A Post-translational Modification-based Strategy For Creating Cyclic Peptide Library And Screening Of Cyclic Peptide Ligands

Peptides are ideal lead molecules for protein analysis and the development of therapeutic agents,but low stability,low affinity and low specificity are the main restrictions.The properties and utility of peptides can be improved by cyclization.For this reason,a variety of cyclization strategies have been developed.Cross-linking cyclization based on small organic compounds is one of the methods that the most widely used.The excellent combination of chemical post-translational modification and phage display technology has provided a robust tool for the directed evolution of cyclic peptide binders against a wide range of protein targets.However,the common cross-linking reagents with thiol-reactive sites usually react without selectivity,which can interfere with the correct pairing of disulfide bonds inherent in wild-type phages,thereby affecting the infectivity.Although the phages with disulfide-free gⅢ protein can be applied instead,however,it can also reduce the infectivity of phages.Despite the cyclization strategies of peptides based on orthogonal reactions used,there are limitations.Such as the reactive groups needed,metal catalysis or organic solvent,high temperature and other harsh conditions which may reduce the phage infectivity.Importantly,the loss of phage infectivity will lead to the loss of some peptide sequences,thereby reducing the diversity of the peptides,which is not conducive to discover cyclic peptide binders.Thus,a chemical post-translational modification approach for creation of cyclic peptide displayed on phage that can perform under biocompatible conditions,site selectivity and no "disulfide-free" phage needed is of great significance for the generation of novel cyclic peptide ligands.In this dissertation,a cyclic peptide library was created based on a site-selective chemical modification strategy without reducing phage infectivity.And then the cyclic peptide ligands with high affinity against different protein targets were isolated by selcetion of this cyclic peptide library.There are three parts in this dissertation and the main contents as follow.Part 1:research background.Firstly,described the application potential of peptides,advantages and disadvantages of peptides;secondly,introduced the role of peptide cyclization,the natural modification sites,several types of biological orthogonal reaction-based cyclization;then reviewed the phage display technology,including the concept,technical process,modification and application of display peptide library;introduced several protein targets;finally,we proposed the research ideas and significance of this dissertation.Part 2:constructing cyclic peptide library based on a chemical post-translational modification strategy.Firstly,a C9C peptide library(fomat CXXXXXXXXXC,X is any amino acid)was designed.The peptide displayed on phage contained an N-terminus and an internal Cys residue.The library capacity was 3.3×109.Then the peptide displayed on phage was cyclized by chemical post-translational modification of ClAc-3,which performed under biocompatible conditions without reducing the infectivity of the phage.The yield of ClAc-3 modification at a concentration of 1.0 mM was 93.03%by capture assay based on streptavidin-coated magnetic beads.Finally,a cADT-C9C phage cyclic peptide library was created.Part 3:screening of cyclic peptide ligands.The cADT-C9C cyclic peptide library was used to screen against each protein target(the intracellular protein Bcl-2,Mdm2 and Keapl)for three rounds,and a series of peptides were isolated.Then some peptides were synthesized in vitro and were determined to showed biologically active that could bind to the target with submicromolar affinities.At last,the research contents were summarized and prospected.