Directed Disulfide Pairing and Folding of Peptides for the De Novo Development of Multicyclic Peptide Libraries.

Disulfide-rich peptides (DRPs) have been an emerging frontier for drug discovery. There have been two DRPs approved as drugs (i.e., Ziconotide and Linaclotide), and many others are undergoing preclinical studies or in clinical trials. All of these DRPs are of nature origin or derived from natural peptides. It is still a challenge to design new DRPs without recourse to natural scaffolds due to the difficulty in handling the disulfide pairing. Here we developed a simple and robust strategy for directing the disulfide pairing and folding of peptides with up to six cysteine residues. Our strategy exploits the dimeric pairing of CPPC (cysteine-proline-proline-cysteine) motifs for directing disulfide formation, and DRPs with different multicyclic topologies were designed and synthesized by regulating the patterns of CPPC motifs and cysteine residues in peptides. As neither sequence manipulations nor unnatural amino acids are involved, the designed DRPs can be used as templates for the de novo development of biosynthetic multicyclic peptide libraries, enabling selection of DRPs with new functions directly from fully randomized sequences. We believe that this work represents as an important step toward the discovery and design of new multicyclic peptide ligands and therapeutics with structures not derived from natural scaffolds.