A glycopeptide phage display approach for the identification of lectin ligands

Boudjelal, Hassan (2023) A glycopeptide phage display approach for the identification of lectin ligands. Doctoral thesis, University of East Anglia.

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Lectins are carbohydrate binding proteins that mediate a wide range of biological processes such as pathogen recognition, cell adhesion and inflammation. The development of inhibitors/agonists for these proteins would therefore be therapeutically valuable in many areas, such as treatment of infectious disease and cancer. However, the development of lectin targeting drugs has proved challenging due to the typically weak binding (Kd = μM to mM range) between lectins and their cognate monosaccharide ligands. The aim of this project was to identify glycopeptides that exhibit high lectin affinity due to the peptide moiety binding adjacently to the lectin carbohydrate recognition domain (CRD). The rationale behind this approach being that the monosaccharide and peptide would act synergistically, providing specificity to the lectin CRD and increased affinity, respectively. Identification of the peptide sequence necessary for increased binding was facilitated through use of phage display, a recombinant screening technique that allows for the selection of high-affinity peptides for a given target molecule.

Three phage libraries were cloned, each displaying >106 random, disulphide-constrained peptides on their surface. All peptides were engineered to contain an Nterminal serine, allowing the libraries to be functionalised with monosaccharides via oxime ligation. Lectin specific glycopeptides could then be selected from the glycopeptide libraries by performing iterative screening and enrichment steps. In this project, several lectins were screened against, including LecB - a virulence factor of the ESKAPE pathogen Pseudomonas aeruginosa that aids biofilm formation. Binding candidates from the screening experiments were identified by Illumina sequencing and the datasets obtained processed using MATLAB and Python scripts. Analysis of the screening output was performed using ligand docking and molecular dynamics to identify glycopeptide leads. These were synthesised and affinity tested using biolayer interferometry, fluorescence polarisation and STD NMR. The glycopeptide M7KQQMannose was determined to bind LecB with a Kd in the nM range, representing a >71-fold increase in affinity compared to the mannose monosaccharide.

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Science > School of Pharmacy
Depositing User: Chris White
Date Deposited: 03 Jul 2024 10:16
Last Modified: 03 Jul 2024 10:28
URI: https://ueaeprints.uea.ac.uk/id/eprint/95773

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