Targeting diverse protein–protein interaction interfaces with α/β-peptides derived from the Z-domain scaffold

Abstract

Peptide-based agents derived from well-defined scaffolds offer an alternative to antibodies for selective and high-affinity recognition of large and topologically complex protein surfaces. Here, we describe a strategy for designing oligomers containing both α-and β-amino acid residues ("α/β-peptides") that mimic several peptides derived from the three-helix bundle "Z-domain" scaffold. We show that α/β-peptides derived from a Z-domain peptide targeting vascular endothelial growth factor (VEGF) can structurally and functionally mimic the binding surface of the parent peptide while exhibiting significantly decreased susceptibility to proteolysis. The tightest VEGF-binding α/β-peptide inhibits the VEGF 165 -induced proliferation of human umbilical vein endothelial cells. We demonstrate the versatility of this strategy by showing how principles underlying VEGF signaling inhibitors can be rapidly extended to produce Z-domain-mimetic α/β-peptides that bind to two other protein partners, IgG and tumor necrosis factor-α. Because wellestablished selection techniques can identify high-affinity Z-domain derivatives from large DNA-encoded libraries, our findings should enable the design of biostable α/β-peptides that bind tightly and specifically to diverse targets of biomedical interest. Such reagents would be useful for diagnostic and therapeutic applications.α/β-peptides | foldamers | protein-protein interactions | inhibitors | molecular recognition D esigned molecules that bind selectively to specific sites on proteins may serve as inhibitors of medically important macromolecular interactions or diagnostic tools for biomarker detection. Small molecules often fail for these applications because of the relatively large and irregularly shaped target surfaces (1-3). In contrast, large polypeptides (e.g., antibodies) can frequently be developed to recognize a protein surface with high affinity and selectivity and represent the state of the art for engineering ligands for specific biomacromolecular targets. Large polypeptides, however, suffer several disadvantages for in vivo applications, including costly production, low storage stability, and/ or low bioavailability because of rapid proteolytic degradation (4, 5).Backbone-modified peptides, an underexplored class of molecules, are proving to be a fruitful source of tight-binding and specific protein ligands. Peptidic oligomers that contain β-amino acid residues interspersed among α-residues ("α/β-peptides") can effectively mimic the recognition surface projected by an α-helix and thereby disrupt or augment protein-protein interactions in which one partner contributes a single helix to the interface (6, 7). The unnatural backbone diminishes α/β-peptide susceptibility to proteolytic degradation relative to conventional peptides (α-residues only, "α-peptides"). As a result, α/β-peptides can exhibit improved pharmacokinetic properties in vivo relative to analogous α-peptides (8, 9). To date, however, the α/β-peptide strategy has been restricted to mimicry of isolated α-helices, ...

High-resolution structures of a heterochiral coiled coil

“…High-resolution data of the type reported here provide a foundation for future efforts to design D polypeptides that can engage in specific and potentially useful interactions with partners comprised of L residues. Given the clinical successes that have been achieved with peptides containing exclusively proteinogenic L-α-amino acid residues (24)(25)(26), and the improvements that can result from minor extensions beyond these 20 building blocks (65), it seems likely that peptidic oligomers with unnatural backbones, such as might be derived from D-α-amino acids, will provide pharmaceutically valuable agents in the future.…”

An α‐Helix‐Mimicking 12,13‐Helix: Designed α/β/γ‐Foldamers as Selective Inhibitors of Protein–Protein Interactions

“…Amongst a multitude of foldamer classes where structural/ conformational determinants have been mapped,1, 2, 3, 4 β‐peptides and hybrid α/β‐peptides, in which β‐amino acids are dispersed along an α‐peptide backbone, can inhibit α‐helix‐mediated protein–protein interactions22, 23, 24, 25, 26, 27, 28, 29 and mimic the structure and the function of protein surfaces 30, 31. Nonetheless foldamers that more accurately mimic the topology and topography of the α‐helix might prove advantageous in comparison to β‐ and α/β‐peptides, which may not fully mimic the spatial presentation of α‐helix side chains.…”

An α‐Helix‐Mimicking 12,13‐Helix: Designed α/β/γ‐Foldamers as Selective Inhibitors of Protein–Protein Interactions

“…Thep roteolytic stability of a/b/g-peptides 1-8 was investigated using a-chymotrypsin (a-CT) as ar epresentative protease,s ince this enzyme selectively hydrolyses the amide bond on the C-terminal side of hydrophobic residues such as Leu, Tr p, and Phe,a ll of which are present in the a/b/gpeptide sequences.T he p53 [15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31] peptide was also studied for comparison (see the Supporting Information). Using HPLC to assess the progress of peptide digestion, it transpired that all eight a/b/g-peptides displayed considerably greater resistance to a-CT than did the native p53 [15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31] (!…”

“…Using HPLC to assess the progress of peptide digestion, it transpired that all eight a/b/g-peptides displayed considerably greater resistance to a-CT than did the native p53 [15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31] (! 32-fold for 2, 4, 6, and 8 and !…”

“…[30,31] Nonetheless foldamers that more accurately mimic the topology and topography of the a-helix might prove advantageous in comparison to b-and a/b-peptides,which may not fully mimic the spatial presentation of a-helix side chains.S everal foldamer scaffolds have been hypothesized to have potential for the inhibition of a-helix-mediated PPIs, [32][33][34][35] but they have not yet been shown to do so experimentally. b/g-Peptide sequences fall into this category:adipeptide of b-a nd gresidues forming a13-membered hydrogen-bonded ring (C = O(i)-NH(i + 3)) is analogous to atripeptide of a-amino acids forming the 13-membered hydrogen-bonded ring (C = O(i)-NH(i + 4)) of the native a-helix.…”