Validation of Aspergillus fumigatus glucosamine-6-phosphate N-acetyltransferase as a potential antifungal target

Abstract

BackgroundAspergillus fumigatus presents a spectrum of clinical, diagnostic, and therapeutic challenges. A new pipeline of novel validated antifungal targets is needed to address limitations in existing agents. The fungal cell wall is essential for survival, with chitin (β(1-4) linked N-acetyl-D-glucosamine [GlcNAc]) forming an integral layer. Enzymes involved in the biosynthesis of its sugar nucleotide precursor, UDP-GlcNAc, are potential antifungal targets. One such enzyme is glucosamine-6-phosphate N-acetyltransferase (GNA1) that N-acetylates glucosamine-6-phosphate to N-acetyl-glucosamine-6-phosphate as an intermediary step. Here we genetically validate A fumigatus GNA1 as an antifungal target and assess whether small-molecule ligands bind to the protein. MethodsWe constructed a gene deletion mutant, based on homologous recombination, by replacing the afugna1 coding sequence with the pyrG selectable marker. Purified GNA1 from A fumigatus (AfGNA1) was chemically biotinylated, and a fragment screen using bio-layer interferometry (Octet Red, Pall Forte Bio, Menlo Park, CA, USA) done to assess the binding of fragments to the target protein. X-ray crystallography was used to verify preliminary hits and determine the binding mode. FindingsThe mutant strain of A fumigatus was unable to grow in vitro without the addition of exogenous GlcNAc. Screening the Dundee Drug Discovery Unit (DDU) fragment library gave a preliminary hit rate of 5·7 % (37/652 with a response rate >0·02 nm). A subset of seven fragments showed stoichiometric binding with equilibrium dissociation constants in the micromolar range. Structural analysis of AfGNA1 in complex with fragment (A) showed that the fragment binds in a groove behind the sugar substrate. InterpretationFragment-based drug discovery provides a complementary and contrasting approach to traditional high-throughput methods by elaborating weakly binding small molecules. Iterative cycles of medicinal chemistry and structural biology are required to optimise this chemical anchor by extending into the active site to generate highly potent drug-like lead molecules. This work suggests that GNA1 is essential for cell viability in A fumigatus and could be a potential novel antifungal target. FundingUK Medical Research Council.