Use of Membrane Proteins as Antifungal Drug Targets

Abstract

Fungal infections represent a much-overlooked threat that has yet to receive its due consideration. Their invasive branch has an underestimated impact on human morbidity and mortality. Despite this, research on antifungal therapies has been stalling for almost two decades now, while resistant strains have emerged on essentially every class of drugs that has been commercially available. Therefore, developing ways to counteract this emerging resistance is of paramount importance if we wish to remain capable of treating invasive fungal infections. New classes of drugs using new mechanisms of action would be highly desirable, especially if they are targeting fungal markers that have not been identified as potential targets before. This thesis project has been carried out in partnership with F2G Ltd, Manchester, UK, regarding the expression of new potential drug targets for antifungal treatments. It focuses on membrane proteins, which are a crucial gateway to the cell and an important source of untargeted markers that could represent very promising alternatives. Two main targets, both enzymatic membrane proteins, have been expressed in Pichia Pastoris yeast cells and solubilised using poly (styrene-co-maleic acid) lipid particles or SMALPs, which enables to retain the membrane protein with its surrounding lipids so that the protein stays in its native conformation. This allowed the protein to remain functional, so that a functional assay could be developed later on in order to test its activity. The final step was to test potential antifungal compounds developed by F2G to inhibit the enzymatic reaction, which was the key for the antifungal activity detected in earlier studies by F2G. Because membrane proteins are much harder to work with than soluble ones, they can sometimes be more difficult to obtain in sufficient amount and purity. Therefore, an attempt at engineering a contaminant-free P. pastoris cell line was carried out, with the goal of removing the main contaminant found during membrane protein production and purification.