Residue-Based Preorganization of BH3-Derived α/ß-Peptides: Modulating Affinity, Selectivity and Proteolytic Susceptibility in α-Helix Mimics.

We report progress toward a general strategy for mimicking the recognition properties of specific α-helices within natural proteins through the use of oligomers that are less susceptible than conventional peptides to proteolysis. The oligomers contain both α- and ß-amino acid residues, with the density of the ß subunits low enough that an α-helix-like conformation can be adopted but high enough to interfere with protease activity. Previous studies with a different protein-recognition system that suggested ring-constrained ß residues can be superior to flexible ß residues in terms of maximizing α/ß-peptide affinity for a targeted protein surface. Here, we use mimicry of the 18-residue Bim BH3 domain to expand the scope of this strategy. Two significant advances have been achieved. First, we have developed and validated a new ring-constrained ß residue that bears an acidic side chain, which complements previously known analogues that are either hydrophobic or basic. Second, we have discovered that placing cyclic ß residues at sites that make direct contact with partner proteins can lead to substantial discrimination between structurally homologous binding partners, the proteins Bcl-xL and Mcl-1. Overall, this study helps to establish that α/ß-peptides containing ring-preorganized ß residues can reliably provide proteolytically resistant ligands for proteins that naturally evolved to recognize α-helical partners.

Extending foldamer design beyond α-helix mimicry: α/ß-peptide inhibitors of vascular endothelial growth factor signaling.

Diverse strategies have been explored to mimic the surface displayed by an α-helical segment of a protein, with the goal of creating inhibitors of helix-mediated protein-protein interactions. Many recognition surfaces on proteins, however, are topologically more complex and less regular than a single α-helix. We describe efforts to develop peptidic foldamers that bind to the irregular receptor-recognition surface of vascular endothelial growth factor (VEGF). Our approach begins with a 19-residue α-peptide previously reported by Fairbrother et al. (Biochemistry 1998, 37, 17754) to bind to this surface on VEGF. Systematic evaluation of αâ†’ß replacements throughout this 19-mer sequence enabled us to identify homologues that contain up to ~30% ß residues, retain significant affinity for VEGF, and display substantial resistance to proteolysis. These α/ß-peptides can block VEGF-stimulated proliferation of human umbilical vein endothelial cells.

Evaluation of diverse α/ß-backbone patterns for functional α-helix mimicry: analogues of the Bim BH3 domain.

Peptidic oligomers that contain both α- and ß-amino acid residues, in regular patterns throughout the backbone, are emerging as structural mimics of α-helix-forming conventional peptides (composed exclusively of α-amino acid residues). Here we describe a comprehensive evaluation of diverse α/ß-peptide homologues of the Bim BH3 domain in terms of their ability to bind to the BH3-recognition sites on two partner proteins, Bcl-x(L) and Mcl-1. These proteins are members of the anti-apoptotic Bcl-2 family, and both bind tightly to the Bim BH3 domain itself. All α/ß-peptide homologues retain the side-chain sequence of the Bim BH3 domain, but each homologue contains periodic α-residue → ß(3)-residue substitutions. Previous work has shown that the ααßαααß pattern, which aligns the ß(3)-residues in a 'stripe' along one side of the helix, can support functional α-helix mimicry, and the results reported here strengthen this conclusion. The present study provides the first evaluation of functional mimicry by ααß and αααß patterns, which cause the ß(3)-residues to spiral around the helix periphery. We find that the αααß pattern can support effective mimicry of the Bim BH3 domain, as manifested by the crystal structure of an α/ß-peptide bound to Bcl-x(L), affinity for a variety of Bcl-2 family proteins, and induction of apoptotic signaling in mouse embryonic fibroblast extracts. The best αααß homologue shows substantial protection from proteolytic degradation relative to the Bim BH3 α-peptide.