Small peptides containing phosphotyrosine and adjacent αMe-phosphotyrosine or its mimetics as highly potent inhibitors of Grb2 SH2 domain

A series of small peptides with the sequence mAZ-pTyr-Xaa-Asn-NH2, where Xa a de notes alpha-methylphosphotyrosine or its carboxylic mimetics, were synthesized as inhibitors of the Grb2 SH2 domain. Peptide 3 with (alpha-Me)pTyr as Xaa has the highest affinity for Grb2 (K-d = 3 +/- 1 nM) and exhibits to date the best inhibitory activity (IC50 = 11 +/- 1 nM) to displace PSpYVNVQN-Grb2 interaction in an ELISA test. The lower affinities of peptides with (a-Me)Tyr, (alpha-Me)Phe(4-CO2H), or (alpha-Me)Phe(4-CH2CO2H) as Xaa demonstrate the importance of a double charged phosphate group at the pY+1 position. Molecular modeling showed additional hydrogen bond interactions provided by the (alpha-Me)pTyr residue with the Grb2 SH2 domain. These results thus show that the effect of hydrophobic pY+1 residues, initially put forth to increased the binding affinities, can be further enhanced by a (-Me)pTyr residue which has both hydrophobic and hydrophilic properties.