RESUMO
Growing antimicrobial resistance is considered a potential threat for human health security by health organizations, such as the WHO, CDC and FDA, pointing to MRSA as an example. New antibacterial drugs and complex derivatives are needed to combat the development of bacterial resistance. Six new copper and cobalt complexes of azole derivatives were synthesized and isolated as air-stable solids and characterized by melting point analyses, elemental analyses, thermogravimetric analyses (TGA), and infrared and ultraviolet/visible spectroscopy. The analyses and spectral data showed that the complexes had 1:1 (M:L) stoichiometries and tetrahedral geometries, the latter being supported by DFT calculations. The antibacterial activities of the metal complexes by themselves and combined with silver nanoparticles (AgNPs; 2 µg mL-1) were assessed in vitro by broth microdilution assays against eight bacterial strains of clinical relevance. The results showed that the complexes alone exhibited moderate antibacterial activities. However, when the metal complexes were combined with AgNPs, their antibacterial activities increased (up to 10-fold in the case of complex 5), while human cell viabilities were maintained. The minimum inhibitory concentration (MIC50) values were in the range of 25-500 µg mL-1. This study thus presents novel approaches for the design of materials for fighting bacterial resistance. The use of azole complexes combined with AgNPs provides a new alternative against bacterial infections, especially when current treatments are associated with the rapid development of antibiotic resistance.
Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Azóis/química , Azóis/farmacologia , Nanopartículas Metálicas/química , Prata/química , Bactérias/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cobalto/química , Coloides , Cobre/química , Humanos , Ligantes , Testes de Sensibilidade Microbiana , Microscopia de Força Atômica , Modelos Moleculares , Estrutura Molecular , Análise Espectral , TermogravimetriaRESUMO
The oomycete Phytophthora infestans is the causal agent of tomato and potato late blight, a disease that causes tremendous economic losses in the production of solanaceous crops. The similarities between oomycetes and the apicomplexa led us to hypothesize that dihydroorotate dehydrogenase (DHODH), the enzyme catalyzing the fourth step in pyrimidine biosynthetic pathway, and a validated drug target in treatment of malaria, could be a potential target for controlling P. infestans growth. In eukaryotes, class 2 DHODHs are mitochondrially associated ubiquinone-linked enzymes that catalyze the fourth, and only redox step of de novo pyrimidine biosynthesis. We characterized the enzymes from both the pathogen and a host, Solanum tuberosum. Plant DHODHs are known to be class 2 enzymes. Sequence analysis suggested that the pathogen enzyme (PiDHODHs) also belongs to this class. We confirmed the mitochondrial localization of GFP-PiDHODH showing colocalization with mCherry-labeled ATPase in a transgenic pathogen. N-terminally truncated versions of the two DHODHs were overproduced in E. coli, purified, and kinetically characterized. StDHODH exhibited a apparent specific activity of 41 ± 1 µmol min-1 mg-1, a kcat app of 30 ± 1 s-1, and a Km app of 20 ± 1 µM for L-dihydroorotate, and a Km app= 30 ± 3 µM for decylubiquinone (Qd). PiDHODH exhibited an apparent specific activity of 104 ± 1 µmol min-1 mg-1, a kcat app of 75 ± 1 s-1, and a Km app of 57 ± 3 µM for L-dihydroorotate, and a Km app of 15 ± 1 µM for Qd. The two enzymes exhibited different activities with different quinones and napthoquinone derivatives, and different sensitivities to compounds known to cause inhibition of DHODHs from other organisms. The IC50 for A77 1726, a nanomolar inhibitor of human DHODH, was 2.9 ± 0.6 mM for StDHODH, and 79 ± 1 µM for PiDHODH. In vivo, 0.5 mM A77 1726 decreased mycelial growth by approximately 50%, after 92 h. Collectively, our findings suggest that the PiDHODH could be a target for selective inhibitors and we provide a biochemical background for the development of compounds that could be helpful for the control of the pathogen, opening the way to protein crystallization.
RESUMO
The importance of pyrimidines lies in the fact that they are structural components of a broad spectrum of key molecules that participate in diverse cellular functions, such as synthesis of DNA, RNA, lipids, and carbohydrates. Pyrimidine metabolism encompasses all enzymes involved in the synthesis, degradation, salvage, interconversion and transport of these molecules. In this review, we summarize recent publications that document how pyrimidine metabolism changes under a variety of conditions, including, when possible, those studies based on techniques of genomics, transcriptomics, proteomics, and metabolomics. First, we briefly look at the dynamics of pyrimidine metabolism during nonpathogenic cellular events. We then focus on changes that pathogen infections cause in the pyrimidine metabolism of their host. Next, we discuss the effects of antimetabolites and inhibitors, and finally we consider the consequences of genetic manipulations, such as knock-downs, knock-outs, and knock-ins, of pyrimidine enzymes on pyrimidine metabolism in the cell.
