because of poor cell penetration and/or lability of the phosphate group on tyrosine.