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Peptide inhibitors of src SH3-SH2-phosphoprotein interactions
T Gilmer1, M Rodriguez, S Jordan
1Glaxo Research Institute, Research Triangle Park, North Carolina 27709.
Abstract:
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.
Insights
Researchers developed an assay to find inhibitors of src SH2-phosphoprotein interactions, crucial for understanding cancer signaling. The peptide Ac-Y*Y*Y*IE emerged as a potent inhibitor, blocking interactions in breast cancer cells.
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- Activated pp60c-src is linked to human cancers like colon carcinoma and breast adenocarcinoma.
- The src SH2 domain's interaction with tyrosine-phosphorylated proteins is vital for src-mediated signal transduction.
- Inhibitors of src SH2 domain-phosphoprotein interactions are key to understanding src's role in signaling pathways.
Purpose of the Study:
- To develop an assay for detecting inhibitors of src SH2 domain-phosphoprotein interactions.
- To identify potent inhibitors of src SH2 domain-phosphoprotein interactions.
- To elucidate structure-activity relationships and structural basis of inhibition.
Main Methods:
- Development of an enzyme-linked immunosorbent assay (ELISA) to measure inhibition of binding between a GST-src SH3-SH2 fusion protein and autophosphorylated EGFR TK domain.
- Screening of phosphopeptide segments and their derivatives for inhibitory activity.
- High-resolution (2 Å) crystal structure determination of human src SH2 complexed with Ac-Y*EEIE.
Main Results:
- The focal adhesion kinase-derived peptide VSETDDY*AEIIDE showed the highest inhibitory activity.
- Structure-activity studies identified Ac-Y*Y*Y*IE as the most potent inhibitor in the ELISA, over 20-fold more active than Ac-Y*EEIE.
- Ac-Y*Y*Y*IE successfully blocked src interactions with cellular phosphoproteins in MDA-MB-468 breast carcinoma cell lysates.
Conclusions:
- A robust ELISA was established for identifying inhibitors of src SH2-phosphoprotein interactions.
- Ac-Y*Y*Y*IE is a highly potent inhibitor of src SH2 domain interactions, with its activity elucidated through structure-activity relationship studies and crystal structure analysis.
- While Ac-Y*Y*Y*IE inhibits src interactions in vitro, its inability to abrogate cancer cell proliferation suggests limitations in cell penetration or stability.