RESUMO
New effective compounds to treat tuberculosis are urgently needed. IQG-607 is an orally active anti-tuberculosis drug candidate, with promising preliminary safety profile and anti-mycobacterial activity in both in vitro and in vivo models of tuberculosis infection. Here, we evaluated the mutagenic and genotoxic effects of IQG-607, and its interactions with CYP450 isoforms. Moreover, we describe for the first time a combination study of IQG-607 in Mycobacterium tuberculosis-infected mice. Importantly, IQG-607 had additive effects when combined with the first-line anti-tuberculosis drugs rifampin and pyrazinamide in mice. IQG-607 presented weak to moderate inhibitory potential against CYP450 isoforms 3A4, 1A2, 2C9, 2C19, 2D6, and 2E1. The Salmonella mutagenicity test revealed that IQG-607 induced base pair substitution mutations in the strains TA100 and TA1535. However, in the presence of human metabolic S9 fraction, no mutagenic effect was detected in any strain. Additionally, IQG-607 did not increase micronucleus frequencies in mice, at any dose tested, 25, 100, or 250 mg/kg. The favorable activity in combination with first-line drugs and mild to moderate toxic events described in this study suggest that IQG-607 represents a candidate for clinical development.
Assuntos
Antibacterianos/farmacologia , Inibidores Enzimáticos/farmacologia , Compostos Ferrosos/efeitos adversos , Compostos Ferrosos/farmacologia , Isoniazida/análogos & derivados , Mycobacterium tuberculosis/efeitos dos fármacos , Salmonella typhimurium/efeitos dos fármacos , Tuberculose/tratamento farmacológico , Animais , Antibacterianos/administração & dosagem , Antibacterianos/efeitos adversos , Aberrações Cromossômicas , Sistema Enzimático do Citocromo P-450/metabolismo , Modelos Animais de Doenças , Quimioterapia Combinada , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/efeitos adversos , Compostos Ferrosos/administração & dosagem , Isoniazida/administração & dosagem , Isoniazida/efeitos adversos , Isoniazida/farmacologia , Masculino , Camundongos , Testes de Sensibilidade Microbiana , Testes de Mutagenicidade , Mycobacterium tuberculosis/genética , Salmonella typhimurium/genética , Tuberculose/microbiologiaRESUMO
Roughly a third of the world's population is estimated to have latent Mycobacterium tuberculosis infection, being at risk of developing active tuberculosis (TB) during their lifetime. Given the inefficacy of prophylactic measures and the increase of drug-resistant M. tuberculosis strains, there is a clear and urgent need for the development of new and more efficient chemotherapeutic agents, with selective toxicity, to be implemented on patient treatment. The component enzymes of the shikimate pathway, which is essential in mycobacteria and absent in humans, stand as attractive and potential targets for the development of new drugs to treat TB. This review gives an update on published work on the enzymes of the shikimate pathway and some insight on what can be potentially explored towards selective drug development.
Assuntos
Antituberculosos/síntese química , Proteínas de Bactérias/antagonistas & inibidores , Inibidores Enzimáticos/síntese química , Regulação Bacteriana da Expressão Gênica , Mycobacterium tuberculosis/efeitos dos fármacos , Ácido Chiquímico/antagonistas & inibidores , Antituberculosos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Humanos , Tuberculose Latente/tratamento farmacológico , Tuberculose Latente/microbiologia , Redes e Vias Metabólicas/efeitos dos fármacos , Redes e Vias Metabólicas/genética , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/crescimento & desenvolvimento , Ácido Chiquímico/química , Ácido Chiquímico/metabolismo , Relação Estrutura-Atividade , Tuberculose Pulmonar/tratamento farmacológico , Tuberculose Pulmonar/microbiologiaRESUMO
IQG-607 is a metal complex previously reported as a promising anti-tuberculosis (TB) drug against isoniazid (INH)-resistant strains of Mycobacterium tuberculosis Unexpectedly, we found that INH-resistant clinical isolates were resistant to IQG-607. Spontaneous mutants resistant to IQG-607 were subjected to whole-genome sequencing, and all sequenced colonies carried alterations in the katG gene. The katG(S315T) mutation was sufficient to confer resistance to IQG-607 in both MIC assays and inside macrophages. Moreover, overexpression of the InhA(S94A) protein caused IQG-607's resistance.
