Assay Development for the Discovery of AQP4 and TRPV4 Inhibitors Towards the Treatment of CNS Oedema

Abstract

CNS oedema affects 60 million people annually. Currently, no pharmacological intervention exists which exposes an urgent, unmet need for new treatments. AQP4 is the primary water channel in the CNS and is responsible for cell swelling in CNS oedema. While many compounds have been cited as AQP4 inhibitors, results vary across different water permeability assays. Trifluoperazine, a calmodulin inhibitor, can inhibit trafficking of AQP4 to the plasma membrane, reducing CNS oedema. Calmodulin is activated by calcium ion binding which occurs through TRPV4. In this thesis, a calcein quenching assay was optimised for high-throughput screening of AQP4 inhibitors. A cell surface biotinylation assay was utilised to identify any compounds that could reduce trafficking of AQP4 and/or TRPV4. An assay to elucidate the effects of AQP4 and TRPV4 on intracellular calcium ion dynamics was also designed. The calcein quenching assay confirmed trifluoperazine as an AQP4 inhibitor. The biotinylation assay revealed co-transfection of AQP4 and TRPV4 increased the basal surface expression of both proteins. It also showed increased surface expression of TRPV4 in response to hypotonicity. Bepridil was able to reduce AQP4 trafficking through unknown mechanisms. The intracellular calcium ion assay showed AQP4 can rapidly increase intracellular calcium ions in response to hypotonicity, more-so than TRPV4. This optimised assay is now established for use as a high-throughput method of identifying intracellular calcium ion modulators. IMAC was used to investigate the physical interaction of AQP4 and TRPV4 but did not show any physical complex. In conclusion, this research has provided new insights into the roles of AQP4 and TRPV4 in water regulation, calcium ion dynamics, trafficking and complex formation. Assays were developed and optimised to elucidate the functions of these proteins. Potential therapeutic compounds have been identified, which may provide relief for the devastating effects of CNS oedema.