De novo design of constrained and sequence-independent peptide scaffolds with topologically-formidable disulfide connectivities.

Yiwu Zheng,Yibing Zhao,Chuanliu Wu,Yaqi Wu,Xiaoting Meng

doi:10.1039/c7sc03956e

Abstract

Disulfide-rich peptides are interesting scaffolds for drug design and discovery. However, peptide scaffolds constrained by disulfide bonds, either naturally occurring or computationally designed, have been suffering from the elusive (oxidative) folding behavior complying with Anfinsen's dogma, which strongly restricts their applicability in bioactive peptide design and discovery; because when primary peptide sequences are extensively manipulated, their disulfide connectivities might become scrambled. Here we present the design of cysteine/penicillamine (C/Pen)-mixed peptide frameworks that are capable of folding into specific regioisomers without dependence on primary amino acid sequences. Even certain folds that are considered to be topologically formidable can be generated in high yields. Currently, almost all disulfide-rich peptide scaffolds are vitally correlated to primary amino acid sequences, but ours are exceptional. These scaffolds should be of particular interest for further designing constrained peptides with new structures and functions, and more importantly, the ultimately designed peptides would not suffer from general oxidative folding problems.

Highlights

A strategy to pull out four speci c isomers from the total of 15 ones forming from the oxidation of a six-cysteine-bearing peptide was demonstrated rst by transforming a model peptide with one CXC motif and four isolated cysteine residues (1) into its triple Pen-substituted analogs (2–5) (Fig. 1a)
It is worth mentioning that the oxidative folding of these C/Pen-mixed peptides into speci c isomers is certainly not driven by primary sequences because all peptides are composed of primarily achiral glycine residues; and some other lysine and tryptophan residues are strategically inserted for facilitating the tryptic digestion analyses
Our strategy to favor the folding of several speci c isomers and yet disfavor many other undesired ones without the recourse to manipulation of primary sequences should be of particular interest to de novo design of proteins, considering that the position of CXC motif, the length of each peptide segment, and the cysteine residues for Pen substitution might all be manipulated arbitrarily

Summary

Introduction

Constrained peptides—lying between larger biologics and small molecules in size, and presumably combining the advantages of both—have been a treasured chemical space for drug developments.[1,2] Among the diverse pharmacologically active peptides constrained with covalent crosslinks, naturally occurring disul de-rich peptides, including plant-derived cyclotides, antimicrobial defensins, and conotoxins from venoms of predatory marine snails, are most extensively explored.[2,3,4,5] Novel bioactive disul de-constrained peptides can be routinely developed by re-engineering these naturally occurring scaffolds using loop gra ing and high-throughput sequence selection.[6,7] there are limits for the structural variety of naturally occurring constrained peptides, which restricts the design of inhibitors to certain targets with a surface topology congenitally complementary to the constrained peptide scaffolds.[8] A strategy to break through this limitation comes from the recent advances in de novo protein design, based on which disul deconstrained peptide scaffolds with new structures can be created without reference to known structures.[8,9] the Previous reports have demonstrated the unique effect of CXC (cysteine-any-cysteine) motifs and the incorporation of penicillamine (Pen) on the oxidative folding of either natural or synthetic peptides.[11,12,13] the essentials of directing the folding of peptide into speci c isomers with up to three disul de bonds have not yet been grasped. In this work we describe our effort of de novo designing C/Pen-mixed

Methods

Results

Conclusion