Macrocyclization in the Design of Grb2 SH2 Domain-Binding Ligands Exhibiting High Potency in Whole-Cell Systems

Abstract

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.

Surface Plasmon Resonance Thermodynamic and Kinetic Analysis as a Strategic Tool in Drug Design. Distinct Ways for Phosphopeptides to Plug into Src- and Grb2 SH2 Domains

“…33,40,41 From molecular modeling, it appeared that our cyclic compound (Chart 1) was able to adopt a similar -turn conformation as the bound linear ligand. However, the assumed favorable entropy due to preorganization in the bound conformation did not result in increased affinity.…”

Probing the Tyrosine Phosphorylation State in Breast Cancer by Src Homology 2 Domain Binding

“…In order to stabilize the ß-turn conformation, the Burke group also attempted to constrain the linear peptide mimetic by macrocyclization [197][198][199]. Surprisingly, although the cyclic analogue exhibited approximately a hundred-fold enhancement in binding potency in vitro relative to its linear counterpart, it was not effective in whole cell assays [197].…”

“…However, when the pTyr a position was modified by a carboxymethyl (CH 3 C0 2 H) functionality (Fig. 3C, compound 1), this compound not only blocked the association of Grb2 with Her2/Neu in whole cell assays, but also displayed anti-mitogenic effects in MDA-MB-453 cells [198,200].…”

“…For example the Nct-3-amino-Z modification improves binding for pTyr-containing ligands, but not for Pmp-containing ligands [209], while the Na-oxalyl functionality is compatible with both pTyr and Pmp [194,209]. It is also remarkable that N-terminal modification by oxalyl or carboxymethyl (CH 2 COOH-) functionalities brings an additional negative charge to the corresponding inhibitors; despite this, such compounds are often active in cell-based assays [197,198]. The affinity improvement observed in in vitro assays may not entirely account for this effect, since the unmodified counterparts, which are inactive in cell based-assays, already bind with high affinity.…”

Structural Examination of Ring-Closing Metathesis-Derived 15-Member Macrocycles as Grb2 SH2 Domain-Binding Tetrapeptide Mimetics

“…A unique feature of our approach has been the joining of C-terminal alkenyl subsituents onto vinyl-and allyl-functionality appended to the β-methylene of N-terminal pTyr mimetics. [10][11][12][13] The open-chain peptide 1 (K D = 5.6 μM) 14 represents a low micromolar affinity starting point for exploring the application of RCM macrocyclization within a unified family of Grb2 SH2 domain-binding inhibitors ( Figure 1). Earlier we reported 2a (K D = 23 nM) and 2b (K D = 55 nM) as the first members of this genre of analogues.…”