The engineering of naturally occurring disulfide-rich peptides (DRPs)
has been significantly hampered by the difficulty of manipulating
disulfide pairing. New DRPs that take advantage of fold-directing motifs
and noncanonical thiol-bearing amino acids are easy-to-fold with
expected disulfide connectivities, representing a new class of scaffolds
for the development of peptide ligands and therapeutics. However, the
limited diversity of the scaffolds and particularly the use of
noncanonical amino acids [e.g., penicillamine (Pen)] that are difficult
to be translated by ribosomes greatly hamper the further development and
application of these DRPs. Here, we designed and synthesized
noncanonical bisthiol motifs bearing sterically obstructed thiol groups
analogous to the Pen thiol to direct the folding of peptides into
specific bicyclic and tricyclic structures. These bisthiol motifs can be
ribosomally incorporated into peptides through a commercially available
PURE system integrated with genetic code reprograming, which enables,
for the first 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 affinity to proteins were successfully
selected. Therefore, this study provides a new, general, and robust
method for discovering de novo DRPs with new structures and functions
not derived from natural peptides, which would greatly benefit the field
of peptide drug discovery.
