N-Terminal carboxyl and tetrazole-containing amides as adjuvants to Grb2 SH2 domain ligand binding Original Research Article

High affinity binding of peptides to Src homology 2 (SH2) domains, often requires the presence of phosphotyrosyl (pTyr) or pTyr-mimicking moieties in the N-terminal position of the binding ligand. Several reports have shown that Nα-acylation of the critical pTyr residue can result in increased SH2 domain binding potency. For Grb2 SH2 domains which recognize pTyr-Xxx-Asn-NH2 motifs, significant potency enhancement can be incurred by Nα-(3-amino)Z derivatization of tripeptides such as pTyr-Ile-Asn-NH2. Using ligands based on the high affinity pY-Ac6c-Asn-(naphthylpropylamide) motif, (where Ac6c=1-aminocyclohexanecarboxylic acid), additional reports have shown moderate potentiating effects of Nα-oxalyl derivatization. The current study examined variations of the Nα-oxalyl theme in the context of a Xxx-Ac6c-Asn-(naphthylpropylamide) platform, where Xxx=the hydrolytically stable pTyr mimetics phosphonomethyl phenylalanine (Pmp) or carboxymethyl phenylalanine (Cmf). The effects of Nα-(3-amino)Z derivatization were also investigated for this platform, to ascertain whether the large binding enhancement reported for tripeptides such as pTyr-Ile-Asn-NH2 could be observed. In ELISA-based extracellular Grb2 SH2 domain binding assays, it was found for the Pmp-based series, that extending the oxalyl carboxyl out by one methylene unit or replacing carboxyl functionality with a tetrazole isostere, resulted in binding potency greater than the parent Nα-acetyl-containing compound, with enhancement approximating that observed for the Nα-oxalyl derivative. When Cmf was used as the pTyr mimetic, only modest differences in IC50 values were observed for the series. Examination of the Nα-(3-amino)Z derivatized Pmp-Ac6c-Asn-(naphthylpropylamide), showed that binding affinity was reduced relative to the parent Nα-acetyl analogue, in contrast to the reported significant enhancement of affinity observed with other peptide ligands. Treatment of MDA-453 tumor cells, which are mitogenically driven through erbB-2 tyrosine kinase-dependent pathways, with Pmp-containing inhibitors resulted in growth inhibition, with the Nα-oxalyl and Nα-malonyl-containing compounds exhibiting IC50 values (4.3 and 4.6 μM, respectively) approximately five-fold lower than the parent Nα-acetyl-containing compound. Tetrazole and Nα-(3-amino)Z-containing inhibitors were from two- to four-fold less potent than these latter analogues in the growth inhibition assays.