RESUMO
Loss-of-function mutations in PTEN-induced kinase 1 (PINK1) are a frequent cause of early-onset Parkinson's disease (PD). Stabilization of PINK1 at the translocase of outer membrane (TOM) complex of damaged mitochondria is critical for its activation. The mechanism of how PINK1 is activated in the TOM complex is unclear. Here, we report that co-expression of human PINK1 and all seven TOM subunits in Saccharomyces cerevisiae is sufficient for PINK1 activation. We use this reconstitution system to systematically assess the role of each TOM subunit toward PINK1 activation. We unambiguously demonstrate that the TOM20 and TOM70 receptor subunits are required for optimal PINK1 activation and map their sites of interaction with PINK1 using AlphaFold structural modeling and mutagenesis. We also demonstrate an essential role of the pore-containing subunit TOM40 and its structurally associated subunits TOM7 and TOM22 for PINK1 activation. These findings will aid in the development of small-molecule activators of PINK1 as a therapeutic strategy for PD.
Assuntos
Proteínas de Transporte da Membrana Mitocondrial , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Proteínas Quinases , Saccharomyces cerevisiae , Proteínas Quinases/metabolismo , Proteínas Quinases/genética , Humanos , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Mitocôndrias/metabolismo , Ligação Proteica , Ativação Enzimática , Modelos Moleculares , Subunidades Proteicas/metabolismo , Subunidades Proteicas/genéticaRESUMO
Aspergillus fumigatus is a devastating opportunistic fungal pathogen causing hundreds of thousands of deaths every year. Phosphoglucose isomerase (PGI) is a glycolytic enzyme that converts glucose-6-phosphate to fructose-6-phosphate, a key precursor of fungal cell wall biosynthesis. Here, we demonstrate that the growth of A. fumigatus is repressed by the deletion of pgi, which can be rescued by glucose and fructose supplementation in a 1:10 ratio. Even under these optimized growth conditions, the Δpgi mutant exhibits severe cell wall defects, retarded development, and attenuated virulence in Caenorhabditis elegans and Galleria mellonella infection models. To facilitate exploitation of A. fumigatus PGI as an antifungal target, we determined its crystal structure, revealing potential avenues for developing inhibitors, which could potentially be used as adjunctive therapy in combination with other systemic antifungals. IMPORTANCE Aspergillus fumigatus is an opportunistic fungal pathogen causing deadly infections in immunocompromised patients. Enzymes essential for fungal survival and cell wall biosynthesis are considered potential drug targets against A. fumigatus. PGI catalyzes the second step of the glycolysis pathway, linking glycolysis and the pentose phosphate pathway. As such, PGI has been widely considered as a target for metabolic regulation and therefore a therapeutic target against hypoxia-related diseases. Our study here reveals that PGI is important for A. fumigatus survival and exhibit pleiotropic functions, including development, cell wall glucan biosynthesis, and virulence. We also solved the crystal structure of PGI, thus providing the genetic and structural groundwork for the exploitation of PGI as a potential antifungal target.
Assuntos
Aspergillus fumigatus , Glucose-6-Fosfato Isomerase , Antifúngicos/farmacologia , Aspergillus fumigatus/metabolismo , Parede Celular/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Glucose-6-Fosfato Isomerase/genética , Glucose-6-Fosfato Isomerase/metabolismo , Humanos , VirulênciaRESUMO
Aspergillus fumigatus is the causative agent of invasive aspergillosis, an infection with mortality rates of up to 50%. The glucan-rich cell wall of A. fumigatus is a protective structure that is absent from human cells and is a potential target for antifungal treatments. Glucan is synthesized from the donor uridine diphosphate glucose, with the conversion of glucose-6-phosphate to glucose-1-phosphate by the enzyme phosphoglucomutase (PGM) representing a key step in its biosynthesis. Here, we explore the possibility of selectively targeting A. fumigatus PGM (AfPGM) as an antifungal treatment strategy. Using a promoter replacement strategy, we constructed a conditional pgm mutant and revealed that pgm is required for A. fumigatus growth and cell wall integrity. In addition, using a fragment screen, we identified the thiol-reactive compound isothiazolone fragment of PGM as targeting a cysteine residue not conserved in the human ortholog. Furthermore, through scaffold exploration, we synthesized a para-aryl derivative (ISFP10) and demonstrated that it inhibits AfPGM with an IC50 of 2 µM and exhibits 50-fold selectivity over the human enzyme. Taken together, our data provide genetic validation of PGM as a therapeutic target and suggest new avenues for inhibiting AfPGM using covalent inhibitors that could serve as tools for chemical validation.
