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

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 VEGF₁₆₅-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 well-established 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.Designed 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, which is a significant limitation given that most protein–protein interactions are mediated by surfaces that are broader than can be covered by a single, regular helix (1–4, 10).Several small proteins have been explored as scaffolds that can be adapted to interact with structurally diverse protein-binding partners (11–13). The defined tertiary structures of such scaffolds allow them to present large binding surfaces that can engage large and complementary surfaces on target proteins. The “Z-domain” or “affibody” scaffold (14) is a widely studied example that is derived from domain B of staphylococcal protein A (15). The parent Z-domain (Z-IgG) (Fig. 1A) is a 58-residue engineered analog of domain B that retains affinity for the Fc portion of IgG, the natural binding partner of protein A (16). Z-IgG adopts a three-helix bundle tertiary structure, with a large surface (>600 Ų buried in the interface with Fc) formed by helices 1 and 2 contributing most of the Fc-contacting residues. Helix 3 stabilizes the Z-domain fold by packing against the other two helices (15, 17).Open in a separate windowFig. 1.Design of α/β-peptides based on the Z-domain scaffold. (A) Sequences of peptides previously derived from the Z-domain scaffold Z-VEGF, Z-IgG, and Z-TNFα targeting VEGF (19), IgG (16), and TNFα (20), respectively. Helices 1, 2, and 3 are indicated by brackets. For Z-VEGF and Z-TNFα, residues on the protein-binding face of helices 1 and 2 that were identified via randomization and selection (including the unintentionally incorporated Ala¹⁴ in Z-VEGF) are shown in red. For Z-IgG, the parent Z-domain, red positions indicate the corresponding residues that contact IgG. Sequences are arranged based on structural alignment of helical regions. (B) Strategy for the design of α/β-peptide mimics of Z-VEGF (shown in yellow and red). Red residues indicate selected residues that contact VEGF_8–109 (shown in gray) in the cocrystal structure. Sites targeted for nonnatural amino acid substitutions shown in teal. Figure is based on PDB ID code 3S1K.The composite surface displayed by helices 1 and 2 of the Z-domain scaffold can be crafted for specific binding to diverse protein partners because the three-helix bundle tertiary structure tolerates substitutions at solvent-exposed positions (18). Combinatorial randomization of as many as 13 solvent-exposed positions on helices 1 and 2, followed by affinity-based selection, has identified Z-domain derivatives that bind to a variety of targets (12, 14), including vascular endothelial growth factor (VEGF) (peptide Z-VEGF; Fig. 1 A and B) (19), tumor necrosis factor-α (TNFα) (peptide Z-TNFα; Fig. 1A) (20), and human epidermal growth factor receptor 2 (HER2) (21). Such Z-domain analogs might represent alternatives to antibodies for selective detection of disease marker proteins or for blocking deleterious signal transduction (11–14). In many cases, selection from a phage library has identified Z-domain derivatives that exhibit dissociation constants (K_D) in the nanomolar range for a chosen protein target. Affinity maturation can enhance binding to K_D values in the picomolar range (21). Recent clinical evaluations of radiolabeled Z-domain derivatives targeting HER2 revealed that these peptides could be safely used to image HER2-overexpressing lesions in breast cancer patients (22), a result that highlights the medical promise of the Z-domain scaffold.The high α-helix content of the Z-domain scaffold led us to envision that α/β-peptide analogs could be developed as binding partners for target proteins (23). We hypothesized that α→β replacements focused at sites distinct from the positions within helices 1 and 2 that mediate target recognition could reduce susceptibility to proteolytic degradation while maintaining high affinity for the partner. This design hypothesis is encouraged by two reports of Z-domain derivatives lacking helix 3 that retained affinity for their designated targets (24–26). Here, we describe the development of α/β-peptides that structurally and functionally mimic Z-VEGF. We demonstrate the versatility of this α/β-peptide strategy by showing how principles revealed in the VEGF-based effort can be extended to achieve functional mimicry of Z-domain peptides (Z-IgG and Z-TNFα) that bind to two other protein partners, IgG and TNFα.