In this PhD thesis I will explore the development of antifungal drugs. Fungal infections are estimated to cause the death of 1.5 million patients each year. There is currently a need for new antifungal drugs as the existing drugs are hampered by lack of broad-spectrum antifungal activity, resistance development and side effects. In an effort to promote the development of new antifungal drugs, I have explored the mechanism of two types of fungal growth inhibitors.
The first project targets the fungal plasma membrane H+-ATPase (Pma1), which is responsible for generating the membrane potential and controlling the intracellular pH. Pma1 is an essential fungal protein that is not present in humans, and it is therefore an obvious drug target. During this project, I have developed methods for studying Pma1 inhibition, and the resulting data combined with data from the literature has revealed that the typical traits for a range of Pma1 inhibitors is characterized by a decrease in membrane potential, decrease in intracellular pH, inhibition of H+-ATPase medium acidification, and a large increase in intracellular ATP (iATP). Most of these observations were expected, but they have now been confirmed with experimental data. One exception, however, is the large increase in iATP observed after Pma1 inhibition with Pma1 inhibitors. This assay was primarily performed to exclude the possibility of an indirect inhibition of Pma1 by a decrease in iATP, which is the energy source for Pma1. The large increase in iATP possible occurs as an effort to upregulate the activity of Pma1 during its inhibition.
In the second project, I have explored a novel series of compounds with broad-spectrum antifungal activity. These compounds were found to modulate fungal zinc homeostasis and were therefore designated as zinc attenuating compounds (ZACs). Zinc is an important micronutrient and the immune system is known to operate with a similar mechanism to the ZACs by scavenging zinc from the site of infection, thus preventing the growth of pathogens through zinc starvation.
In addition to the observations made about the ZAC compounds, extracellular addition of zinc and glucose was also found to modify the zinc homeostasis in Candida albicans. It was found that zinc was dynamically released from intracellular membranes such as the ER in response to the extracellular stimuli and this could be reversed by applying EDTA, which chelates extracellular zinc. The glucose-induced intracellular release of zinc was found to be dependent on the cyclic AMP-PKA pathway, while the presence of extracellular zinc did not induce this pathway. In mammalian cells, zinc is an established signaling molecule, but similar reports have not yet emerged for the fungal kingdom. The results generated in this PhD project strongly suggest that zinc also functions as a signaling molecule within C. albicans, and this offers a potential additional explanation for the antifungal activity of the ZACs. It remains to be investigated whether or not differences in zinc signaling between fungal and mammalian cells can explain the apparent preference of ZACs for inhibiting fungal cells.