Assuntos
Aspergilose , Aspergillus fumigatus , Antifúngicos/farmacologia , Aspergilose/tratamento farmacológico , Aspergilose/microbiologia , Aspergillus fumigatus/enzimologia , Aspergillus fumigatus/genética , Glucanos/metabolismo , Humanos , Fosfoglucomutase/genética , Fosfoglucomutase/metabolismoRESUMO
Autosomal recessive mutations in the PINK1 gene are causal for Parkinson's disease (PD). PINK1 encodes a mitochondrial localized protein kinase that is a master-regulator of mitochondrial quality control pathways. Structural studies to date have elaborated the mechanism of how mutations located within the kinase domain disrupt PINK1 function; however, the molecular mechanism of PINK1 mutations located upstream and downstream of the kinase domain is unknown. We have employed mutagenesis studies to define the minimal region of human PINK1 required for optimal ubiquitin phosphorylation, beginning at residue Ile111. Inspection of the AlphaFold human PINK1 structure model predicts a conserved N-terminal α-helical extension (NTE) domain forming an intramolecular interaction with the C-terminal extension (CTE), which we corroborate using hydrogen/deuterium exchange mass spectrometry of recombinant insect PINK1 protein. Cell-based analysis of human PINK1 reveals that PD-associated mutations (e.g. Q126P), located within the NTE : CTE interface, markedly inhibit stabilization of PINK1; autophosphorylation at Serine228 (Ser228) and Ubiquitin Serine65 (Ser65) phosphorylation. Furthermore, we provide evidence that NTE and CTE domain mutants disrupt PINK1 stabilization at the mitochondrial Translocase of outer membrane complex. The clinical relevance of our findings is supported by the demonstration of defective stabilization and activation of endogenous PINK1 in human fibroblasts of a patient with early-onset PD due to homozygous PINK1 Q126P mutations. Overall, we define a functional role of the NTE : CTE interface towards PINK1 stabilization and activation and show that loss of NTE : CTE interactions is a major mechanism of PINK1-associated mutations linked to PD.
Assuntos
Proteínas Quinases , Ubiquitina , Ativação Enzimática , Humanos , Fosforilação , Conformação Proteica em alfa-Hélice , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Aspergillus fumigatus is an opportunistic mold responsible for severe life-threatening fungal infections in immunocompromised patients. The cell wall, an essential structure composed of glucan, chitin, and galactomannan, is considered to be a target for the development of antifungal drugs. The nucleotide sugar donor GDP-mannose (GDP-Man) is required for the biosynthesis of galactomannan, glycosylphosphatidylinositol (GPI) anchors, glycolipid, and protein glycosylation. Starting from fructose-6-phosphate, GDP-Man is produced by the sequential action of the enzymes phosphomannose isomerase, phosphomannomutase (Pmm), and GDP-mannose pyrophosphorylase. Here, using heterokaryon rescue and gene knockdown approaches we demonstrate that the phosphomannomutase encoding gene in A. fumigatus (pmmA) is essential for survival. Reduced expression of pmmA is associated with significant morphological defects including retarded germination, growth, reduced conidiation, and abnormal polarity. Moreover, the knockdown strain exhibited an altered cell wall organization and sensitivity toward cell wall perturbing agents. By solving the first crystal structure of A. fumigatus phosphomannomutase (AfPmmA) we identified non-conservative substitutions near the active site when compared to the human orthologues. Taken together, this work provides a genetic and structural foundation for the exploitation of AfPmmA as a potential antifungal target.
