Screening Of Bicyclic Peptide Ligands By Phage Display Technology

Bicyclic peptides have been promising scaffolds for the development of potent protein binders and new therapeutics.This class of peptides mainly includes disulfide-rich peptides from nature and synthetic peptides cross-linked by small organic molecules.Many novel protein binders have been developed in the last few years using natural disulfide-rich peptides as scaffolds,but it is still a challenge to repurpose disulfide-rich peptide bicycles for binding new targets due to conservativeness of their sequences.And the structural rigidity of peptides cross-linked by small organic molecules may decrease with the increase of loop lengths,especially for those lack ofα-helical and β-sheet secondary structures and hydrophobic cores.This would hamper the development of bicyclic peptide ligands to some challenging targets and limit their binding affinity.Thus,peptide bicycles with ordered structures and amenable to extensive sequence manipulations should be attractive scaffolds for the design of highly potent protein binders and new therapeutics.In this paper,we discovered a class of bicyclic peptide scaffolds with ordered but irregular secondary structures from phage selection against target protein MDM2.These peptides had a conserved cysteine/proline framework for directing peptides oxidative folding into the fused bicyclic structure consisting of four irregular turns and a 3₁₀ helix.This paper was divided into three chapters and the main contents are as follows:Chapter 1:Firstly,we introduced the potential of peptides in targeting protein-protein interaction and the advantages brought by the constrained structure of cyclic peptides and the development of cyclic peptide drugs.Secondly,we summarized the structural characteristics and pharmacological activities of natural disulfide-rich peptides.Then,two methods for developing novel cyclic peptide binding ligands were introduced:molecular grafting of epitopes onto natural disulfide-rich peptide scaffolds and phage selection of affinity reagents.Finally,put forward the research ideas and significance of this paper on the basis of the relevant researches.Chapter 2:The target protein MDM2 was screened by phage display bicyclic peptide library（GCXCX5CX5C）.A group of peptides with conserved sequence was obtained after sequencing the single colonys of the experimental group.Randomly selected some sequences and synthesized them in vitro.After oxidation,the binding affinity of bicyclic peptide products to MDM2 was determined by fluorescence polarization competition assays.The results showed that the peptides containing four cysteine can regio-selectively fold into a single isomer（yield>90%）.Most of them displayed a submicromolar binding affinity（Ki=62～1450 nM）.Chapter 3:Mutational analyses and NMR structural characterizations unveiled a key role for proline-stabilized structures in the precise pairing of cysteine residues and the ultimate ordered bicyclic structures.At the same time,the NMR results of pb-13 and pb-18 showed that their structures contain a 3₁₀ helix and four irregular turns.Their structures showed overall rigid and similar conformations with high convergence.The rigid conformations should be driven together by two disulfide bonds,four backbone hydrogen bonds and the well onserved prolines.Finally,the research contents in this paper were summarized and prospected.