Construction And Screening Of Phage-displayed Disulfide-rich Peptide Libraries

Cyclic peptides have emerged as a class of therapeutic molecules that combines the high binding affinity of biologics and some of the biophysical properties of smallmolecule drugs.Multicyclic structure exhibits high affinity and selectivity due to its well-defined topology and multiple binding loops.Disulfide bond as an important translation modification,disulfide-rich peptides(DRPs)scaffolds(such as cyclotide)have been progressively optimized for specific functions over millions of years of evolution.Inspired by the potency of these compounds,there have been considerable efforts to generate new bioactive molecules by re-engineering existing constrained peptides using loop grafting,sequence randomization and selections.Despite extensive efforts,the generation of designer DRPs with novel activity and specificity against pharmaceutical targets has proven to be challenging.For example,some strategies that grafting new peptide epitomes into the loops of cyclotides scaffolds to generate variants with novel target tropism.However,these new epitomes are not necessarily optimal for cyclotides,and the resulting molecules have consequently exhibited relatively moderate levels of activity.Besides,peptide display-based selection is an alternative approach to discover ligands for pharmaceutical targets from library of sequence-diverse cyclotides.One of the key obstacles is the complex nature of the DRPs scaffold,with difficulties in high-throughput production of DRPs libraries placing a limitation on their accessible sequence or chemical diversity.Although the theoretical diversities of the E.coli or yeast display systems could be up to 109 variants,it is difficult to accurately estimates the expression of correctly folded cyclotide variants in such cellular systems.This is because that some sequences do not support the correct disulfide formation,yet resulting in a lower diversity than the theoretical value.To solve these problems,some efforts have been obtained to promote polypeptides into their favoring specific disulfides connectivities by inserting non-canonical amino acids(such as selenocysteine and penicillamine).However,these strategies are restricted in the display systems mentioned above.Consequently,evolutionary processes(exploring new functional cyclic peptides based on natural evolutionary cyclotides templates)are not a good guide for its exploration.Instead,if we can trace the source to solve the chemical problems of disulfide pairing,the connection of disulfide bonds is not subject to the control of the primary sequence,it should become possible to obtain a wide polypeptide sequence space from de novo protein design.Based on this,the following work has been done in this paper:Chapter 1:the first chapter summarizes the classification,properties,applications and natural evolution of disulfide-rich cyclic peptides in nature,and introduces different strategies to develop drug ligands using disulfide-rich cyclic peptides in nature.Then it describes how to explore new functional cyclic peptides in a broader sequence space.Based on the above review,the significance and ideas of this paper are also proposed.Chapter 2:CPPC(cysteine-proline-proline-cysteine)motifs have the property of orthogonality parallel pairing.When applied to binary and ternary cyclic peptides,it can still effectively regulate the pairing of disulfide bonds.Based on this property,it was successfully applied to the construction of phage cyclic peptide libraries,which did not involve the operation of primary sequence and insertion of unnatural amino acids,and specific binding polypeptide ligands were screened for target proteins.Therefore,this work will greatly benefit the discovery and design of polycyclic peptide ligands and drugs.Chapter 3:The orthogonality antiparallel pairing property of a novel biscysteine motif was studied.When the biscysteine motif was applied to tricyclic peptides,the inherent property was still retained and the disulfide pairing pattern could be effectively regulated.The peptide folding regulation method developed in this chapter provides an information path for the subsequent work of phage library construction and polycyclic peptide molecular design.Chapter 4:Based on the concept of biscysteine motif-guided peptide oxidative folding proposed in Chapter 3,phage cyclic peptide libraries were successfully constructed,and a series of intracellular and extracellular targets were screened,and specific binding cyclic peptide ligand was obtained.In this peptide library,only cysteine and proline residues are reserved,achieving an unprecedented degree of sequence randomization.It is easier to obtain peptide ligands that are completely complementary to the target binding pocket by screening the target proteins of interest in such a huge sequence space.Therefore,it is easier to obtain tricyclic peptides with excellent performance by using this method.Chapter 5:At last,the work of this paper is summarized and prospected.