Assuntos
Antituberculosos/farmacologia , Farmacorresistência Bacteriana/efeitos dos fármacos , Compostos Ferrosos/farmacologia , Isoniazida/análogos & derivados , Mycobacterium tuberculosis/efeitos dos fármacos , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Proteínas de Bactérias/genética , DNA Bacteriano/genética , Farmacorresistência Bacteriana/genética , Humanos , Isoniazida/farmacologia , Mutação/genética , Mycobacterium tuberculosis/genética , Sequenciamento Completo do Genoma/métodosRESUMO
In the present study, we evaluated the safety and the possible toxic effects of IQG-607 after acute and 90-day repeated administrations in rats. Single oral administration of IQG-607 (300 or 2000 mg/kg) on female rats did not result in any mortality. No gross lesions were observed in the animals at necropsy. Ninety-day administration test resulted in 20% of deaths, in both male and female rats administered with the highest dose of IQG-607, 300 mg/kg. Repeated administration of the IQG 607 (25, 100 and 300 mg/kg) did not result in any significant body mass alteration, or changes in food and water consumption. The most important clinical sign observed was salivation in both sexes. Importantly, long-term treatment with IQG-607 did not induce alterations in any hematological (for both sex) and serum biochemical (for female) parameters evaluated, even at the highest dose tested. Treatment of male rats with 100 or 300 mg/kg of IQG-607 decreased total cholesterol levels, while animals treated with 100 mg/kg also presented reduction on triglyceride levels. Of note, no treatment induced significant histopathological alterations in tissues of all organs and glands analyzed, even in that group that received the highest dose of IQG-607.
Assuntos
Compostos Ferrosos/toxicidade , Isoniazida/análogos & derivados , Administração Oral , Animais , Índice de Massa Corporal , Ingestão de Líquidos/efeitos dos fármacos , Ingestão de Alimentos/efeitos dos fármacos , Feminino , Compostos Ferrosos/administração & dosagem , Isoniazida/administração & dosagem , Isoniazida/toxicidade , Masculino , Ratos , Salivação/efeitos dos fármacos , Testes de Toxicidade Aguda/métodosRESUMO
BACKGROUND: Annexin V, a 35.8 kDa intracellular protein, is a Ca⺲-dependent phospholipid binding protein with high affinity to phosphatidylserine (PS), which is a well-known hallmark of apoptosis. Annexin V is a sensitive probe for PS exposure upon the cell membrane, and used for detection of apoptotic cells both in vivo and in vitro. Large-scale production of recombinant human annexin V is worth optimization, because of its wide use in nuclear medicine, radiolabeled with (99m)Tc, for the evaluation of cancer chemotherapy treatments, and its use in identification of apoptotic cells in histologic studies. Here we describe the high-yield production of a tag-free version of human annexin V recombinant protein by linear fed-batch cultivation in a bioreactor. RESULTS: We cloned the human ANXA5 coding sequence into the pET-30a (+) expression vector and expressed rhANXA5 in batch and fed-batch cultures. Using E. coli BL21 (DE3) in a semi-defined medium at 37°C, pH 7 in fed-batch cultures, we obtained a 45-fold increase in biomass production, respective to shaker cultivations. We developed a single-step protocol for rhANXA5 purification using a strong anion-exchange column (MonoQ HR16/10). Using these procedures, we obtained 28.5 mg of homogeneous, nontagged and biologically functional human annexin V recombinant protein from 3 g wet weight of bacterial cells from bioreactor cultures. The identity and molecular mass of rhANXA5 was confirmed by mass spectrometry. Moreover, the purified rhANXA5 protein was functionally evaluated in a FITC-annexin V binding experiment and the results demonstrated that rhANXA5 detected apoptotic cells similarly to a commercial kit. CONCLUSIONS: We describe a new fed-batch method to produce recombinant human annexin V in large scale, which may expand the commercial utilities for rhANXAV to applications such as in vivo imaging studies.
