Bicyclic peptides generated through directed evolution by using phage display offer an attractive ligand format for the development of therapeutics. Being nearly 100-fold smaller than antibodies, they promise advantages such as access to chemical synthesis, efficient diffusion into tissues, and needle-free application. However, unlike antibodies, they do not have a folded structure in solution and thus bind less well. We developed bicyclic peptides with hydrophilic chemical structures at their center to promote noncovalent intramolecular interactions, thereby stabilizing the peptide conformation. The sequences of the peptides isolated by phage display from large combinatorial libraries were strongly influenced by the type of small molecule used in the screen, thus suggesting that the peptides fold around the small molecules. X-ray structure analysis revealed that the small molecules indeed formed hydrogen bonds with the peptides. These noncovalent interactions stabilize the peptide-protein complexes and contribute to the high binding affinity.

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 β-catenin. We succeeded in developing ligands with Kd'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.

Bicyclic peptides generated through directed evolution by using phage display offer an attractive ligand format for the development of therapeutics. Being nearly 100-fold smaller than antibodies, they promise advantages such as access to chemical synthesis, efficient diffusion into tissues, and needlefree application. However, unlike antibodies, they do not have a folded structure in solution and thus bind less well. We developed bicyclic peptides with hydrophilic chemical structures at their center to promote noncovalent intramolecular interactions, thereby stabilizing the peptide conformation. The sequences of the peptides isolated by phage display from large combinatorial libraries were strongly influenced by the type of small molecule used in the screen, thus suggesting that the peptides fold around the small molecules. X-ray structure analysis revealed that the small molecules indeed formed hydrogen bonds with the peptides. These noncovalent interactions stabilize the peptide-protein complexes and contribute to the high binding affinity.

Sortases are transpeptidase enzymes that anchor surface proteins, including virulence factors, to the cell wall of Gram-positive bacteria, and they are potential targets for the development of anti-infective agents. While several large compound libraries were searched by high-throughput screening, no high-affinity inhibitors of sortases could be developed to date. Here, we applied phage display to screen billions of peptide macrocycles against sortase A (SrtA) of Staphylococcus aureus (S. aureus). We were able to identify potent and selective inhibitors of SrtA that blocked SrtA-mediated anchoring of synthetic substrates to the surface of live S. aureus cells. A region present in all inhibitory peptides (Leu-Pro-Pro) resembled the natural substrates of SrtA (Leu-Pro-Xaa-Thr-Gly), suggesting that the macrocycles bind to the enzyme's active site and that they form similar molecular contacts as natural substrates. The evolved peptide macrocycles may be used as lead structures for the development of potent peptidomimetic SrtA inhibitors.

Upregulation of β-catenin, the primary mediator of the Wnt signaling pathway, plays an important role in the tumorigenesis of several types of human cancer. Targeting β-catenin to interfere with its ability to serve as a translational co-activator is considered an attractive therapeutic approach. However, the development of inhibitors has been challenging because of the lack of obvious binding pockets for ligands, and because inhibitors should not interfere with other β-catenin functions. Only two ligands with known molecular interactions with β-catenin have been developed so far, and are based on stabilized α-helical peptides. In this study, we screened a large combinatorial library of bicyclic peptides by phage display. Binders to different surface regions of β-catenin were identified. The binding site of one group of ligands was mapped to the interaction region of the translational Wnt inhibitor ICAT (inhibitor of β-catenin and Tcf), which is a prime target site on β-catenin for therapeutic intervention, and to which no ligands could be developed before.