Design and Ribosomal Incorporation of Noncanonical Disulfide-Directing Motifs for the Development of Multicyclic PeptideLibraries br

The engineering of naturally occurring disulfide-rich peptides (DRPs) has been significantly hampered by thedifficulty of manipulating disulfide pairing. New DRPs that takeadvantage of fold-directing motifs and noncanonical thiol-bearingamino acids are easy-to-fold with expected disulfide connectivities,representing a new class of scaffolds for the development ofpeptide ligands and therapeutics. However, the limited diversity ofthe scaffolds and particularly the use of noncanonical amino acids[e.g., penicillamine (Pen)] that are difficult to be translated byribosomes greatly hamper the further development and applicationof these DRPs. Here, we designed and synthesized noncanonicalbisthiol motifs bearing sterically obstructed thiol groups analogousto the Pen thiol to direct the folding of peptides into specific bicyclic and tricyclic structures. These bisthiol motifs can beribosomally incorporated into peptides through a commercially available PURE system integrated with genetic code reprograming,which enables, for thefirst time, the in vitro expression of bicyclic peptides with two noncanonical and orthogonal disulfide bonds.We further constructed a bicyclic peptide library encoded by mRNA, with which new bicyclic peptide ligands with nanomolar affinityto proteins were successfully selected. Therefore, this study provides a new, general, and robust method for discovering de novoDRPs with new structures and functions not derived from natural peptides, which would greatly benefit thefield of peptide drugdiscovery.