Phage Selection of Chemically Stabilized α-Helical Peptide Ligands

Short α-helical peptides stabilized by linkages between constituent amino acids offer an attractive format for ligand development.In recent years, a range of excellent ligands based on stabilized α-helices were generated by rational design using α-helical peptides of natural proteins as templates.Herein, we developed a method to engineer chemically stabilized α-helical ligands in a combinatorial fashion.In brief, peptides containing cysteines in position i and i + 4 are genetically encoded by phage display, the cysteines are modified with chemical bridges to impose α-helical conformations, and binders are isolated by affinity selection.We applied the strategy to affinity mature an α-helical peptide binding βcatenin.We succeeded in developing ligands with K d 's as low as 5.2 nM, having >200-fold improved affinity.The strategy is generally applicable for affinity maturation of any α-helical peptide.Compared to hydrocarbon stapled peptides, the herein evolved thioether-bridged peptide ligands can be synthesized more easily, as no unnatural amino acids are required and the cyclization reaction is more efficient and yields no stereoisomers.A further advantage of the thioether-bridged peptide ligands is that they can be expressed recombinantly as fusion proteins.