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Towards light induced apoptosis - investigating the application of a red-shifted photoswitch to induce cell death

Ubler, Martin 2021. Towards light induced apoptosis - investigating the application of a red-shifted photoswitch to induce cell death. PhD Thesis, Cardiff University.
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Abstract

Manipulating protein-protein interactions of the tightly regulated Bcl-2 network with peptides can restore the apoptotic process in immortal cancerous cells. Chemical staples on BH3-peptides have previously been used to enforce α-helical structures to disrupt protein-protein interactions of the Bcl-2 family. However, these staples are either permanently active or require UV-light to control apoptotic events in a spatiotemporal manner. A tetra-ortho-chloro substituted azobenzene staple has recently been reported to reversibly stabilise α-helical peptide structures upon irradiation with red light (light state) and was expected to improve existing BH3-stapled peptide systems. The reported photochromic staple and a number of apoptosis-inducing peptides were synthesised. Solid-phase peptide synthesis (SPPS) was used to generate the pro-apoptotic and cell-penetrating peptides and an enzymatic ligation of the peptides was explored. A solid phase stapling approach was investigated to attach the linker to the peptide prior to cleavage from the resin. Successfully stapled peptide constructs were tested and characterised using biophysical assays in vitro including fluorescence polarization (FP) and protein-NMR binding assays. Furthermore, cell death induction was tested on live mammalian cells in vivo. Characterisation of the majority of constructs in vitro proved challenging due to poor solubility of the azobenzene moieties under assay conditions. Modification of the peptides with hydrophilic sequences did not improve the solubility. In FP assays, all tested peptides showed specific binding to the anti-apoptotic protein Bcl-xL to some degree, although with large margins of error. Peptides containing water-soluble linker or solubilizing sequences showed significantly lower margins of error suggesting that these were a product of the limited solubility. However, no significant difference between dark (trans) and light (cis) state could be observed for any of the stapled constructs. Binding of the constructs with the target protein was further investigated using protein-NMR techniques but no specific binding was observed for the stapled peptides. The dark state of the constructs showed higher cytotoxicity against mammalian cells than the light state, this was hypothesised to be a result of the physical properties of the linker. This work has demonstrated the use of solid phase peptide synthesis for the generation of pro-apoptotic-CPP constructs and chemical modification of such constructs prior to cleavage from the solid support. In addition, specific binding of the constructs to the anti-apoptotic protein Bcl-xL has been demonstrated. Challenges in solubility and control by light have been highlighted and it is hoped that these findings will lead to improved designs in the future.

Item Type: Thesis (PhD)
Date Type: Completion
Status: Unpublished
Schools: Chemistry
Date of First Compliant Deposit: 13 August 2021
Last Modified: 13 Aug 2022 01:30
URI: https://orca.cardiff.ac.uk/id/eprint/143369

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