Structural requirements for Tyr in the consensus sequence Y-E-N of a novelnonphosphorylated inhibitor to the Grb2-SH2 domain

The phage library derived, nonphosphorylated and thioether-cyclized peptide , termed G1TE, cyclo(CH2CO-Glu(1)-Leu-Tyr(3)-Glu-Asn-Val-Gly-Met-Tyr-Cys(10 ))-amide, represents a new structural motif that binds to the Grb2-SH2 doma in in a pTyr-independent manner, with an IC50 of 20 mu M. The retention of binding affinity is very sensitive with respect to peptide ring-size altera tions and Ala mutations. We demonstrated previously that the Glu(1) side ch ain and its closely related analogs partially compensate for the absence of the phosphate functionality on Tyr(3), and, based on molecular modeling, t hese acidic side-chains complex with the Arg67 and Arg86 sidechains of the protein in the binding cavity. In this study we judiciously altered and inc orporated various natural and unnatural amino acids as Tyr replacements wit hin the -YEN- motif, and me demonstrate the functional importance and struc tural requirement of Tyr(3) for effective binding of this novel non-phospho rylated ligand to the Grb2-SH2 domain. The phenyl side-chain moiety and a p olar functional group with specific orientation in position Y-3 of the pept ide are particularly required. Using SPR binding assays, a submicromolar in hibitor (IC 0.70 mu M) was obtained when Glu(1) was replaced with alpha-ami noadipate and Tyr(3) was replaced with 4-carboxymethyl-Phe, providing pepti de 14, G1TE(Adi(1), cmPhe(3)). Peptide 14 also inhibited Grb2/p185(erbB-2) protein association in cell homogenates of erb-2-overexpressing MDA-MA-453 cancer cells at near one micromolar concentrations, (C) 1999 Academic Press .