| Due to the advantages of structural stability,high bioavailability and high affinity with target proteins,disulfide-rich peptides(DRPs)have become a active research in the field of protein ligands and drug discovery.However,DRPs with stable threedimensional structure are usually natural cyclic peptides or engineering peptides developed by natural cyclic peptides.Thus,it is important to develop a variety of DRPs with excellent performance for peptide drugs.The design of DRP is difficult,because disulfide tend to undergo oxidative rearrangement to produce multiple isomers in some environments and the orthogonal strategy between disulfide bonds can effectively reduce the formation of isomers.Computer aided design(CAD)has been used in the design of DRPs,but it needs a lot of information about proteins or peptides and requires higher professional knowledge.Unnatural amino acids(penicillamine or selenocysteine)have been reported to improve the orthogonality of disulfide,but it makes biosynthesis more difficult.Although these methods have promoted the development of DRPs,there are still some shortcomings.Therefore,more new methods for the development of DRPs are still worth exploring.Here,based on previous work,we designed a new method for the development of DRPs.This research paper consists of four chapters as follows:In the first chapter,the development of peptide drugs was introduced,including the differences between cyclic peptides and linear peptides in ligands and drug applications and the advantages of cyclic peptides in peptide drugs.Then,the progress in the synthesis and design of disulfide rich peptides was summarized.Then the phage display technology was described.Finally,the research ideas and main contents of this paper are introduced.In the second chapter,more CPPC-guided disulfide rich peptides(CPPC-DRPs)with new three-dimensional structures were obtained by phage display technology and the structural space of CPPC-DRPs was further discussed.A new CPPC-DRPs phage hybrid library was constructed by changing the spacing between cysteine residues,and the ligands of two target proteins(MDM2 and Keapl)were screened.The results showed that CPPC-DRPs with new structure and function could be identified by screening peptide library of protein targets with different types of binding pockets,and CPPC-DRPs scaffolds could adapt to the changes of 3D structure.In the third chapter,the design and application space of CPPC-DRPs 3D structure are explored.(1)CPPC-DRPs with new structure were designed by epitope grafting with special sequence structure;(2)Design new functions of CPPC-DRPS that interact with Keapl by replacing disulfide constrained translocation in CPPC-DRPs with active sequence epitopes;(3)Based on the modifiability of CPPC-DRPs scaffolds,the secondary phage library was constructed by grafting epitope active sequences and affinity design to explore CPPC-DRPs with improved affinity to Keapl.The results showed that epitope grafting and library screening were feasible to find new CPPCDRPs.The fourth chapter focuses on the structure-directed design of CPPC-DRPs phage library and the application of CPPC-DRPs in the field of skeleton development and drug discovery.First,a peptide library was constructed that could display a-helical structure on the phage surface and a cyclic peptide ligand of CD28 was obtained by screening.Then,a secondary peptide library with a-helical amino acid randomization was constructed to screen CD28 for CPPC-DRPs,and affinity and cell specificity tests were performed.To assess the contribution of the invariable sequence region in the peptide to the CD28 binding and to increase the binding affinity of the peptide,a secondary library with all invariable residues in the primary library replaced by random amino acids was prepared and panned against CD28.The results showed that the structure-guided design of CPPC-DRPs libraries can utilize the sequential sequence evolution strategy to develop peptide ligands with high binding affinity and selectivity. |
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