Assuntos
Anexina A5/metabolismo , Técnicas de Cultura Celular por Lotes , Anexina A5/química , Anexina A5/genética , Biomassa , Cromatografia por Troca Iônica , Clonagem Molecular , Escherichia coli/metabolismo , Fluoresceína-5-Isotiocianato/química , Humanos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
PURPOSE: 5-fluorouracil (5-FU) has been broadly used to treat solid tumors for more than 50 years. One of the major side effects of fluoropyrimidines therapy is oral and intestinal mucositis. Human uridine phosphorylase (hUP) inhibitors have been suggested as modulators of 5-FU toxicity. Therefore, the present study aimed to test the ability of hUP blockers in preventing mucositis induced by 5-FU. METHODS: We induced intestinal mucositis in Wistar rats with 5-FU, and the intestinal damage was evaluated in presence or absence of two hUP1 inhibitors previously characterized. We examined the loss of weight and diarrhea following the treatment, the villus integrity, uridine levels in plasma, and the neutrophil migration by MPO activity. RESULTS: We found that one of the compounds, 6-hydroxy-4-methyl-1H-pyridin-2-one-3-carbonitrile was efficient to promote intestinal mucosa protection and to inhibit the hUP1 enzyme, increasing the uridine levels in the plasma of animals. However, the loss of body weight, diarrhea intensity or neutrophil migration remained unaffected. CONCLUSION: Our results bring support to the hUP1 inhibitor strategy as a novel possibility of prevention and treatment of mucositis during the 5-FU chemotherapy, based on the approach of uridine accumulation in plasma and tissues.
Assuntos
Antimetabólitos Antineoplásicos/efeitos adversos , Fluoruracila/efeitos adversos , Enteropatias/tratamento farmacológico , Mucosite/tratamento farmacológico , Piridonas/uso terapêutico , Uridina Fosforilase/antagonistas & inibidores , Animais , Inibidores Enzimáticos/uso terapêutico , Feminino , Humanos , Enteropatias/induzido quimicamente , Enteropatias/metabolismo , Enteropatias/patologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/patologia , Intestino Delgado/efeitos dos fármacos , Intestino Delgado/metabolismo , Intestino Delgado/patologia , Mucosite/induzido quimicamente , Mucosite/metabolismo , Mucosite/patologia , Peroxidase/metabolismo , Ratos Wistar , Uridina/sangueRESUMO
Acute liver injury was induced in male BALB/c mice by coadministering isoniazid and rifampicin. In this work, the effects of resveratrol (1) were investigated in the hepatotoxicity caused by isoniazid-rifampicin in mice. Compound 1 was administered 30 min prior to isoniazid-rifampicin. Serum biochemical tests, liver histopathological examination, oxidative stress, myeloperoxidase activity, cytokine production (TNF-α, IL-12p70, and IL-10), and mRNA expression of SIRT1-7 and PPAR-γ/PGC1-α were evaluated. The administration of 1 significantly decreased aspartate transaminase and alanine aminotransferase levels, myeloperoxidase activity, and cytokine levels. Furthermore, 1 reverted the decrease of catalase and glutathione activities and ameliorated the histopathological alterations associated with antituberculosis drugs. Modulation of SIRT1 and PPAR-γ/PGC1-α expression is likely involved in the protective effects of 1. The results presented herein show that 1 was able to largely prevent the hepatotoxicity induced by isoniazid and rifampicin in mice, mainly by modulating SIRT1 mRNA expression.
