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Bioresponsive polymer therapeutics containing coiled-coil motifs

Deacon, Samuel Philip Edward 2009. Bioresponsive polymer therapeutics containing coiled-coil motifs. PhD Thesis, Cardiff University.

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Polyethyleneglycol (PEG) conjugates of peptides, proteins and an aptamer are in routine clinical use as first generation nanomedicines. Here a new family of polymer therapeutics based on PEG conjugates containing a coiled-coil peptide motif as a molecular switch are proposed. The coiled-coil motif is adopted by many naturally occurring proteins/peptides, including transcription factors key to cancer progression (E2F1/AP-1) and Ebola virus proteins (VP35/GP2). These were chosen as the first targets, however there is potentially a much wider role for this novel family of therapeutics. First studies selected coiled-coil motif peptide sequences (using computational prediction software and published literature) that were then synthesised using a solid phase approach, purified and characterised. To facilitate subsequent PEGylation, peptides were engineered to include an N-terminal cysteine residue. mPEG-maleimide (-5,500 g mol 1) was then conjugated site-specifically via the cysteine thiol. A purification method optimised using cation-exchange chromatography enabled the removal of both unreacted mPEG-maleimide and free peptide purity was > 95 % for each conjugate. Proof of concept was obtained with mPEG-FosWc, which was designed to inhibit coiled-coil heterodimerisation of native c-Jun and c-Fos proteins (AP-1). 1H, 15N HSQC spectroscopy confirmed target hybridisation of heterodimeric coiled-coils FosWc : c-Jun and mPEG-FosWc : c-Jun. In addition, both NMR and CD spectroscopy showed that both heterodimers adopted very similar structures under physiological conditions, irrespective of the presence or absence of PEG. Further studies using fluorescently labelled conjugates investigated cellular uptake in MCF-7 cells, and biological activity was assessed using the MTT assay with and without the use of a cationic transfection reagent. These studies demonstrate the potential of mPEG-coiled-coil motifs as therapeutic agents. However, demonstrating reproducible biological activity was not possible with the intracellular targets. Investigating the biological activity of the conjugates designed to target the extracellular Ebola virus fusion proteins remains an exciting prospect.

Item Type: Thesis (PhD)
Status: Unpublished
Schools: Pharmacy
Subjects: R Medicine > RS Pharmacy and materia medica
ISBN: 9781303189173
Date of First Compliant Deposit: 30 March 2016
Last Modified: 03 Dec 2014 16:42

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