Abstract

Infections caused by the opportunistic fungal pathogen Aspergillus fumigatus pose a serious public health system burden. The inherent limitations in existing antifungal drugs in conjunction with a rising emergence of antifungal resistance emphasizes an urgent need to identify and target alternative pathways crucial to survival and virulence. Targeting the fungal mannitol biosynthesis enzymes provides a promising avenue in the development of new antifungals due to the multifaceted roles mannitol fulfils in the fungal life cycle. However, a distinct lack of available structural information for these enzymes has hindered drug discovery efforts. We report the first crystal structure of mannitol-2-dehydrogenase from A. fumigatus in an unbound monomeric state (1.8 [A]) and bound to its co-factor NADH (2.1 [A]), via. a large, central cavity lined with positively charged residues that readily accommodates NADH. This interaction is further stabilised by a network of hydrogen bond interactions and ?-? stacking between Phe45 and the nicotinamide ring of NADH. Furthermore, rigorous kinetic characterisation of A. fumigatus mannitol-2-dehydrogenase demonstrates the dose-dependent inhibitory activity of 1,4-benzoquinone, a cysteine-modifying small molecule inhibitor (IC50 = 1.2 {+/-} 0.2 nM). In addition, intact MS and proteomic analysis further reveal that 1,4-benzoquinone modifies up to five cysteine residues of mannitol-2-dehydrogenase and displays antifungal activity against A. fumigatus, which is enhanced in combination with a front-line antifungal voriconazole. From this work, we have established the foundations for a novel antifungal drug discovery avenue that targets the fungal mannitol biosynthesis pathway to better treat aspergillosis and related pathogenic infections.