ABSTRACT
Aspergillus fumigatus is a human opportunistic fungal pathogen whose cell wall protects it from the extracellular environment including host defenses. Chitin, an essential component of the fungal cell wall, is synthesized from UDP-GlcNAc produced in the hexosamine biosynthetic pathway. As this pathway is critical for fungal cell wall integrity, the hexosamine biosynthesis enzymes represent potential targets of antifungal drugs. Here, we provide genetic and chemical evidence that glucosamine 6-phosphate N-acetyltransferase (Gna1), a key enzyme in this pathway, is an exploitable antifungal drug target. GNA1 deletion resulted in loss of fungal viability and disruption of the cell wall, phenotypes that could be rescued by exogenous GlcNAc, the product of the Gna1 enzyme. In a murine model of aspergillosis, the Δgna1 mutant strain exhibited attenuated virulence. Using a fragment-based approach, we discovered a small heterocyclic scaffold that binds proximal to the Gna1 active site and can be optimized to a selective submicromolar binder. Taken together, we have provided genetic, structural, and chemical evidence that Gna1 is an antifungal target in A. fumigatus.
Subject(s)
Antifungal Agents/pharmacology , Aspergillus fumigatus/enzymology , Biosynthetic Pathways/drug effects , Glucosamine 6-Phosphate N-Acetyltransferase/antagonists & inhibitors , Hexosamines/metabolism , Animals , Antifungal Agents/chemistry , Aspergillosis/drug therapy , Aspergillosis/metabolism , Aspergillosis/microbiology , Aspergillus fumigatus/drug effects , Aspergillus fumigatus/metabolism , Catalytic Domain/drug effects , Cell Wall/drug effects , Cell Wall/metabolism , Chitin/metabolism , Crystallography, X-Ray , Glucosamine 6-Phosphate N-Acetyltransferase/chemistry , Glucosamine 6-Phosphate N-Acetyltransferase/metabolism , Male , Mice , Models, Molecular , Molecular Targeted Therapy , Protein Conformation/drug effects , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacologyABSTRACT
Apicomplexa form a phylum of obligate parasitic protozoa of great clinical and veterinary importance. These parasites synthesize glycoconjugates for their survival and infectivity, but the enzymatic steps required to generate the glycosylation precursors are not completely characterized. In particular, glucosamine-phosphate N-acetyltransferase (GNA1) activity, needed to produce the essential UDP-N-acetylglucosamine (UDP-GlcNAc) donor, has not been identified in any Apicomplexa. We scanned the genomes of Plasmodium falciparum and representatives from six additional main lineages of the phylum for proteins containing the Gcn5-related N-acetyltransferase (GNAT) domain. One family of GNAT-domain containing proteins, composed by a P. falciparum sequence and its six apicomplexan orthologs, rescued the growth of a yeast temperature-sensitive GNA1 mutant. Heterologous expression and in vitro assays confirmed the GNA1 enzymatic activity in all lineages. Sequence, phylogenetic and synteny analyses suggest an independent origin of the Apicomplexa-specific GNA1 family, parallel to the evolution of a different GNA1 family in other eukaryotes. The inability to disrupt an otherwise modifiable gene target suggests that the enzyme is essential for P. falciparum growth. The relevance of UDP-GlcNAc for parasite viability, together with the independent evolution and unique sequence features of Apicomplexa GNA1, highlights the potential of this enzyme as a selective therapeutic target against apicomplexans.
Subject(s)
Glucosamine 6-Phosphate N-Acetyltransferase/genetics , Glycoconjugates/biosynthesis , Plasmodium falciparum/enzymology , Trypanosoma brucei brucei/enzymology , Amino Acid Sequence , Base Sequence , CRISPR-Cas Systems , Crystallography, X-Ray , Evolution, Molecular , Glucosamine 6-Phosphate N-Acetyltransferase/antagonists & inhibitors , Glucosamine 6-Phosphate N-Acetyltransferase/chemistry , Glucosamine 6-Phosphate N-Acetyltransferase/metabolism , Mutation , Protein Structure, Secondary , Substrate SpecificityABSTRACT
Glucosamine 6-phosphate N-acetyltransferase (GNA1; EC 2.3.1.4) is required for the de novo synthesis of N-acetyl-d-glucosamine-6-phosphate (GlcNAc-6P), which is an essential precursor in Uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) biosynthesis pathway. Therefore, GNA1 is indispensable for the viability of organisms. Here, a novel cell-free expression strategy was developed to efficiently produce large amounts of human GNA1(HsGNA1) and HsGNA1-sGFP for throughput inhibitor screening. The binding site of inhibitor glucose-6-phosphate (G6P) to hGNA was identified by simulated annealing. Subtle differences to the binding site of Aspergillius GNA1(AfGNA1) can be harnessed for inhibitor design. HsGNA1 may be also useful as an antimicrobial and chemotherapeutic target against cancer. Additionally HsGNA1 inhibitors/modulators can possibly be administered with other drugs in the next generation of personalized medicine.
