Phage Selection of Chemically Stabilized alpha-Helical Peptide Ligands
Short alpha-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 alpha-helices were generated by rational design using alpha-helical peptides of natural proteins as templates. Herein, we developed a method to engineer chemically stabilized alpha-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 alpha-helical conformations, and binders are isolated by affinity selection. We applied the strategy to affinity mature an alpha-helical peptide binding beta-catenin. 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 alpha-helical peptide. Compared to hydrocarbon stapled peptides, the herein evolved thioether-bridged peptide ligands can be synthesized more easily, 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.