Targeting cancer via peptide-based inhibition of DNA licensing
File(s)
Author(s)
Tomkins, Joshua Robert
Type
Thesis or dissertation
Abstract
DNA licensing is the tightly regulated process by which eukaryotes prepare their DNA for replication and generate the core of the DNA helicase in G1 phase. This process has been well characterised in budding yeast but is not fully understood within human cells due to the lack of a fully reconstituted human DNA licensing system. Nevertheless, inhibition of DNA licensing has previously resulted in the S-phase arrest and apoptosis of human cancer cells, whilst merely arresting non-cancerous cells in G1 phase.
This research aimed to synthesise peptides that inhibit the protein-protein interactions (PPIs) involved in DNA licensing as a novel anti-cancer strategy. Peptides were primarily designed to mimic the regulatory protein Geminin, which inhibits DNA licensing outside of G1 phase. Additionally, peptide inhibitors targeting key DNA licensing intermediates were also designed. Peptide efficacies were evaluated in vitro using an unpublished fully reconstituted DNA licensing assay developed in the Speck group, and the ability of Geminin mimetic peptides to inhibit cancer cell proliferation was examined using human U2 osteosarcoma (U2OS) cells. Since the mechanism by which Geminin inhibits DNA licensing is currently unclear, Geminin mimetic peptides were also used to characterise the binding of Geminin to its protein target, Cdt1, and propose a mechanism whereby Geminin sterically inhibits the incorporation of Cdt1 within a key DNA licensing intermediate. Geminin residues 96-135 were found to be the minimum binding sequence, binding Cdt1158-546 with an affinity of 22 ± 7 nM. Despite this, further N-terminal and C terminal extensions were required to inhibit DNA licensing, resulting in functional peptides of 60-70 residues. Further optimisation is required to reduce the length and improve the pharmacokinetic properties of these peptides, however, the improved understanding of Geminin mechanism and of promising DNA licensing targets will aid the future development of DNA licensing inhibitors as anti-cancer therapeutics.
This research aimed to synthesise peptides that inhibit the protein-protein interactions (PPIs) involved in DNA licensing as a novel anti-cancer strategy. Peptides were primarily designed to mimic the regulatory protein Geminin, which inhibits DNA licensing outside of G1 phase. Additionally, peptide inhibitors targeting key DNA licensing intermediates were also designed. Peptide efficacies were evaluated in vitro using an unpublished fully reconstituted DNA licensing assay developed in the Speck group, and the ability of Geminin mimetic peptides to inhibit cancer cell proliferation was examined using human U2 osteosarcoma (U2OS) cells. Since the mechanism by which Geminin inhibits DNA licensing is currently unclear, Geminin mimetic peptides were also used to characterise the binding of Geminin to its protein target, Cdt1, and propose a mechanism whereby Geminin sterically inhibits the incorporation of Cdt1 within a key DNA licensing intermediate. Geminin residues 96-135 were found to be the minimum binding sequence, binding Cdt1158-546 with an affinity of 22 ± 7 nM. Despite this, further N-terminal and C terminal extensions were required to inhibit DNA licensing, resulting in functional peptides of 60-70 residues. Further optimisation is required to reduce the length and improve the pharmacokinetic properties of these peptides, however, the improved understanding of Geminin mechanism and of promising DNA licensing targets will aid the future development of DNA licensing inhibitors as anti-cancer therapeutics.
Version
Open Access
Date Issued
2024-03-25
Date Awarded
2024-09-01
Copyright Statement
Creative Commons Attribution NonCommercial Licence
License URL
Advisor
Barnard, Anna
Barr, Alexis
Speck, Christian
Sponsor
Engineering and Physical Sciences Research Council
Grant Number
CHTP_G98726
Publisher Department
Chemistry
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)