N-terminal carboxyl and tetrazole-containing amides as adjuvants to Grb2 SH2 domain ligand binding

High affinity binding of peptides to Src homology 2 (SH2) domains, often re quires the presence of phosphotyrosyl (pTyr) or pTyr-mimicking moieties in the N-terminal position of the binding ligand. Several reports have shown t hat N-alpha-acylation of the critical pTyr residue can result in increased SH2 domain binding potency. For Grb2 SH2 domains which recognize pTyr-Xxx-A sn-NH2 motifs, significant potency enhancement can be incurred by N-alpha-( 3-amino)Z derivatization of tripeptides such as pTyr-Xxe-Asn-NH2. Using lig ands based on the high affinity pY-Ac(6)c-Asn-(naphthylpropylamide) motif, (where Ac(6)c = 1-aminocyclohex-anecarboxylic acid), additional reports hav e shown moderate potentiating effects of N-alpha-oxalyl derivatization. The current study examined variations of the N-alpha-oxalyl theme in the conte xt of a Xxx-Ac(6)c-Asn-(naphthylpropylamide) platform, where Xxx = the hydr olytically stable pTyr mimetics phosphonomethyl phenylalanine (Pmp) or carb oxymethyl phenylalanine (Cmf). The effects of N-alpha-(3-amino)Z derivatiza tion were also investigated for this platform, to ascertain whether the lar ge binding enhancement reported for tripeptides such as pTyr-Ile-Asn-NH2 co uld be observed. In ELISA-based extracellular Grb2 SH2 domain binding assay s, it was found for the Pmp-based series, that extending the oxalyl carboxy l out by one methylene unit or replacing carboxyl functionality with a tetr azole isostere, resulted in binding potency greater than the parent N-alpha -acetyl-containing compound, with enhancement approximating that observed f or the N-alpha-oxalyl derivative. When Cmf was used as the pTyr mimetic, on ly modest differences in IC50 values were observed for the series. Examinat ion of the N-alpha-(3-amino)Z derivatized Pmp-Ac(6)c-Asn-(naphthylpropylami de), showed that binding affinity was reduced relative to the parent N-alph a-acetyl analogue, in contrast to the reported significant enhancement of a ffinity observed with other peptide ligands. Treatment of MDA-453 tumor cel ls, which are mitogenically driven through erbB-2 tyrosine kinase-dependent pathways, with Pmp-containing inhibitors resulted in growth inhibition, wi th the N-alpha-oxalyl and N-alpha-malonyl-containing compounds exhibiting I C50 values (4.3 and 4.6 muM, respectively) approximately five-fold lower th an the parent N-alpha-acetyl-containing compound. Tetrazole and N-alpha-(3- amino)Z-containing inhibitors were from two- to four-fold less potent than these latter analogues in the growth inhibition assays. (C) 2001 Elsevier S cience Ltd. All rights reserved.