Development of a new system for screening potential drug candidates to treat fasciolosis

• Main Author: Moore, C. M.
• Published: Queen's University Belfast 2013
• Subjects: 616.963
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.580098

Fasciolosis is a parasitic infection which infects 17 million people world-wide with a further 180 million at risk. Its prevalence is rising so new drugs are needed, but drug discovery is time-consuming, expensive, and has a high rate of attrition. P-type calcium ATPases are considered good drug targets as they are essential for cellular processes and are in an exposed position in the cell. The aim of this study was to develop a yeast-based screening system which could identify specific inhibitors of the parasite's Plasma Membrane Calcium ATPase (PMCA). The parasite PMCA coding-region was obtained using PCR, and the protein was localised to the parenchyma beneath the tegument by immunohistochemistry. Fluorescence experiments showed that the PMCA interacts with the F. hepatica calmodulin-like protein FhCaMl but not FhCaM2 or FhCaM3. The F. hepatica PMCA was heterologously expressed in yeast which restored viability in a PMCA-deleted strain. A screening assay was chosen and optimised which relied on growth curves in a 96-well plate format. Controls included strains with orthologous genes of the target deleted, and strains functionally expressing the mammalian orthologue. The assay proved to be quick, sensitive and specific to known inhibitors. Functional expression of a proposed target in yeast provides a screening assay which expedites the process of drug discovery, by eliminating cytotoxic or cell-membrane impermeable compounds early, thus reducing attrition at the later, more expensive stages.