Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.584694
Title: Bioresponsive polymer therapeutics containing coiled-coil motifs
Author: Deacon, Samuel Philip Edward
Awarding Body: Cardiff University
Current Institution: Cardiff University
Date of Award: 2009
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Abstract:
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.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.584694  DOI: Not available
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