Assuntos
Células/metabolismo , Pirimidinas/metabolismo , Animais , Células/citologia , Células/patologia , Biologia Computacional , Humanos , Infecções/metabolismo , Infecções/patologiaRESUMO
A small number of cetaceans have adapted to an entirely freshwater environment, having colonized rivers in Asia and South America from an ancestral origin in the marine environment. This includes the 'river dolphins', early divergence from the odontocete lineage, and two species of true dolphins (Family Delphinidae). Successful adaptation to the freshwater environment may have required increased demands in energy involved in processes such as the mitochondrial osmotic balance. For this reason, riverine odontocetes provide a compelling natural experiment in adaptation of mammals from marine to freshwater habitats. Here we present initial evidence of positive selection in the NADH dehydrogenase subunit 2 of riverine odontocetes by analyses of full mitochondrial genomes, using tests of selection and protein structure modeling. The codon model with highest statistical support corresponds to three discrete categories for amino acid sites, those under positive, neutral, and purifying selection. With this model we found positive selection at site 297 of the NADH dehydrogenase subunit 2 (dN/dS>1.0,) leading to a substitution of an Ala or Val from the ancestral state of Thr. A phylogenetic reconstruction of 27 cetacean mitogenomes showed that an Ala substitution has evolved at least four times in cetaceans, once or more in the three 'river dolphins' (Families Pontoporidae, Lipotidae and Inidae), once in the riverine Sotalia fluviatilis (but not in its marine sister taxa), once in the riverine Orcaella brevirostris from the Mekong River (but not in its marine sister taxa) and once in two other related marine dolphins. We located the position of this amino acid substitution in an alpha-helix channel in the trans-membrane domain in both the E. coli structure and Sotalia fluviatilis model. In E. coli this position is located in a helix implicated in a proton translocation channel of respiratory complex 1 and may have a similar role in the NADH dehydrogenases of cetaceans.
Assuntos
Adaptação Fisiológica , Golfinhos/genética , Genoma Mitocondrial , NAD/genética , Seleção Genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Golfinhos/classificação , Dados de Sequência Molecular , NAD/química , Filogenia , Subunidades Proteicas/química , Subunidades Proteicas/genéticaRESUMO
The oomycete Phytophthora infestans, causal agent of the tomato and potato late blight, generates important economic and environmental losses worldwide. As current control strategies are becoming less effective, there is a need for studies on oomycete metabolism to help identify promising and more effective targets for chemical control. The pyrimidine pathways are attractive metabolic targets to combat tumors, virus and parasitic diseases but have not yet been studied in Phytophthora. Pyrimidines are involved in several critical cellular processes and play structural, metabolic and regulatory functions. Here, we used genomic and transcriptomic information to survey the pyrimidine metabolism during the P. infestans life cycle. After assessing the putative gene machinery for pyrimidine salvage and de novo synthesis, we inferred genealogies for each enzymatic domain in the latter pathway, which displayed a mosaic origin. The last two enzymes of the pathway, orotate phosphoribosyltransferase and orotidine-5-monophosphate decarboxylase, are fused in a multi-domain enzyme and are duplicated in some P. infestans strains. Two splice variants of the third gene (dihydroorotase) were identified, one of them encoding a premature stop codon generating a non-functional truncated protein. Relative expression profiles of pyrimidine biosynthesis genes were evaluated by qRT-PCR during infection in Solanum phureja. The third and fifth genes involved in this pathway showed high up-regulation during biotrophic stages and down-regulation during necrotrophy, whereas the uracil phosphoribosyl transferase gene involved in pyrimidine salvage showed the inverse behavior. These findings suggest the importance of de novo pyrimidine biosynthesis during the fast replicative early infection stages and highlight the dynamics of the metabolism associated with the hemibiotrophic life style of pathogen.
Assuntos
Phytophthora infestans/genética , Phytophthora infestans/metabolismo , Phytophthora infestans/patogenicidade , Pirimidinas/biossíntese , Processamento Alternativo , Clonagem Molecular , Di-Hidro-Orotase/genética , Di-Hidro-Orotase/metabolismo , Orotato Fosforribosiltransferase/genética , Orotato Fosforribosiltransferase/metabolismo , Orotidina-5'-Fosfato Descarboxilase/genética , Orotidina-5'-Fosfato Descarboxilase/metabolismo , Filogenia , Pirimidinas/metabolismo , Solanum/microbiologiaRESUMO
The pyrimidine biosynthesis pathway in the protozoan pathogen Toxoplasma gondii is essential for parasite growth during infection. To investigate the properties of dihydroorotate dehydrogenase (TgDHOD), the fourth enzyme in the T. gondii pyrimidine pathway, we expressed and purified recombinant TgDHOD. TgDHOD exhibited a specific activity of 84U/mg, a k(cat) of 89s(-1), a K(m)=60µM for l-dihydroorotate, and a K(m)=29µM for decylubiquinone (Q(D)). Quinones lacking or having short isoprenoid side chains yielded lower k(cat)s than Q(D). As expected, fumarate was a poor electron acceptor for this family 2 DHOD. The IC(50)s determined for A77-1726, the active derivative of the human DHOD inhibitor leflunomide, and related compounds MD249 and MD209 were, 91µM, 96µM, and 60µM, respectively. The enzyme was not significantly affected by brequinar or TTFA, known inhibitors of human DHOD, or by atovaquone. DSM190, a known inhibitor of Plasmodium falciparum DHOD, was a poor inhibitor of TgDHOD. TgDHOD exhibits a lengthy 157-residue N-terminal extension, consistent with a potential organellar targeting signal. We constructed C-terminally c-myc tagged TgDHODs to examine subcellular localization of TgDHOD in transgenic parasites expressing the tagged protein. Using both exogenous and endogenous expression strategies, anti-myc fluorescence signal colocalized with antibodies against the mitochondrial marker ATPase. These findings demonstrate that TgDHOD is associated with the parasite's mitochondrion, revealing this organelle as the site of orotate production in T. gondii. The TgDHOD gene appears to be essential because while gene tagging was successful at the TgDHOD gene locus, attempts to delete the TgDHOD gene were not successful in the KU80 background. Collectively, our study suggests that TgDHOD is an excellent target for the development of anti-Toxoplasma drugs.