De Novo Discovery of Cysteine Frameworks for Developing Multicyclic Peptide Libraries for Ligand Discovery.

Conserved cysteine frameworks are essential components of disulfide-rich peptides (DRPs), which dominantly define the structural diversity of both naturally occurring and de novo-designed DRPs. However, there are only very limited numbers of conserved cysteine frameworks, and general methods enabling de novo discovery of cysteine frameworks with robust foldability are still not available. Here, we devised a "touchstone"-based strategy that relies on chasing oxidative foldability between two individual disulfide-rich folds on the phage surface to discover new cysteine frameworks from random sequences. Unique cysteine frameworks with a high degree of compatibility with phage display systems and broad sequence tolerance were successfully identified, which were subsequently exploited for the development of multicyclic DRP libraries, enabling the rapid discovery of new peptide ligands with low-nanomolar and picomolar binding affinity. This study provides an unprecedented method for exploring and exploiting the sequence and structure space of DRPs that is not readily accessible by existing strategies, holding the potential to revolutionize the study of DRPs and significantly advance the design and discovery of multicyclic peptide ligands and drugs.

Disulfide-Directed Multicyclic Peptide Libraries for the Discovery of Peptide Ligands and Drugs.

Multicyclic peptides with stable 3D structures are a kind of novel and promising peptide formats for drug design and discovery as they have the potential to combine the best characteristics of small molecules and proteins. However, the development of multicyclic peptides is largely limited to naturally occurring products. It remains a big challenge to develop multicyclic peptides with new structures and functions without recourse to the existing natural scaffolds. Here, we report a general and robust method relying on the utility of new disulfide-directing motifs for designing and discovering diverse multicyclic peptides with potent protein-binding capability. These peptides, referred to as disulfide-directed multicyclic peptides (DDMPs), are tolerant to extensive sequence manipulations and variations of disulfide-pairing frameworks, enabling the development of de novo DDMP libraries useful for ligand and drug discovery. This study opens a new avenue for creating a new generation of multicyclic peptides in sequence and structure space inaccessible by natural scaffolds, thus would greatly benefit the field of peptide drug discovery.

Design and Ribosomal Incorporation of Noncanonical Disulfide-Directing Motifs for the Development of Multicyclic Peptide Libraries.

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.

Ribosomal Incorporation of Lithocholic Acid into Peptides for the De Novo Discovery Of Peptide-Lithocholic Acid Hybrid Macrocyclic Peptides.

Peptide-bile acid hybrids offer promising drug candidates due to enhanced pharmacological properties, such as improved protease resistance and oral bioavailability. However, it remains unknown whether bile acids can be incorporated into peptide chains by the ribosome to produce a peptide-bile acid hybrid macrocyclic peptide library for target-based de novo screening. In this study, we achieved the ribosomal incorporation of lithocholic acid (LCA)-d-tyrosine into peptide chains. This led to the construction of a peptide-LCA hybrid macrocyclic peptide library, which enabled the identification of peptides TP-2C-4L3 (targeting Trop2) and EP-2C-4L5 (targeting EphA2) with strong binding affinities. Notably, LCA was found to directly participate in binding to EphA2 and confer on the peptides improved stability and resistance to proteases. Cell staining experiments confirmed the high specificity of the peptides for targeting Trop2 and EphA2. This study highlights the benefits of LCA in peptides and paves the way for de novo discovery of stable peptide-LCA hybrid drugs.