Assuntos
Aspergillus fumigatus/genética , Guanosina Difosfato Manose/metabolismo , Fosfotransferases (Fosfomutases)/genética , Fosfotransferases (Fosfomutases)/metabolismo , Antifúngicos/farmacologia , Aspergilose/tratamento farmacológico , Aspergilose/patologia , Aspergillus fumigatus/efeitos dos fármacos , Aspergillus fumigatus/metabolismo , Parede Celular/metabolismo , Deleção de Genes , Humanos , Virulência/genéticaRESUMO
O-GlcNAcylation is a post-translational modification catalysed by O-GlcNAc transferase (OGT). Missense mutations in OGT have been associated with developmental disorders, OGT-linked congenital disorder of glycosylation (OGT-CDG), which are characterized by intellectual disability. OGT relies on the hexosamine biosynthetic pathway (HBP) for provision of its UDP-GlcNAc donor. We considered whether mutations in UDP-N-acetylhexosamine pyrophosphorylase (UAP1), which catalyses the final step in the HBP, would phenocopy OGT-CDG mutations. A de novo mutation in UAP1 (NM_001324114:c.G685A:p.A229T) was reported in a patient with intellectual disability. We show that this mutation is pathogenic and decreases the stability and activity of the UAP1 isoform AGX1 in vitro. X-ray crystallography reveals a structural shift proximal to the mutation, leading to a conformational change of the N-terminal domain. These data suggest that the UAP1A229T missense mutation could be a contributory factor to the patient phenotype.
Assuntos
Deficiências do Desenvolvimento/genética , Galactosiltransferases/genética , Hexosaminas/biossíntese , Mutação de Sentido Incorreto , Sequência de Aminoácidos , Animais , Cristalografia por Raios X , Estabilidade Enzimática , Galactosiltransferases/química , Galactosiltransferases/metabolismo , Humanos , Processamento de Proteína Pós-Traducional , Homologia de Sequência de AminoácidosRESUMO
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.
Assuntos
Antifúngicos/farmacologia , Aspergillus fumigatus/enzimologia , Vias Biossintéticas/efeitos dos fármacos , Glucosamina 6-Fosfato N-Acetiltransferase/antagonistas & inibidores , Hexosaminas/metabolismo , Animais , Antifúngicos/química , Aspergilose/tratamento farmacológico , Aspergilose/metabolismo , Aspergilose/microbiologia , Aspergillus fumigatus/efeitos dos fármacos , Aspergillus fumigatus/metabolismo , Domínio Catalítico/efeitos dos fármacos , Parede Celular/efeitos dos fármacos , Parede Celular/metabolismo , Quitina/metabolismo , Cristalografia por Raios X , Glucosamina 6-Fosfato N-Acetiltransferase/química , Glucosamina 6-Fosfato N-Acetiltransferase/metabolismo , Masculino , Camundongos , Modelos Moleculares , Terapia de Alvo Molecular , Conformação Proteica/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
UDP-N-acetylglucosamine pyrophosphorylase (UAP1) catalyses the last step in eukaryotic biosynthesis of uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc), converting UTP and GlcNAc-1P to the sugar nucleotide. Gene disruption studies have shown that this gene is essential in eukaryotes and a possible antifungal target, yet no inhibitors of fungal UAP1 have so far been reported. Here we describe the crystal structures of substrate/product complexes of UAP1 from Aspergillus fumigatus that together provide snapshots of catalysis. A structure with UDP-GlcNAc, pyrophosphate and Mg2+ provides the first Michaelis complex trapped for this class of enzyme, revealing the structural basis of the previously reported Mg2+ dependence and direct observation of pyrophosphorolysis. We also show that a highly conserved lysine mimics the role of a second metal observed in structures of bacterial orthologues. A mechanism-inspired UTP α,ß-methylenebisphosphonate analogue (meUTP) was designed and synthesized and was shown to be a micromolar inhibitor of the enzyme. The mechanistic insights and inhibitor described here will facilitate future studies towards the discovery of small molecule inhibitors of this currently unexploited potential antifungal drug target.
