Structural and biochemical studies on three Aspergillus fumigatus proteins that present as targets for novel antifungal drugs

Abstract

The increasing use of aggressive immunosuppressive regimes over the last half-century has been accompanied by an increased incidence of opportunistic invasive fungal infections, with Aspergillus fumigatus emerging as one of the main etiologic agents. A. fumigatus is now the major airborne fungal pathogen and is a pathogen to be feared; it is difficult to diagnose early and accurately, a detailed understanding of the factors regulating its growth and pathogenesis is lacking, and invasive infections are associated with high mortality rates, longer hospital stays and high treatment costs. In addition, the number of antifungals effective against it is limited and resistance is increasing, highlighting the need for the characterisation of novel targets for new antifungals. A detailed knowledge of the structure of a protein is essential for understanding its underlying molecular mechanism and enabling structure-based drug design. This dissertation reports the application a structural genomics-style approach to a pilot set of proteins that are involved in a range of processes essential for the growth, cellular homeostasis and survival of A. fumigatus in the human host. Following on from preliminary experiments to identify proteins amenable to study by X-ray crystallography, three A. fumigatus proteins were selected for comprehensive structural and biochemical characterisation: (1) Crystal structures of cytosolic thiolase (afCT), which catalyses the first step in isoprenoid synthesis, were solved to resolutions between 1.7 and 2.25 Å. One of these crystals trapped two previously unobserved tetrahedral reaction intermediates, providing the first direct structural observation of the full thiolase reaction cycle. Unexpectedly, afCT is more similar structurally and biochemically to human mitochondrial thiolase II (hT2) than it is to human cytosolic thiolase (hCT). Here it is shown that, like hT2, afCT is strongly activated by monovalent cations, with a preference for K⁺ ions. Additionally, structural comparisons and in silico docking experiments suggest that afCT has a substrate specificity more similar to hT2 than hCT. (2) The structure of proliferating cell nuclear antigen (AfumPCNA), which is essential for DNA replication and repair, was solved to 2.6 Å. Strong structural similarities between AfumPCNA and human PCNA at their main protein-protein interaction site suggest that the structural basis by which PCNA functions is conserved. Consistent with this, it is shown herein that the PCNA-interacting peptide of the human tumour-suppressor protein p21 binds AfumPCNA with only ten-fold lower affinity than for hPCNA, presenting AfumPCNA as a target for potential peptide-based antifungals. (3) Thioredoxin reductase (AfTrxR) catalyses the transfer of reducing equivalents from NADPH to a number of important biosynthetic and redox-active enzymes. The structure of AfTrxR was solved to 3.2 Å, and shows high structural similarity to bacterial, plant and yeast TrxRs. It is also shown herein that ebselen (EbSe), a small drug-like molecule, is a nanomolar inhibitor of AfTrxR in vitro and a potent inhibitor of A. fumigatus growth. Further investigation by mass spectrometry revealed that EbSe interacts covalently with a redox-active cysteine at the AfTrxR catalytic site. In silico docking experiments were used to define key enzyme-inhibitor interactions that will be important to consider when designing potent and specific antifungal drugs targeting TrxR in the future.