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Spiropiperidines have been labelled as 'privileged structures' in regard to their ability to provide ligands for G-protein coupled receptors (GPCRs). These GPCRs are a superfamily of cell regulators implicated in the control of a vast number of disorders that range from arthritis and atherosclerosis to anxiety and depression. The work in this thesis describes the synthesis of a novel spiro [1-benzofuran-2,4'piperidine]- 3-one scaffold, aimed at providing efficient access to lead compounds within the GPCR family. Derivatisation of the spiropiperidine core has been achieved both in single chemoselective reactions, and later in the c:omposition of multi-step telescope reaction matrices. In the latter case, the compounds synthesised exhibit physical characteristics consistent with guidelines for sound drug-like properties. Synthesis of an analogous indoline-spiropiperidine was also accomplished. Taking advantage of sulfur's ability to undergo neighbouring group participation led to the optimisation of a 5-elldo-tet cyclisation to afford another privileged spiropiperidine core with the potential to serve as a novel scaffold to target GPCRs.

K+ channels mediating the rapid delayed rectifier current (lKr) are encoded by human ether-a-go-go-related gene (hERG) and play an important role in determining cardiac action potential (AP) repolarisation and the QT interval of the electrocardiogram. A gain-of-function hERG mutation (N588K) is associated with variant 1 of the Short QT syndrome (SQT1), which is characterised by short QT intervals «320 ms) and increased risk of cardiac arrhythmias and sudden death. Using whole-cell patch-clamp recordings ofhERG current (lhERG) from Chinese Hamster Ovary cells expressing WildType (WT) or N588K-hERG at 37°C, I investigated the effects of the N588K mutation on hERG channel electrophysiology and pharmacology. The N588K mutation produced a -+60 mV shift in IhERG availability, without concomitant alterations ofthe voltage-dependence of activation or deactivation kinetics OfIhERG. N588K IhERG peaked much earlier than did WT IhERG during guinea-pig and human ventricular AP command waveforms. The N588K mutation also resulted in IhERG peaking earlier during atrial and Purkinje fibre AP commands. MiRP1 co-expression with hERG had little effect on the timing ofpeak repolarising current during AP commands, but did affect maximal IhERG density. Results of experiments using paired AP waveforms raise the possibility that the potentially protective role ofhERG against premature depolarisations may to an extent be compromised for N588K-hERG. The N588K mutation differentially attenuated IhERG block of selected antiarrhythmic drugs. My data indicate that in addition to quinidine, disopyramide and amiodarone may be useful pharmacological treatments for SQT1. Co-expression ofhERG-1a with hERG-1b accelerated WT and N588K IhERG deactivation kinetics and for N588K increased attenuation of inactivation compared to hERG-1a alone. The differences in IhERG-Iallb kinetics as a result of the N588K mutation did not greatly influence currents during AP command waveforms, except that peak repolarising current occurred earlier during the AP than for hERG 1a alone.

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ELISAs were used to investigate the effects of a range of opioid ligands on the internalisation of the T7-tagged μ-opioid receptor (MOR) expressed in HEK 293 cells. The different opioid ligands were seen to vary in their abilities to induce MOR internalisation. The effect of the MOR agonists, DAMGO and morphine on MOR phosphorylation mutants (MOR-T180A and MOR-S363A) expressed in HEK 293 cells was also examined by ELISA. The ability of DAMGO to induce internalisation was significantly inhibited in the MOR-T180A in comparison to the wild type MOR. An N-terminal superecliptic pHluorin-tagged μ-opioid receptor (pHMOR) was generated to study MOR trafficking. Superecliptic pHluorin is a pH-sensitive variant of GFP which emits fluorescence at physiological extracellular pH (7.4) but not in acidic environments such as that occurring in intracellular organelles. In HEK 293 cells expressing pHMOR, fluorescence from pHluorin was only observed at the plasma membrane and this was abolished by lowering the extracellular pH to 6. Raising the intracellular pH with NH4Cl (50 mM) revealed fluorescence in intracellular compartments. The pHMOR construct was shown to be functional as indicated by the robust activation of GIRK channels as well as the rapid decrease in the level of cell surface pHMOR fluorescence following treatment with DAMGO. Total internal reflection fluorescence (TIRF) microscopy demonstrated that the surface expressed pHMORs were very dynamic and displayed characteristics which were not detected by confocal microscopy experiments such as receptor clustering following treatment with DAMGO or morphine. The agonist-activated receptors were also seen to colocalise with DsRed-tagged clathrin. The TIRF microscopy technique together with the pHluorin tagged MOR presents a very useful and promising approach to investigate dynamic changes in cell surface MOR expression in live cells.

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