MOrPH-PhD: An Integrated Phage Display Platform for the Discovery of Functional Genetically Encoded Peptide Macrocycles

[Image: see text] 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 10(5)- to 10(8)-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.