Abstract
Natural disulfide-rich peptides (DRPs) are valuable scaffolds for the development of new bioactive molecules and therapeutics. However, there are only a limited number of topologically distinct DRP folds in nature, and most of them suffer from the problem of in vitro oxidative folding. Thus, strategies to design DRPs with new constrained topologies beyond the scope of natural folds are desired. Herein we report a general evolution-inspired strategy to design new DRPs with diverse disulfide frameworks, which relies on the incorporation of two cysteine residues and a random peptide sequence into a precursor disulfide-stabilized fold. These peptides can spontaneously fold in redox buffers to the expected tricyclic topologies with high yields. Moreover, we demonstrated that these DRPs can be used as templates for the construction of phage-displayed peptide libraries, enabling the discovery of new DRP ligands from fully randomized sequences. This study thus paves the way for the development of new DRP ligands and therapeutics with structures not derived from natural DRPs.
Highlights
We have recently discovered a unique bicyclic scaffold with a CPCX2PX2CPX4C (C, P and X are cysteine, proline and any residue respectively; the digits are numbers of the residue X) framework from a peptide library screening, which consists of four irregular turns and a short 310 helix.[34]
We examined the oxidative folding of these peptides in redox buffers of glutathione (GSH)/oxidized GSH (GSSG) using HPLC (Fig. S2†)
Eleven de-rich peptides (DRPs) scaffolds with different disul de connectivities were designed, and almost all of them can spontaneously fold in redox buffers into the expected tricyclic structures as the major products
Summary
Disul de-rich peptides (DRPs) represent a unique class of constrained peptides that are widely distributed in nature.[1,2] These peptides, including conotoxins, cyclotides and knottins, have many unique structural and functional features, which can regulate cell signaling and immunity by inhibiting proteases, blocking channels and binding receptors.[3,4,5,6] To generate new bioactive DRPs, these naturally occurring peptides can be reengineered through epitope gra ing or library screening.[7,8,9,10] The newly designed DRPs usually possess the merits of their precursor scaffolds in terms of stability and bioavailability.[7,11,12,13,14] there are only a limited variety of disul de-stabilized peptide folds in nature, signi cantly limiting the development of potent DRPs for new targets. We describe a general and robust method to design a class of new DRPs with diverse disul de frameworks that are easy to fold and amenable to random peptide library design and screening.
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