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Title: Designing novel modulators selective for the intermediate conductance calcium activated potassium channel
Author: Delaforce, Christopher
ISNI:       0000 0004 8502 8976
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2018
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Reduction of systemic vascular resistance by dilation of resistance arteries, is a powerful method of blood pressure reduction. Endothelium derived hyperpolarisation is a major vasorelaxant pathway in resistance arteries, dependant on the activation of endothelium Ca2+ activated K+ (KCa) channels. Existing positive modulators of KCa channels act by stabilising the protein-protein interaction of the KCa channel gating complex, involving the Ca2+ sensing protein, calmodulin (CaM). Herein describes an investigation of the KCa channel positive modulator binding cavity, the rational design of next generation positive modulators, and developments towards a biophysical screening assay. A homology model of the intermediate conductance (IKCa3.1) channel positive modulator binding cavity was generated, docked with existing positive modulators and scrutinised for the rational design of novel next generation analogues. A set of modulator analogues, capable of hydrogen bonding to an IKCa3.1 specific serine residue, were designed and synthesised. These analogues were tested for KCa channel modulation via their vasorelaxant activities by ex vivo artery wire myography. During this assessment, a novel and unknown vasorelaxant effect, unrelated to KCa channels, was discovered. The IKCa3.1 gating complex proteins, CaM and the channel CaM binding domain (CaMBD), were expressed for biophysical investigation. Several techniques were designed, developed and evaluated as a modulator screening assay. In differential scanning fluorimetry (DSF), existing KCa channel positive modulators produced a significant effect, potentially enabling the observation of direct gating complex stabilisation. Native protein mass spectrometry observed oligomerisation of the gating complex in solution and could be used in future to confirm modulator binding and so the DSF results. This would be the first biophysical assay able to identify KCa channel ligands; a powerful tool for the development of increasingly potent and selective positive modulators.
Supervisor: Conway, Stuart ; Donohoe, Timothy ; Garland, Christopher Sponsor: British Heart Foundation Centre of Research Excellence
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available