PEPTIDE INHIBITORS OF SRC SH3-SH2-PHOSPHOPROTEIN INTERACTIONS

Activated pp60(c-src) has been implicated in a number of human maligna ncies including colon carcinoma and breast adenocarcinoma. Association of the src SH2 do main 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 t o detect inhibitors of src SH2-phosphoprotein interactions, This assay measures inhibition of binding of a fusion construct (glutathione S-t ransferase src SH3-SH2) with autophosphorylated epidermal growth facto r receptor tyrosine kinase domain, Activities of phosphopeptide segmen ts derived from potential src SH2 cognate phosphoprotein partners were determined, with the focal adhesion kinase-derived segment VSETDDYAE IIDE yielding the highest inhibitory activity, Structure activity stud ies starting from acetyl (Ac)-YEEIE have identified Ac-Y*Y*Y*IE as th e most active compound screened ill the ELISA. This compound is at lea st all-fold more active than the parent peptide Ac-YEEIE. A high reso lution (2 Angstrom) crystal structure of human src SH2 complexed with Ac-YEEIE was obtained and provided a useful framework for understandi ng the structure-activity relationships, Additionally, Ac-YEEIE was a ble to block interactions between src and its cellular phosphoprotein partners in vanadate-treated cell lysates from MDA-MB-468 breast carci noma 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.