RESUMO
N-acetylphosphoglucosamine mutase (AGM1) is a key component of the hexosamine biosynthetic pathway that produces UDP-GlcNAc, an essential precursor for a wide range of glycans in eukaryotes. AGM belongs to the α-d-phosphohexomutase metalloenzyme superfamily and catalyzes the interconversion of N-acetylglucosamine-6-phosphate (GlcNAc-6P) to N-acetylglucosamine-1-phosphate (GlcNAc-1P) through N-acetylglucosamine-1,6-bisphosphate (GlcNAc-1,6-bisP) as the catalytic intermediate. Although there is an understanding of the phosphoserine-dependent catalytic mechanism at enzymatic and structural level, the identity of the requisite catalytic base in AGM1/phosphoglucomutases is as yet unknown. Here, we present crystal structures of a Michaelis complex of AGM1 with GlcNAc-6P and Mg2+, and a complex of the inactive Ser69Ala mutant together with glucose-1,6-bisphosphate (Glc-1,6-bisP) that represents key snapshots along the reaction co-ordinate. Together with mutagenesis, these structures reveal that the phosphate group of the hexose-1,6-bisP intermediate may act as the catalytic base.
Assuntos
Acetilglucosamina/análogos & derivados , Aspergillus fumigatus/enzimologia , Proteínas Fúngicas/química , Glucose-6-Fosfato/análogos & derivados , Fosfoglucomutase/química , Acetilglucosamina/química , Acetilglucosamina/metabolismo , Catálise , Proteínas Fúngicas/metabolismo , Glucose-6-Fosfato/química , Glucose-6-Fosfato/metabolismo , Fosfoglucomutase/metabolismoRESUMO
Mutations in the human kinase PINK1 (hPINK1) are associated with autosomal recessive early-onset Parkinson's disease (PD). hPINK1 activates Parkin E3 ligase activity, involving phosphorylation of ubiquitin and the Parkin ubiquitin-like (Ubl) domain via as yet poorly understood mechanisms. hPINK1 is unusual amongst kinases due to the presence of three loop insertions of unknown function. We report the structure of Tribolium castaneum PINK1 (TcPINK1), revealing several unique extensions to the canonical protein kinase fold. The third insertion, together with autophosphorylation at residue Ser205, contributes to formation of a bowl-shaped binding site for ubiquitin. We also define a novel structural element within the second insertion that is held together by a distal loop that is critical for TcPINK1 activity. The structure of TcPINK1 explains how PD-linked mutations that lie within the kinase domain result in hPINK1 loss-of-function and provides a platform for the exploration of small molecule modulators of hPINK1.
Assuntos
Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Tribolium/enzimologia , Animais , Sítios de Ligação , Cristalografia por Raios X , Células HeLa , Humanos , Modelos Moleculares , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Doença de Parkinson/fisiopatologia , Ligação Proteica , Conformação Proteica , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Ubiquitina/metabolismoRESUMO
Treatment of filamentous fungal infections relies on a limited repertoire of antifungal agents. Compounds possessing novel modes of action are urgently required. N-myristoylation is a ubiquitous modification of eukaryotic proteins. The enzyme N-myristoyltransferase (NMT) has been considered a potential therapeutic target in protozoa and yeasts. Here, we show that the filamentous fungal pathogen Aspergillus fumigatus possesses an active NMT enzyme that is essential for survival. Surprisingly, partial repression of the gene revealed downstream effects of N-myristoylation on cell wall morphology. Screening a library of inhibitors led to the discovery of a pyrazole sulphonamide compound that inhibits the enzyme and is fungicidal under partially repressive nmt conditions. Together with a crystallographic complex showing the inhibitor binding in the peptide substrate pocket, we provide evidence of NMT being a potential drug target in A. fumigatus.
