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Title: Biophysical and structural characterisation of protein-protein interactions, HIF-1α/p300 and eIF4E/eIF4G to inform inhibitor rational design
Author: Kyle, Hannah Frances
ISNI:       0000 0004 5369 1968
Awarding Body: University of Leeds
Current Institution: University of Leeds
Date of Award: 2015
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Protein-protein interactions (PPIs) are an important class of therapeutic target, however due to their large interaction interface they are considered difficult to inhibit. The two PPIs of interest in this thesis are the HIF-1α (hypoxia inducible factor-1α)/p300 and eIF4E (eukaryotic initiation factor 4E)/eIF4G interactions, both of which have been shown to be involved in many different cancers and are hypothesised to be good targets for targeted therapy. A rational design approach is favoured for PPIs, however for this to be possible a detailed understanding of the binding interface is required. Biophysical assessment of the HIF-1α CTAD/p300 CH1 interface has revealed a key binding “hot-spot” of p300 where the helix three region of HIF-1α binds to p300, this area has subsequently been targeted using oligoamide α-helix mimetics to disrupt the interaction. This binding site was found by two approaches, used to probe the HIF-1α binding surface on p300, first, by analysis of the binding of shorter HIF-1α peptide fragments; and second, by phage display experiments. The HIF-1α fragment study demonstrated that HIF-1α helix 3 region binds to p300 with a higher affinity than any of the short (<20 amino acids) peptide regions of HIF-1α and a peptide containing helices 2 and 3 binds with a higher affinity than a peptide containing helices 1 and 2, this importance of the helix 3 binding site was validated using mutagenesis. The phage display experiment found 12mer peptides that bind to p300 with a higher affinity than any short peptide region of HIF-1α. Structural techniques and mutagenesis were the used to verify that this binding site was similar to that of HIF-1α helix 3. The rationally designed mimetics of HIF-1α helix 3 were able to disrupt the interaction with low micromolar affinity. A second phage display experiment found Adhirons which bound with low nanomolar affinity and were able to disrupt the interaction with low micromolar affinity, docking of the crystal structure of this Adhiron indicated that it may be acting at a different site to the HIF-1α helix 3 region. This is a proof of principle that a detailed understanding of an interaction, using both biophysical and structural techniques, can directly lead to the development of inhibitors of challenging and therapeutically relevant PPIs.
Supervisor: Edwards, Thomas A. ; Wilson, Andrew J. ; Nelson, Adam N. Sponsor: EPSRC
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available