The University of Southampton
University of Southampton Institutional Repository

Towards the next generation of small molecule inhibitors of CtBP dimerisation

Towards the next generation of small molecule inhibitors of CtBP dimerisation
Towards the next generation of small molecule inhibitors of CtBP dimerisation
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+.
University of Southampton
Mardle, Charlotte
a85cbf7c-10b3-4c41-acc3-94f98ef5c0c5
Mardle, Charlotte
a85cbf7c-10b3-4c41-acc3-94f98ef5c0c5
Blaydes, Jeremy
e957f999-fd91-4f77-ad62-5b4ef069b15b
Tavassoli, Ali
d561cf8f-2669-46b5-b6e1-2016c85d63b2

Mardle, Charlotte (2015) Towards the next generation of small molecule inhibitors of CtBP dimerisation. University of Southampton, Doctoral Thesis, 289pp.

Record type: Thesis (Doctoral)

Abstract

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+.

Text
CM thesis corrected final - Version of Record
Available under License University of Southampton Thesis Licence.
Download (198MB)

More information

Published date: September 2015

Identifiers

Local EPrints ID: 434992
URI: http://eprints.soton.ac.uk/id/eprint/434992
PURE UUID: 458edc62-665e-406d-9eac-2412ae3e4a2e
ORCID for Jeremy Blaydes: ORCID iD orcid.org/0000-0001-8525-0209
ORCID for Ali Tavassoli: ORCID iD orcid.org/0000-0002-7420-5063

Catalogue record

Date deposited: 17 Oct 2019 16:30
Last modified: 20 Feb 2020 05:01

Export record

Download statistics

Downloads from ePrints over the past year. Other digital versions may also be available to download e.g. from the publisher's website.

View more statistics

Atom RSS 1.0 RSS 2.0

Contact ePrints Soton: eprints@soton.ac.uk

ePrints Soton supports OAI 2.0 with a base URL of http://eprints.soton.ac.uk/cgi/oai2

This repository has been built using EPrints software, developed at the University of Southampton, but available to everyone to use.

We use cookies to ensure that we give you the best experience on our website. If you continue without changing your settings, we will assume that you are happy to receive cookies on the University of Southampton website.

×