Assuntos
Antituberculosos/farmacologia , Isoniazida/farmacologia , Rifampina/farmacologia , Sirtuína 1/metabolismo , Estilbenos/farmacologia , Alanina Transaminase/sangue , Alanina Transaminase/efeitos dos fármacos , Animais , Aspartato Aminotransferases/sangue , Aspartato Aminotransferases/efeitos dos fármacos , Doença Hepática Induzida por Substâncias e Drogas , Glutationa/metabolismo , Interleucina-10/análise , Interleucina-10/metabolismo , Fígado/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Estrutura Molecular , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , PPAR gama/efeitos dos fármacos , Peroxidase/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Resveratrol , Sirtuína 1/efeitos dos fármacos , Sirtuína 1/genética , Fatores de Transcrição/efeitos dos fármacos , Fator de Necrose Tumoral alfa/análise , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
There has been a dramatic increase in the identification of non-canonical translation and a significant expansion of the protein-coding genome. Among the strategies used to identify unannotated small Open Reading Frames (smORFs) that encode microproteins, Ribosome profiling (Ribo-Seq) is the gold standard for the annotation of novel coding sequences by reporting on smORF translation. In Ribo-Seq, ribosome-protected footprints (RPFs) that map to multiple genomic sites are removed since they cannot be unambiguously assigned to a specific genomic location. Furthermore, RPFs necessarily result in short (25-34 nucleotides) reads, increasing the chance of multi-mapping alignments, such that smORFs residing in these regions cannot be identified by Ribo-Seq. Moreover, it has been challenging to identify protein evidence for Ribo-Seq. To solve this, we developed Rp3, a pipeline that integrates proteogenomics and Ribosome profiling to provide unambiguous evidence for a subset of microproteins missed by current Ribo-Seq pipelines. Here, we show that Rp3 maximizes proteomics detection and confidence of microprotein-encoding smORFs.
Assuntos
Fases de Leitura Aberta , Proteogenômica , Ribossomos , Ribossomos/metabolismo , Ribossomos/genética , Proteogenômica/métodos , Fases de Leitura Aberta/genética , Biossíntese de Proteínas , Humanos , Proteômica/métodos , Proteínas/genética , Proteínas/metabolismo , Perfil de RibossomosRESUMO
Tuberculosis is a disease caused by a single pathogen that leads to a death toll estimated to be more than a million per year. Mycobacterium tuberculosis (Mtb), which affects mainly the lungs, spreads by airborne transmission when infectious respiratory particles from an infected human enter the respiratory tract of another person. Despite diagnosis and treatment being well established, the rise of cases of patients infected with Mtb strains with multidrug resistance to the antibiotics used in the regimen against the disease is alarming. Indole used as a core molecule has been described as a promising structure to treat several diseases. 5-Fluoroindole (5-FI) compound, evaluated in the free base and in the hydrochloride (5-FI.HCl) forms, inhibited the growth of pan-sensitive Mtb H37Rv strain in the same range (4.7-29.1 µM) of clinical isolates that have resistance to at least two first-line drugs. Although 5-FI showed no cytotoxicity in Vero and HepG2 cells, high permeability (2.4.10-6 cm/s) in the PAMPA assay, and high metabolic stability (Clint 9.0 mL/min/kg) in rat liver microsomes, limited solubility at plasmatic and intestinal pH values prompted formation and employment of its salt form (5-FI.HCl). Although the 5-FI.HCl compound showed increased solubility at pH values of 7.4 and 9.1 and increased stability in aqueous solutions, data for intrinsic clearance (Clint = 48 mL/min/kg) and a half-life (t 1/2 = 12 min) showed decreased metabolic stability. As 5-FI.HCl showed both good absorption and ability to reach the systemic circulation of animals without the need to use vehicles containing cosolvents or surfactants, it was chosen to evaluate its effectiveness in the model of tuberculosis in mice. The in vivo results showed the concentration of the compound in plasma increasing within 30 min in the systemic circulation and the capacity of reducing the Mtb burden in the lungs at the concentration of 200 µmol/kg after 21 days of infection, with no toxicity in mice.
