Mode-of‐action of PAF26 and the discovery of more active and stable cyclic PAF26 derivatives
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The significance of fungal infections has been grossly underestimated. Only a few drugs are clinically available to treat life-‐threatening fungal infections, and resistance against these drugs is rising. Antifungal peptides (AFPs) are being actively explored as novel pharmaceuticals. PAF26 is a de novo designed hexapeptide possessing N-‐terminal cationic and C-‐terminal hydrophobic regions. Previously the roles of each of these motifs in the antifungal mode-‐of-‐action of PAF26 have indicated that it involves three stages: interaction with the plasma membrane, internalisation, and cell killing. The overall aim of my project was to obtain further insights into its mode-‐of-‐action and develop more active antifungal derivatives of PAF26. Three experimental fungal systems were used in this study: the model Neurospora crassa, the human pathogen Aspergillus fumigatus and the plant/human pathogen Fusarium oxysporum. The first objective of the study was to evaluate the impact of different fluorescent labels on the intracellular localisation and antifungal properties of PAF26. For this purpose a library of PAF26 labelled with 13 different fluorophores was synthesised. This library contained PAF26 conjugates of broad chemical and spectral diversity. These fluorescent PAF26 conjugates were analysed by live-‐cell imaging and tested for their antifungal activities. The different fluorescent labels were found to have significant impacts both on intracellular localisation and antifungal activities. TMR, carboxyfluorescein, NBD and DMN were found to be the best labels for live-‐cell imaging because they had the least influence on the intracellular localisation and antifungal activity of PAF26. The second objective was to identify target proteins of PAF26 in N. crassa cells. A large number of proteins were identified as binding to PAF26 from a protein pull-‐down and mass spectroscopy analysis using TMR-‐ and fluorescein-‐labelled PAF26. One of these proteins was the highly abundant plasma membrane ATPase PMA-‐1. An in-‐silico analysis showed that PMA-‐1 is likely to be a major target protein of PAF26. The final objective was to develop novel antifungals based on PAF26 with improved activities and stability. Novel cyclic derivatives of PAF26 were designed in-‐silico against PMA-‐1. These peptides were synthesised and tested against N. crassa, A. fumigatus and F. oxysporum and were found to have higher activities (at the sub-‐micromolar level) and greater stability than the linear PAF26. Overall this study has provided novel mechanistic insights into the mode-‐of-‐action of PAF26 and discovered novel highly active antifungal peptides with clinical potential as therapeutics.