Structural and Biochemical Insights into Antifungal Drug Targets from Aspergillus fumigatus
Date
2021
Authors
Nguyen, Stephanie
Editors
Advisors
Bruning, John
Shearwin, Keith
Jovcevski, Blagojce
Shearwin, Keith
Jovcevski, Blagojce
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Thesis
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Abstract
Aspergillus fumigatus, the leading cause of invasive aspergillosis, has been recognised as a priority fungal pathogen by the World Health Organisation due to its impact on human health and the emergence of resistance against existing antifungal therapeutics. Ubiquitously found in the natural environment, A. fumigatus produces air-borne fungal spores that are routinely inhaled into the respiratory system where the initial infection is established. In severe cases, the fungus can subsequently invade multiple organs, causing invasive aspergillosis. Current approaches to treating invasive fungal infections require aggressive and expensive antifungal therapy with limited success due to misdiagnosis, poor drug pharmacokinetics and pharmacodynamics, high drug toxicity and low efficacy of existing drugs against resistant strains. As the mortality rate associated with invasive aspergillosis remains unacceptably high and primary therapy failure occurs in 40% - 70% for invasive aspergillosis patients, there is a need to identify new targets for the development of novel classes of antifungal therapeutics. In order to effectively embark on a drug discovery project, intimate structural and biochemical knowledge of the target is required. Together, this information provides insights into protein function and furthers our understanding of their contribution to fungal survival and virulence. Furthermore, the structural data will be essential in guiding rational drug design efforts. This thesis focuses on four A. fumigatus enzymes from the purine biosynthesis, mannitol biosynthesis and glycolysis pathways that have been identified as promising antifungal drug targets. The structure and kinetic characteristics of each enzyme has been comprehensively studied using X-ray crystallography, analytical size-exclusion chromatography, native mass spectrometry and in vitro activity assays. Findings from this work has provided a necessary foundation for future antifungal drug discovery projects that target nucleoside diphosphatekinase (NDK), guanosine monophosphate (GMP) synthase, mannitol-2-dehydrogenase (M2DH) and enolase. (1) Seven crystal structures of NDK (1.6 Å – 2.3 Å) were solved, either in an unbound form, or bound to one of the six possible nucleoside triphosphate substrates used in its reaction. Analysis of the kinetic properties of the enzyme revealed the following order of preference: adenosine > guanosine > inosine > uridine > thymidine > cytidine substrates. By combining the structural and kinetic data obtained, the structural determinants that govern nucleoside selectivity in A. fumigatus NDK were determined. (2) The first structure of a fungal GMP synthase enzyme from A. fumigatus was solved to a resolution of 2.3 Å. Analysis of this structure in comparison to the existing structure of the human homologue has revealed significant differences. Despite both being from eukaryotic species, the A. fumigatus GMP synthase lacks a D1 dimerisation domain that is observed in the human counterpart. Although denoted as the D1 dimerisation domain in the literature, this domain is known to prevent dimerisation. As a result, GMP synthase forms a dimeric complex, which is contrary to the monomeric human homologue. Analysis of binding-pocket residues in combination with rigorous analysis of the kinetic properties have revealed species-specific differences exist between fungal and human GMP synthases. This data will be imperative in the design of inhibitors that selectively target the fungal homologue. (3) The crystal structure of A. fumigatus M2DH in an unbound state (1.8 Å) and bound to NADH (2.1 Å) were solved. Analysis of these structures have revealed a central binding cavity that is located between the N- and C-terminal domains of the enzyme. This binding pocket is predominantly positively charged that readily accommodates the negative charges derived from the phosphate groups of NADH. Kinetic analysis of the substrates and co-factors used in the interconversion reaction have revealed that M2DH is likely to play a supporting role to the enzyme mannitol-1-phosphate-5-dehydrogenase in the biosynthesis of mannitol in fungi. As a proof of concept, we have shown that the activity of M2DH can be modulated by the small molecule 1,4-benzoquinone and have determined that the mechanism of inhibition is achieved by the covalent modification of all 5 cysteine residues. (4) Although the canonical function of cytoplasmic enolase as a glycolysis enzyme is well understood, its ‘moonlighting’ functions as a virulence factor have not been explored in molecular detail. Enolase, when expressed on the surface of pathogenic bacteria and fungi, can function as a receptor for human proteins, including plasminogen. This interaction drives tissue invasion and facilitates nutrient acquisition, both of which function to accelerate infection. A novel approach to developing antifungal drugs aims to prevent the formation of the enolase-plasminogen complex. In order to better understand this interaction, the crystal structure of A. fumigatus enolase, unbound (2.0 Å) and bound to its endogenous substrates, 2-phosphoglycerate (1.9 Å) and phosphoenolpyruvate (2.3 Å) were determined. Enolase from A. fumigatus forms a dimeric complex in which both monomers are arranged in an anti-parallel orientation. Using the solved crystal structure of A. fumigatus enolase and the existing crystal structure of human plasminogen, we have developed a model of this complex using protein: protein docking software. These data provide insights to better understand this interaction and provides foundations to guide the design of peptidomimetics or small molecules that could potentially disrupt this interaction.
School/Discipline
School of Biological Sciences
Dissertation Note
Thesis (Ph.D.) -- University of Adelaide, School of Biological Sciences, 2021
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