Triscysteine disulfide-directing motifs enabling design and discovery of multicyclic peptide binders.

Peptides are valuable for therapeutic development, with multicyclic peptides showing promise in mimicking antigen-binding potency of antibodies. However, our capability to engineer multicyclic peptide scaffolds, particularly for the construction of large combinatorial libraries, is still limited. Here, we study the interplay of disulfide pairing between three biscysteine motifs, and designed a range of triscysteine motifs with unique disulfide-directing capability for regulating the oxidative folding of multicyclic peptides. We demonstrate that incorporating these motifs into random sequences allows the design of disulfide-directed multicyclic peptide (DDMP) libraries with up to four disulfide bonds, which have been applied for the successful discovery of peptide binders with nanomolar affinity to several challenging targets. This study encourages the use of more diverse disulfide-directing motifs for creating multicyclic peptide libraries and opens an avenue for discovering functional peptides in sequence and structural space beyond existing peptide scaffolds, potentially advancing the field of peptide drug discovery. Multicyclic peptides have the potential to mimic the antigen-binding potency of the complementarity-determining regions of antibodies, enabling the development of potent peptide binders to challenging targets, but the scaffolds of multicyclic peptides are difficult to engineer. Here, the authors design a range of triscysteine motifs with disulfide-directing capability for regulating the oxidative folding of multicyclic peptides and employ them in the design of disulfide-directed multicyclic peptide libraries for discovery of potent peptide binders. [ABSTRACT FROM AUTHOR]