Fluorescent ligands have found numerous applications for studying interactions of drug molecules with their target and as a probe of biological systems. A common approach when designing and synthesising a fuorescent ligand is to separate the fluorophore and pharmacophore via a linker. One novel approach is to utilise click chemistry to allow the coupling of fluorophore to a pharmacophore. This thesis reports the results of an investigation into utilising click chemistry, specifically the alkyne-azide copper (I) cycloaddition to synthesis novel fluorescent GPCR ligands. Targets included the β1, β2 adrenoceptor and the muscarinic M3 receptor. Investigations into the introduction of a 1,2,3-triazole within the linker to the fluorophore resulted in 14 novel fluorescent antagonists active at the β1 and β2 adrenoceptor. The most promising ligand had log Ki values of -6.77 ± 0.20 (β1) and -7.32 ± 0.05 (β2). These ligands were used in a confocal microscopy studies to visualise the β1 and β2 adrenoceptors on the surface of CHO cells. However the ligands internalistion, and receptor visualisation was not possible. A range of structural modifications were made to reduce this with the introduction of a polar linker but this did not reduce the intracellular accumulation. The change to a longer wavelength fluorophore stopped intracellular accumulation but reduced the binding log Ki to - 5.16 ± 0.06 (β1) -5.96 ± 0.20 (β2). Twenty two novel fluorescent M3 ligands were synthesised and their inhibitory properties were investigated. An initial screen showed four promising ligands and further study into the binding affinities showed the ligands to have high potency (log Kb -7.97 ± 0.07 to -8.89 ± 0.11). These ligands were studied with confocal microscopy and intracellular accumulation did not occur. Structural changes to include a polar side chain or a sulfonic acid onto the fluorophore were investigated and led to three novel fluorescent ligands that had reduced lipophilicity. With this reduced lipophilicity, binding affinities were also reduced by ten fold compared to the original fluorescent ligand. The seven ligands were fully profiled physiochemically and kinetically. The physioschemical properties of these seven ligands gave a wide variety of lipophilic values. The kinetic profiles of the ligands exhibited very similar dissociation properties to those of the parent ligand with varying association rates. The Muscarinic M3 ligands synthesised show great binding affinities for fluorescent ligands and kinetic profiles that are extremely similar to the parent ligand. These fluorescent ligands hold characteristics that can be used to further examine the pharmacology of muscarinic receptors and be used to replace radioligands for binding studies.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:588316 |
| Date | January 2013 |
| Creators | Speed, Daniel |
| Publisher | University of Nottingham |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://eprints.nottingham.ac.uk/12933/ |
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