The University of Southampton
University of Southampton Institutional Repository

Cyclic peptide inhibitors of the eukaryotic translation initiation factor 4E and 4G interaction

Cyclic peptide inhibitors of the eukaryotic translation initiation factor 4E and 4G interaction
Cyclic peptide inhibitors of the eukaryotic translation initiation factor 4E and 4G interaction
Cancerous tumors require a range of oncogenic proteins to promote cellular proliferation and inhibit apoptotic signals. The effective cap-dependent translation of oncogenic mRNA displays an absolute requirement for certain eukaryotic translation initiation factors (eIF). In particular the interaction of eukaryotic translation initiation factor 4E (eIF4E) with eukaryotic initiation factor 4G (eIF4G) is absolutely required to maintain the malignant phenotype. By contrast the eIF4E / eIF4G interaction is not required for the cap-independent translation of mRNAs that encode homeostatic proteins within healthy cells. As such, the inhibition of cap-dependent translation via disruption of the eIF4E / eIF4G interaction has become an attractive strategy towards the development of novel cancer drugs.

A reverse two-hybrid system was constructed with binding fragments of eIF4E and eIF4G within Escherichia coli, in order to mimic the oncogenic PPI found within malignant tissue. Split intein circularization of peptides and protein technology was then utilized to screen a library of DNA encoded cyclic peptides against the eIF4E / eIF4G interaction. Screening isolated four potential cyclic peptide inhibitors of the oncogenic protein-protein interaction that disrupted the eIF4E / eIF4G interaction in Escherichia coli. Putative cyclic peptide inhibitors of the eIF4E / eIF4G interaction were subsequently synthesized by 9-fluorenylmethoxycarbonyl solid phase peptide synthesis in preparation for testing against immortalized cancerous cell lines.

A series of assays were devised to determine the effect of compound treatment on translation. Screening of cyclic peptides by MTT cell viability assay determined that two putative inhibitors of the eIF4E / eIF4G interaction elicited a dose-dependent response in MCF7 cells. Further testing of cyclic peptides by a luciferase reporter assay suggested that cyclic peptide treatment had little effect on cap-dependent or cap-independent translation in HeLa cells. This finding was confirmed by 35S labelled methioinine/ cysteine incorporation, which suggested that cyclic peptide treatment had little effect on global translation rates in HeLa cells. m7 GTP pull-down experiments subsequently revealed that cyclic peptides did not disrupt the eIF4E / eIF4G interaction within HeLa cells or within HeLa cell lysate. It is therefore probable that SICLOPPS screening failed to isolate cyclic peptide inhibitors of this oncogenic PPI.
Hoose, Alex
fc7182aa-e84a-4d44-aea1-043ac32f1040
Hoose, Alex
fc7182aa-e84a-4d44-aea1-043ac32f1040
Tavassoli, Ali
d561cf8f-2669-46b5-b6e1-2016c85d63b2

(2016) Cyclic peptide inhibitors of the eukaryotic translation initiation factor 4E and 4G interaction. University of Southampton, Department of Chemistry, Doctoral Thesis, 278pp.

Record type: Thesis (Doctoral)

Abstract

Cancerous tumors require a range of oncogenic proteins to promote cellular proliferation and inhibit apoptotic signals. The effective cap-dependent translation of oncogenic mRNA displays an absolute requirement for certain eukaryotic translation initiation factors (eIF). In particular the interaction of eukaryotic translation initiation factor 4E (eIF4E) with eukaryotic initiation factor 4G (eIF4G) is absolutely required to maintain the malignant phenotype. By contrast the eIF4E / eIF4G interaction is not required for the cap-independent translation of mRNAs that encode homeostatic proteins within healthy cells. As such, the inhibition of cap-dependent translation via disruption of the eIF4E / eIF4G interaction has become an attractive strategy towards the development of novel cancer drugs.

A reverse two-hybrid system was constructed with binding fragments of eIF4E and eIF4G within Escherichia coli, in order to mimic the oncogenic PPI found within malignant tissue. Split intein circularization of peptides and protein technology was then utilized to screen a library of DNA encoded cyclic peptides against the eIF4E / eIF4G interaction. Screening isolated four potential cyclic peptide inhibitors of the oncogenic protein-protein interaction that disrupted the eIF4E / eIF4G interaction in Escherichia coli. Putative cyclic peptide inhibitors of the eIF4E / eIF4G interaction were subsequently synthesized by 9-fluorenylmethoxycarbonyl solid phase peptide synthesis in preparation for testing against immortalized cancerous cell lines.

A series of assays were devised to determine the effect of compound treatment on translation. Screening of cyclic peptides by MTT cell viability assay determined that two putative inhibitors of the eIF4E / eIF4G interaction elicited a dose-dependent response in MCF7 cells. Further testing of cyclic peptides by a luciferase reporter assay suggested that cyclic peptide treatment had little effect on cap-dependent or cap-independent translation in HeLa cells. This finding was confirmed by 35S labelled methioinine/ cysteine incorporation, which suggested that cyclic peptide treatment had little effect on global translation rates in HeLa cells. m7 GTP pull-down experiments subsequently revealed that cyclic peptides did not disrupt the eIF4E / eIF4G interaction within HeLa cells or within HeLa cell lysate. It is therefore probable that SICLOPPS screening failed to isolate cyclic peptide inhibitors of this oncogenic PPI.

PDF
Alex Hoose PLR FINAL.thesis.pdf - Other
Available under License University of Southampton Thesis Licence.
Download (15MB)

More information

Published date: April 2016
Organisations: University of Southampton, Chemistry

Identifiers

Local EPrints ID: 403847
URI: http://eprints.soton.ac.uk/id/eprint/403847
PURE UUID: 40c2d0c5-3313-4552-99dd-875f3e2d141f
ORCID for Ali Tavassoli: ORCID iD orcid.org/0000-0002-7420-5063

Catalogue record

Date deposited: 14 Dec 2016 15:30
Last modified: 24 Oct 2019 04:01

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.

×