Design and characterization of non-phosphopeptide inhibitors for Src family SH2 domains

The development of novel non-phosphopeptide inhibitors for the Src family SH2 domain is described. Several commercially available hydroxyl aromatic acids have been appended off the N-terminus of pYEEIE and the potent phosphopeptide inhibitors of GST-Lck-SH2 were identified via ELISA. The most potent inhibitor, caffeic acid-pYEEIE, exhibited approximately 30-fold more binding activity than Ac-pYEEIE. Non-phosphopeptides were synthesized by replacing phosphotyrosine of caffeic acid-pYEEIE with tyrosine or 3,4-dihydroxyphenylalanine (DOPA). Caffeic acid-DOPA-EEIE that did not contain phosphotyrosine and its isosteres exhibited less than 20 times decreased binding affinity for GST-Lck-SH2 than Ac-pYEEIE. Moreover, it had a similar binding affinity for the GST-Lck-SH2, GST-Src-SH2, and GST-Fyn-SH2 domains. This study showed that the pY-1 positions of the phosphopeptide inhibitors and of the non-phosphopeptide inhibitors played an important role in the binding for the SH2 domain and that the non-phosphopeptide inhibitor must be a new lead in the development of SH2 inhibitors.