Macrocyclic peptides represent attractive scaffolds for targeting protein-protein interactions, making methods for the diversification and functional selection of these molecules highly valuable for molecular discovery purposes. Here, we report the development of a novel strategy for the generation and high-throughput screening of combinatorial libraries of macrocyclic peptides constrained by a nonreducible thioether bridge. In this system, spontaneous, posttranslational peptide cyclization by means of a cysteine-reactive noncanonical amino acid was integrated with M13 bacteriophage display, enabling the creation of genetically encoded macrocyclic peptide libraries displayed on phage particles. This platform, named MOrPH-PhD, was successfully applied to produce and screen 105- to 108-member libraries of peptide macrocycles against three different protein targets, resulting in the discovery of a high-affinity binder for streptavidin (K D: 20 nM) and potent inhibitors of the therapeutically relevant proteins Kelch-like ECH-associated protein 1 (K D: 40 nM) and Sonic Hedgehog (K D: 550 nM). This work introduces and validates an efficient and general platform for the discovery and evolution of functional, conformationally constrained macrocyclic peptides useful for targeting proteins and protein-mediated interactions.
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