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Title: Towards the next generation of small molecule inhibitors of CtBP dimerisation
Author: Mardle, Charlotte
ISNI:       0000 0004 8501 6967
Awarding Body: University of Southampton
Current Institution: University of Southampton
Date of Award: 2015
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C-terminal binding proteins (CtBP1 and CtBP2) are transcriptional co-repressors whose activity can promote tumour cell survival and the migration of tumours. It is CtBP homo- and hetero-dimer complexes that recruit the transcription factors necessary to mediate transcription and CtBPs are particularly driven to dimerise in tumour cells by the increase in NADH caused by aerobic glycolysis. Therefore, CtBPs link cancer cell metabolism to other drivers of malignancy and have been identified as potential targets for novel cancer therapeutics. A cyclic peptide inhibitor of CtBP1 homo-dimerisation, labelled CP61, has previously been identified and could form the basis of novel molecules to target CtBPs. In this project, in vitro assays, including a FRET and a dehydrogenase assay were developed to investigate the activity and mechanism of this inhibitor. A thermal shift assay using a truncated version of CtBP1 showed that CP61 bound to either the core nucleotide or substrate binding domain of the protein and had a destabilising effect. Alanine scanning of CP61 was completed using split-intein circular ligation of proteins and peptides (SICLOPPS) within a bacterial reverse-two hybrid system (RTHS) and the thermal shift assay. The aromatic amino acid side-chains were shown to be essential for the activity of this peptide. A screen to find CtBP2 specific inhibitors, which identified peptide sequences containing a high prevalence of aromatic residues, supported this. Finally, the thermal shift assay and mutant versions of recombinant CtBP were used to elucidate the possible binding orders of CtBP substrates and inhibitors, and their effects on protein stability. This included the ordered binding of NAD+ and MTOB and that CtBP1 has a higher affinity for NADH that NAD+.
Supervisor: Blaydes, Jeremy ; Tavassoli, Ali Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available