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

• Main Author: Hoose, Alex
• Format: Dissertation
• Language: English
• Published: University of Southampton 2016

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.