Assuntos
Aciltransferases/antagonistas & inibidores , Antifúngicos/farmacologia , Aspergillus fumigatus/efeitos dos fármacos , Parede Celular/efeitos dos fármacos , Proteínas Fúngicas/antagonistas & inibidores , Processamento de Proteína Pós-Traducional , Aciltransferases/química , Aciltransferases/metabolismo , Aminopiridinas/química , Aminopiridinas/farmacologia , Antifúngicos/química , Aspergillus fumigatus/enzimologia , Aspergillus fumigatus/genética , Aspergillus fumigatus/crescimento & desenvolvimento , Domínio Catalítico , Parede Celular/química , Parede Celular/enzimologia , Cristalografia por Raios X , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Cinética , Testes de Sensibilidade Microbiana , Ligação Proteica , Estrutura Secundária de Proteína , Pirazóis/química , Pirazóis/farmacologia , Relação Estrutura-Atividade , Sulfonamidas/química , Sulfonamidas/farmacologiaRESUMO
Aspergillus fumigatus is the causative agent of IA (invasive aspergillosis) in immunocompromised patients. It possesses a cell wall composed of chitin, glucan and galactomannan, polymeric carbohydrates synthesized by processive glycosyltransferases from intracellular sugar nucleotide donors. Here we demonstrate that A. fumigatus possesses an active AfAGM1 (A. fumigatus N-acetylphosphoglucosamine mutase), a key enzyme in the biosynthesis of UDP (uridine diphosphate)-GlcNAc (N-acetylglucosamine), the nucleotide sugar donor for chitin synthesis. A conditional agm1 mutant revealed the gene to be essential. Reduced expression of agm1 resulted in retarded cell growth and altered cell wall ultrastructure and composition. The crystal structure of AfAGM1 revealed an amino acid change in the active site compared with the human enzyme, which could be exploitable in the design of selective inhibitors. AfAGM1 inhibitors were discovered by high-throughput screening, inhibiting the enzyme with IC50s in the low µM range. Together, these data provide a platform for the future development of AfAGM1 inhibitors with antifungal activity.
Assuntos
Aspergillus fumigatus/enzimologia , Proteínas Fúngicas/química , Fosfotransferases (Fosfomutases)/química , Antifúngicos/química , Aspergillus fumigatus/ultraestrutura , Domínio Catalítico , Parede Celular/enzimologia , Cristalografia por Raios X , Proteínas Fúngicas/antagonistas & inibidores , Proteínas Fúngicas/genética , Humanos , Concentração Inibidora 50 , Cinética , Magnésio , Testes de Sensibilidade Microbiana , Viabilidade Microbiana , Modelos Moleculares , Fosfotransferases (Fosfomutases)/antagonistas & inibidores , Fosfotransferases (Fosfomutases)/genética , Estrutura Secundária de ProteínaRESUMO
Uridine diphosphate N-acetylglucosamine pyrophosphorylase (UAP) catalyzes the final reaction in the biosynthesis of UDP-GlcNAc, an essential metabolite in many organisms including Trypanosoma brucei, the etiological agent of Human African Trypanosomiasis. High-throughput screening of recombinant T. brucei UAP identified a UTP-competitive inhibitor with selectivity over the human counterpart despite the high level of conservation of active site residues. Biophysical characterization of the UAP enzyme kinetics revealed that the human and trypanosome enzymes both display a strictly ordered bi-bi mechanism, but with the order of substrate binding reversed. Structural characterization of the T. brucei UAP-inhibitor complex revealed that the inhibitor binds at an allosteric site absent in the human homologue that prevents the conformational rearrangement required to bind UTP. The identification of a selective inhibitory allosteric binding site in the parasite enzyme has therapeutic potential.
Assuntos
Nucleotidiltransferases/antagonistas & inibidores , Tripanossomicidas/química , Tripanossomicidas/farmacologia , Trypanosoma brucei brucei/efeitos dos fármacos , Trypanosoma brucei brucei/enzimologia , Regulação Alostérica/efeitos dos fármacos , Domínio Catalítico , Humanos , Nucleotidiltransferases/química , Nucleotidiltransferases/metabolismo , Conformação Proteica , Trypanosoma brucei brucei/crescimento & desenvolvimento , Tripanossomíase Africana/tratamento farmacológico , Tripanossomíase Africana/parasitologia , Uridina Difosfato N-Acetilglicosamina/metabolismoRESUMO
The sugar nucleotide UDP-N-acetylglucosamine (UDP-GlcNAc) is an essential metabolite in both prokaryotes and eukaryotes. In fungi, it is the precursor for the synthesis of chitin, an essential component of the fungal cell wall. UDP-N-acetylglucosamine pyrophosphorylase (UAP) is the final enzyme in eukaryotic UDP-GlcNAc biosynthesis, converting UTP and N-acetylglucosamine-1-phosphate (GlcNAc-1P) to UDP-GlcNAc. As such, this enzyme may provide an attractive target against pathogenic fungi. Here, we demonstrate that the fungal pathogen Aspergillus fumigatus possesses an active UAP (AfUAP1) that shows selectivity for GlcNAc-1P as the phosphosugar substrate. A conditional mutant, constructed by replacing the native promoter of the A. fumigatusâ uap1 gene with the Aspergillus nidulansâ alcA promoter, revealed that uap1 is essential for cell survival and important for cell wall synthesis and morphogenesis. The crystal structure of AfUAP1 was determined and revealed exploitable differences in the active site compared with the human enzyme. Thus AfUAP1 could represent a novel antifungal target and this work will assist the future discovery of small molecule inhibitors against this enzyme.
