McCarthy, Stephen Edward Daniel;
(2020)
Chemical Biology Approaches to Understanding the Structure and Function of Protoxin-II.
Doctoral thesis (Ph.D), UCL (University College London).
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Abstract
Chronic pain affects many millions of people worldwide, with significant social and economic costs. Existing treatments for chronic pain have side effects, such as dependency and adaptation, that undermine their effectiveness in patients. New treatments with novel mechanisms of action are therefore required. The voltage-gated sodium channel Nav1.7 has recently been discovered to play an important role in the sensation of pain, and inhibitors of this protein are urgently sought as potential drug leads. However, a major challenge in designing inhibitory drugs for Nav1.7 is off-target effects arising from concurrent inhibition of homologous voltage-gated sodium channel subtypes. Potential drug leads must therefore be both potent and also highly specific for Nav1.7. The peptide Protoxin-II is isolated from the venom of the Peruvian Green Velvet Tarantula, and has attracted interest as a potent (0.3 nM) and specific (100-fold) inhibitor of Nav1.7 over other subtypes. This peptide, and others from its class of disulfide-rich peptides (the inhibitor cystine knot peptides) are promising leads in the treatment of chronic pain. However, exactly how Protoxin-II is able to achieve such potency and specificity remains poorly understood. This thesis demonstrates the chemical synthesis of correctly-folded Protoxin-II and investigates the conditions under which it forms its three disulfide bonds. The three- dimensional structure of the peptide is investigated by X-ray crystallography, NMR, and ion-mobility mass spectrometry, and its inhibitory potency against Nav1.7 is investigated by patch-clamp electrophysiology. The structure of Nav1.7 was not available until very recently, and so homology modelling of the protein was created in order to investigate possible peptide-protein interactions by computational docking. Analogues of Protoxin-II containing functionalised handles have also been synthesised and tested for activity against Nav1.7. Finally, the Protoxin-II/Nav1.7 complex is studied by in vivo crosslinking mass spectrometry.
| Type: | Thesis (Doctoral) |
|---|---|
| Qualification: | Ph.D |
| Title: | Chemical Biology Approaches to Understanding the Structure and Function of Protoxin-II |
| Event: | UCL |
| Open access status: | An open access version is available from UCL Discovery |
| Language: | English |
| Additional information: | Copyright © The Author 2020. Original content in this thesis is licensed under the terms of the Creative Commons Attribution 4.0 International (CC BY 4.0) Licence (https://creativecommons.org/licenses/by/4.0/). Any third-party copyright material present remains the property of its respective owner(s) and is licensed under its existing terms. Access may initially be restricted at the author’s request. |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices UCL > Provost and Vice Provost Offices > UCL BEAMS UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Maths and Physical Sciences UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Maths and Physical Sciences > Dept of Chemistry |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10097026 |
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