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.