Assuntos
Aspergillus fumigatus/enzimologia , Proteínas Fúngicas/química , Nucleotidiltransferases/química , Acetilglucosamina/análogos & derivados , Acetilglucosamina/química , Aspergillus fumigatus/genética , Aspergillus fumigatus/ultraestrutura , Parede Celular/ultraestrutura , Clonagem Molecular , Proteínas Fúngicas/genética , Genes Essenciais , Mutação , Nucleotidiltransferases/genética , Regiões Promotoras Genéticas , Estrutura Terciária de Proteína , Especificidade por SubstratoRESUMO
African sleeping sickness or human African trypanosomiasis, caused by Trypanosoma brucei spp., is responsible for approximately 30,000 deaths each year. Available treatments for this disease are poor, with unacceptable efficacy and safety profiles, particularly in the late stage of the disease when the parasite has infected the central nervous system. Here we report the validation of a molecular target and the discovery of associated lead compounds with the potential to address this lack of suitable treatments. Inhibition of this target-T. brucei N-myristoyltransferase-leads to rapid killing of trypanosomes both in vitro and in vivo and cures trypanosomiasis in mice. These high-affinity inhibitors bind into the peptide substrate pocket of the enzyme and inhibit protein N-myristoylation in trypanosomes. The compounds identified have promising pharmaceutical properties and represent an opportunity to develop oral drugs to treat this devastating disease. Our studies validate T. brucei N-myristoyltransferase as a promising therapeutic target for human African trypanosomiasis.
Assuntos
Aciltransferases/antagonistas & inibidores , Antiparasitários/farmacologia , Antiparasitários/uso terapêutico , Trypanosoma brucei brucei/efeitos dos fármacos , Trypanosoma brucei brucei/enzimologia , Tripanossomíase Africana/tratamento farmacológico , Tripanossomíase Africana/parasitologia , Aciltransferases/metabolismo , Aminopiridinas/química , Aminopiridinas/metabolismo , Aminopiridinas/farmacologia , Aminopiridinas/uso terapêutico , Animais , Antiparasitários/química , Antiparasitários/metabolismo , Ensaios Enzimáticos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Feminino , Humanos , Camundongos , Estrutura Molecular , Pirazóis/química , Pirazóis/metabolismo , Pirazóis/farmacologia , Pirazóis/uso terapêutico , Ratos , Sulfonamidas/química , Sulfonamidas/metabolismo , Sulfonamidas/farmacologia , Sulfonamidas/uso terapêutico , Fatores de Tempo , Trypanosoma brucei brucei/crescimento & desenvolvimentoRESUMO
Aspergillus fumigatus is the causative agent of aspergillosis, a frequently invasive colonization of the lungs of immunocompromised patients. GNA1 (D-glucosamine-6-phosphate N-acetyltransferase) catalyses the acetylation of GlcN-6P (glucosamine-6-phosphate) to GlcNAc-6P (N-acetylglucosamine-6-phosphate), a key intermediate in the UDP-GlcNAc biosynthetic pathway. Gene disruption of gna1 in yeast and Candida albicans has provided genetic validation of the enzyme as a potential target. An understanding of potential active site differences between the human and A. fumigatus enzymes is required to enable further work aimed at identifying selective inhibitors for the fungal enzyme. In the present study, we describe crystal structures of both human and A. fumigatus GNA1, as well as their kinetic characterization. The structures show significant differences in the sugar-binding site with, in particular, several non-conservative substitutions near the phosphate-binding pocket. Mutagenesis targeting these differences revealed drastic effects on steady-state kinetics, suggesting that the differences could be exploitable with small-molecule inhibitors.