Structure-guided design of CPPC-paired disulfide-rich peptide libraries for ligand and drug discovery

Peptides constrained through multiple disulfides (or disulfide-rich peptides, DRPs) have been an emerging frontier for ligand and drug discovery. Such peptides have the potential to combine the binding capability of biologics with the stability and bioavailability of smaller molecules. However, DRPs with stable three-dimensional (3D) structures are usually of natural origin or engineered from natural ones. Here, we report the discovery and identification of CPPC (cysteine–proline–proline–cysteine) motif-directed DRPs with stable 3D structures (i.e., CPPC–DRPs). A range of new CPPC–DRPs were designed or selected from either random or structure–convergent peptide libraries. Thus, for the first time we revealed that the CPPC–DRPs can maintain diverse 3D structures by taking advantage of constraints from unique dimeric CPPC mini-loops, including irregular structures and regular α-helix and β-sheet folds. New CPPC–DRPs that can specifically bind the receptors (CD28) on the cell surface were also successfully discovered and identified using our DRP-discovery platform. Overall, this study provides the basis for accessing an unconventional peptide structure space previously inaccessible by natural DRPs and computational designs, inspiring the development of new peptide ligands and therapeutics.