The design, synthesis and evaluation of peptides and small molecules targeting the dyskerin-dyskerin protein-protein interaction

Van Wier, Suzanne Pauline (2023) The design, synthesis and evaluation of peptides and small molecules targeting the dyskerin-dyskerin protein-protein interaction. Doctoral thesis, University of East Anglia.

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One of the hallmarks of cancers is their ability to replicate limitlessly making them immortal. In 80-90% of cancer cells this is due to the reactivation of telomerase, a protein complex which elongates telomeres at the end of chromosomes, protecting the chromosomes from degradation and preventing cell senescence. The Cryo-EM structure of telomerase published in 2021 provided an opportunity to identify new ways to target telomerase. In patients with
Dyskeratosis Congenita, a disease characterised by shortened telomeres, the structure showed that genetic mutations are transcribed to the dyskerin-dyskerin protein-protein interaction (PPI) in telomerase.

Herein, we investigate the inhibition of the dyskerin-dyskerin PPI with peptides and small molecules. Based on the telomerase Cryo-EM structure, linear, hydrocarbon and lactam stapled peptides were designed and synthesised. Using recombinantly-expressed MBPdyskerin, fluorescence polarisation and surface plasmon resonance binding assays were developed to investigate the binding affinity of the peptides towards dyskerin. The peptides were further evaluated in a Homogeneous Time Resolved Fluorescence assay to assess their ability to disrupt the dimer formation with six peptides found to disrupt the dyskerin-dyskerin PPI at 200 μM.

When compared to small molecule drugs, peptide therapeutics can have drawbacks such as their limited stability and cell permeability. Therefore, peptide-directed binding was used to go from peptide to small molecule inhibitor by computationally identifying fragments able to replace parts of the peptide. Nineteen small molecules were synthesised with six compounds able to disrupt the dyskerin-dyskerin dimer at 100 μM.

Both the peptides and small molecules were further evaluated for their cytotoxicity in an MTS assay. The peptides were also explored for their cellular uptake and proteolytic stability, two properties which could limit their cellular effects.

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Science > School of Pharmacy
Depositing User: Nicola Veasy
Date Deposited: 11 Apr 2024 14:53
Last Modified: 11 Apr 2024 14:53

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