The growth factor receptor-bound protein 2 (Grb2) is an SH2 domain-containing docking module that participates in the signaling of numerous oncogenic growth factor receptor protein-tyrosine kinases (PTKs). Presented herein is a 5-methylindolyl-containing macrocyclic tetrapeptide mimetic (5) that binds to Grb2 SH2 domain protein with K-d = 75 pM. This represents the highest affinity yet reported for a synthetic inhibitor against any SH2 domain. In whole cell assays this novel analogue is able to effectively block the association of Grb2 to cognate cytoplasmic erbB-2 at IC50 < 10nM without prodrug derivatization or the addition of carrier peptide motifs. Anti-mitogenic effects against erbB-2-dependent breast cancers are achieved at non-cytotoxic concentrations (IC50 = 0.6 muM). Macrocycle 5 may be representative of a new class of therapeutically relevant Grb2 SH2 domain-directed agents. Published by Elsevier Inc.