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While most SH2 domains bind phosphotyrosyl (pTyr) containing peptides in extended fashion, the growth factor receptor-bound protein 2 (Grb2) SH2 domain preferentially binds ligands in bend conformations. Accordingly, incorporation of bend-inducing functionality into synthetic ligands could potentially enhance their affinity for this SH2 domain. A macrocyclic tripeptide mimetic that contains a simplified pTyr surrogate lacking an alpha-nitrogen has recently been shown to exhibit high Grb2 SH2 domain-binding affinity in extracellular ELISA-based assays. However, the same compound is largely ineffective in whole-cell assays. It is known that acidic functionality originating from the alpha-nitrogen of pTyr residues or from the alpha-position of P0 pTyr mimetics not only increases binding affinity of peptides to Grb2 SH2 domains in extracellular assays but also enhances potency in cell-based systems. Such functionality is absent from the previously reported macrocycle. Therefore, the current study was undertaken to examine the effects of introducing carboxylic functionality at the pTyr mimetic alpha-position of macrocyclic ligands. It was found that such a modification not only enhanced Grb2 SH2 domain binding in extracellular assays but also conferred high efficacy in whole-cell systems. The most potent compound of the current study exhibited an IC(50) value of 0.002 microM in an extracellular ELISA-based assay, and in MDA-MB-453 cells, it both inhibited the association of Grb2 with p185(erbB-2) and exhibited antimitogenic effects with submicromolar IC50 values.

Mimetics of phosphotyrosine (pTyr) such as phosphonomethylphenylalanine (Pmp) have traditionally retained alpha-amino functionality. However, beta-amino acids represent isomeric variants, which may exhibit properties that are distinct from the parent. Reported herein is the first beta-amino pTyr mimetic (Pmp(beta)) bearing protection suitable for peptide synthesis. Preparation of Pmp(beta) was accomplished enantioselectively in 43% overall yield from commercially available 4-vinylbenzyl chloride.

The diastereoselective preparation of(2S)-2-fluoro-4-[[bis(1,1-dimethylethoxy)phosphinyl]methyl] benzenepropanoic acid(5) is presented as new phosphotyrosyl(pTyr) mimetic suitably protected for incorporation into peptides. Synthesis of 5 utilized electrophilic fluorination under chiral induction provided by the commercially available Evans auxiliary,(S)-(+)-4-phenyl-2-oxazolidinone. In order to demonstrate its synthetic utility, analogue 5 was employed to prepare a signal transduction-directed tripeptide. The design considerations for this peptide and its preliminary biological evaluation are included. [ABSTRACT FROM AUTHOR]

Without the presence of a phosphotyrosyl group, a phage library derived non-phosphorylated cyclic peptide ligand of Grb2-SH2 domain attributed its high affinity and specificity to well-defined and highly favored interactions of its structural elements with the binding pocket of the protein. We have disclosed a significant compensatory role of the Glu2− sidechain for the absence of the phosphate functionality on Tyr0 in the peptide ligand, cyclo(CH2CO-Glu2−-Leu-Tyr0-Glu-Asn-Val-Gly-Met5+-Tyr-Cys)-amide (termed G1TE). In this study, we report the importance of hydrophobic residue at the Tyr + 5 site in G1TE. Both acidic and basic amino acid substitutes are disfavored at this position, and replacement of Met with β-tert-butyl-Ala was found to improve the antagonist properties. Besides, the polarity of the cyclization linkage was implicated as important in stabilizing the favored binding conformation. Oxidation of the thioether linkage into sulfoxide facilitated the binding to Grb2-SH2 markedly. Simultaneous modification of the three distant sites within G1TE provided the best agent with an IC50 of 220 nM, which is among the most potent non-phosphorous- and non-phosphotyrosine-mimic containing Grb2-SH2 domain inhibitors yet reported. This potent peptidomimetic provides a novel template for the development of chemotherapeutic agents for the treatment of erbB2-related cancer. Biological assays on G1TE(Gla2−) in which the original residue of Glu2− was substituted by γ-carboxyglutamic acid (Gla) indicated that it could inhibit the interaction between activated GF receptor and Grb2 protein in cell homogenates of MDA-MB-453 breast cancer cells at the 2 μM level. More significantly, both G1TE(Gla2−) alone and the conjugate of G1TE(Gla2−) with a peptide carrier can effectively inhibit intracellular association of erbB2 and Grb2 in the same cell lines with IC50 of 50 and 2 μM, respectively. [Copyright &y& Elsevier]

Blocking the interaction between phosphotyrosine (pTyr)-containing activated receptors and the Src homology 2 (SH2) domain of the growth factor receptor bound protein 2 (Grb2) is considered to be an effective and non-cytotoxic strategy to develop new anti-proliferative agents due to its potential to shut down the Ras activation pathway. Generally, the pTyr-X-Asn minimal binding motif is required for a high-affinity ligand binding to the Grb2-SH2 domain. Using phage-display techniques, we discovered a non-pTyr-containing cyclic peptide G1 with moderate binding affinity from 107 different sequences. To understand the structural basis for the high-affinity binding of these novel non-phosphorus-containing inhibitors to the Grb2-SH2 domain, we extensively studied herein the unique functional requirements of the acidic side chain in Tyr-2 position due to the absence of the phosphate group in these non-phosphorylated peptides. A comprehensive SAR study was also carried out to develop potent Grb2-SH2 domain antagonists based upon this novel template. With both the peptidomimetic optimization of the amino acid side-chains and the constraint of the backbone conformation guided by molecular modeling, we developed several potent antagonists with low nanomolar range binding affinity, such as cyclic peptide 20 with an IC50=0.026 μM, which is one of the most potent non-phosphorus-containing Grb2-SH2 antagonists reported to date. Whole cell assays indicate that peptide 20 can penetrate the cell membranes and inhibit the association of Grb2 with p185erbB2 in erbB2-overexpressing MDA-MA-453 cancer cells at low micromolar concentrations. [Copyright &y& Elsevier]

Phosphonomethylphenylalanine (Pmp, 2) has shown wide utility as a hydrolytically stable phosphtyrosyl (pTyr, 1) mimetic, particularly in Src homology 2 (SH2) domain-binding peptides. (2S,3R)-3-(4-(phosphonomethyl)phenyl)pipecolic acid (3) represents a variant of Pmp having φ and χ1 torsion angles constrained through incorporation into the piperidinyl ring structure contained within pipecolic acid. Reported herein is the enantioselective preparation of 3, in an orthogonally protected form (4) suitable for use in peptide synthesis. Stereochemistries at both the 2- and 3-positions are derived inductively from a single chiral center provided by the commercially available Evans chiral auxiliary, (4S)-4-benzyl-1,3-oxazolidin-2-one. Incorporation of 4 into a Grb2 SH2 domain-directed tripeptide (18) showed that Grb2 SH2 domain-binding affinity was reduced relative to the parent Pmp-containing tripeptide (19). Although conformational constraint did not enhance affinity in this case, novel amino acid analogue 4 may serve as a useful tool for the induction of defined phosphotyrosyl geometry in peptides directed at other signal transduction targets. [Copyright &y& Elsevier]