AbstractAspergillus fumigatus is a filamentous fungus and considered a feared opportunistic pathogen in highly immunosuppressed patients. The spectrum and burden of clinical disease due to A. fumigatus is becoming increasingly complicated. Therapeutic management is limited to three antifungal classes (polyenes, triazoles and echinocandins) all of which have significant contraindications in terms of toxicity, drug-drug interactions and/or efficacy. With no new antifungal classes in late stage clinical trials what is required is the characterisation of new targets possessing novel modes of action. The work presented in this thesis addresses this evolving area of unmet clinical need using a target-based approach to assess a miniature portfolio of enzymes that are involved in the synthesis and remodelling of the A. fumigatus cell wall as potential antifungal targets.
The cell wall of A. fumigatus is predominantly composed of chitin, β-(1,3) glucan and galactomannan and is essential for survival and structural stability. Chitin, a linear polymer of β-(1,4) linked N-acetylglucosamine (GlcNAc), forms a rigid inner layer that is also partially hydrolysed during morphogenesis by glycoside hydrolase family 18 plant-type chitinases. In contrast, exploitation of glucosamine-6-phosphate N-acetyltransferase (GNA1) an enzyme in the highly conserved UDP-GlcNAc biosynthetic pathway is an elegant means of targeting chitin synthesis upstream by inhibition of the sugar nucleotide substrate UDPGlcNAc. Finally, UDP-galactopyranose mutase (UGM) is the sole source of the galactofuranose sugar that forms short crosslinks in the outer layer of galactomannan.
The underlying hypothesis is that gene deletion of A. fumigatus (i) plant-type chitinases, (ii) GNA1 and (iii) UGM produce distinctive biological phenotypes that are reproducible using small-molecule inhibitors and therefore fulfil the criteria of a “druggable” antifungal target.
This work describes the discovery of novel, low micromolar chemical inhibitors of A. fumigatus plant-type chitinase A1 by high-throughput screening. Analysis of the binding mode by X-ray crystallography confirmed competitive inhibition and further kinetic studies identified two compounds with selectivity towards A. fumigatus plant-type chitinases. The lead compound and a sub-micromolar derivative reduced biofilm formation and provide new chemical tools to further probe pan-chitinase inhibition on A. fumigatus biofilm growth and virulence, presenting attractive starting points for potentially developing further potent drug-like molecules.
An A. fumigatus Δgna1 null mutant and reconstituted strain were constructed and demonstrated that GNA1 is essential for in vitro cell viability under standard laboratory nutritional requirements. A. fumigatus Δgna1 null mutants wereavirulent in a mini host invertebrate model of infection and preliminary experiments in a murine model of inhalational aspergillosis were suggestive of attenuated virulence. In addition, a crystallographic complex of GNA1 with a ligand efficient compound derived from a fragment screen identified a previously uncharacterised fungal specific binding pocket adjacent to the sugar binding site. This discovery provides new insights into future selective small-molecule inhibitor development.
An important aspect of early-stage drug discovery is target prioritisation. Although UGM did not proceed beyond an initial chemical and structural exploration, the work described herein provides original contributions to the evidence base that further investment in A. fumigatus GNA1 and plant-type chitinases as novel antifungal targets is merited.
|Date of Award||2014|
|Sponsors||Medical Research Council|
|Supervisor||Daan van Aalten (Supervisor)|