Motif-Directed Oxidative Folding to Design and Discover Multicyclic Peptides for Protein Recognition

Multicyclic peptides that are constrained through covalent cross-linkers can usually maintain stable three-dimensional (3D) structures without the necessity of incorporating noncovalently interacting cores. This configuration allows for a greater utilization of residues for functional purposes compared to larger proteins, rendering multicyclic peptides attractive molecular modalities for the development of chemical tools and therapeutic agents. Even smaller multicyclic peptides, which may lack stable 3D structures due to limited sequence-driven folding capabilities, can still benefit from the specific conformations stabilized by covalent cross-linkers to facilitate target binding. Disulfide-rich peptides (DRPs) are a class of particularly significant multicyclic peptides that are primarily composed of disulfide bonds in their interior. However, the structural diversity of DRPs is limited to a few naturally occurring and designer scaffolds, which significantly impedes the development of multicyclic peptide ligands and therapeutics. To address this issue, we developed a novel method that utilizes disulfide-directing motifs to design and discover DRPs with new structures and functions in random sequence space. Compared with traditional DRPs, these new DRPs that incorporate disulfide-directing motifs exhibit more precise oxidative folding regarding disulfide pairing and demonstrate greater tolerance to sequence manipulations. Thus, we designated these peptides as disulfide-directed multicyclic peptides (DDMPs).Over the past decade, we have developed a new class of multicyclic peptides by leveraging disulfide-directing motifs, including biscysteine motifs such as CPXXC, CPPC, and CXC (C: cysteine; P: proline; X: any amino acid), as well as triscysteine motifs that rationally combine two biscysteine motifs (e.g., CPPCXC and CPXXCXC) to direct the oxidative folding of peptides. This leads to the introduction of a novel concept known as motif-directed oxidative folding, which is valuable for the construction of peptides with multiple disulfide bonds. A large diversity of DDMPs have been designed by simply altering the disulfide-directing motifs, the arrangement of cysteine residues (i.e., cysteine patterns), and the number of random residues separating them. As the oxidative folding of DDMPs is primarily determined by disulfide-directing motifs, these peptides are intrinsically more tolerant of extensive sequence manipulations compared to traditional DRPs. Consequently, multicyclic peptide libraries with an unprecedented high degree of sequence randomization have been developed by utilizing commonly used biological display systems such as phage display. We have validated the applicability of these libraries by successfully discovering DDMPs with unique protein-like 3D structures and high affinity and specificity to various cell-surface receptors, including tumor-associated antigens, immune costimulatory receptors, and G protein-coupled receptors (GPCRs). Currently, multicyclic peptides used in clinical settings are of natural origin or derived from natural DRPs. Our studies have opened up the possibility of developing multicyclic peptides without relying on natural scaffolds, representing a pivotal breakthrough in the field of peptide ligand and drug discovery. Further investigations will facilitate the application of our DDMPs in broader fields such as bioanalysis, chemical biology, and biomedicine.