RESUMO
Cytidine monophosphate kinase from Mycobacterium tuberculosis (MtCMK) likely plays a role in supplying precursors for nucleic acid synthesis. MtCMK catalyzes the ATP-dependent phosphoryl group transfer preferentially to CMP and dCMP. Initial velocity studies and Isothermal titration calorimetry (ITC) measurements showed that MtCMK follows a random-order mechanism of substrate (CMP and ATP) binding, and an ordered mechanism for product release, in which ADP is released first followed by CDP. The thermodynamic signatures of CMP and CDP binding to MtCMK showed favorable enthalpy and unfavorable entropy, and ATP binding was characterized by favorable changes in enthalpy and entropy. The contribution of linked protonation events to the energetics of MtCMK:phosphoryl group acceptor binary complex formation suggested a net gain of protons. Values for the pKa of a likely chemical group involved in proton exchange and for the intrinsic binding enthalpy were calculated. The Asp187 side chain of MtCMK is suggested as the likely candidate for the protonation event. Data on thermodynamics of binary complex formation were collected to evaluate the contribution of 2'-OH group to intermolecular interactions. The data are discussed in light of functional and structural comparisons between CMP/dCMP kinases and UMP/CMP ones.
Assuntos
Trifosfato de Adenosina/metabolismo , Monofosfato de Citidina/metabolismo , Desoxicitidina Monofosfato/metabolismo , Mycobacterium tuberculosis/enzimologia , Núcleosídeo-Fosfato Quinase/metabolismo , Sequência de Aminoácidos , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/metabolismo , Núcleosídeo-Fosfato Quinase/química , Ligação Proteica , Alinhamento de Sequência , Especificidade por Substrato , TermodinâmicaRESUMO
BACKGROUND: Isoprenoids are the most diverse and abundant group of natural products. In Plasmodium falciparum, isoprenoid synthesis proceeds through the methyl erythritol diphosphate pathway and the products are further metabolized by farnesyl diphosphate synthase (FPPS), turning this enzyme into a key branch point of the isoprenoid synthesis. Changes in FPPS activity could alter the flux of isoprenoid compounds downstream of FPPS and, hence, play a central role in the regulation of a number of essential functions in Plasmodium parasites. METHODS: The isolation and cloning of gene PF3D7_18400 was done by amplification from cDNA from mixed stage parasites of P. falciparum. After sequencing, the fragment was subcloned in pGEX2T for recombinant protein expression. To verify if the PF3D7_1128400 gene encodes a functional rPfFPPS protein, its catalytic activity was assessed using the substrate [4-14C] isopentenyl diphosphate and three different allylic substrates: dimethylallyl diphosphate, geranyl diphosphate or farnesyl diphosphate. The reaction products were identified by thin layer chromatography and reverse phase high-performance liquid chromatography. To confirm the product spectrum formed of rPfFPPS, isoprenic compounds were also identified by mass spectrometry. Apparent kinetic constants KM and Vmax for each substrate were determined by Michaelis-Menten; also, inhibition assays were performed using risedronate. RESULTS: The expressed protein of P. falciparum FPPS (rPfFPPS) catalyzes the synthesis of farnesyl diphosphate, as well as geranylgeranyl diphosphate, being therefore a bifunctional FPPS/geranylgeranyl diphosphate synthase (GGPPS) enzyme. The apparent KM values for the substrates dimethylallyl diphosphate, geranyl diphosphate and farnesyl diphosphate were, respectively, 68 ± 5 µM, 7.8 ± 1.3 µM and 2.06 ± 0.4 µM. The protein is expressed constitutively in all intra-erythrocytic stages of P. falciparum, demonstrated by using transgenic parasites with a haemagglutinin-tagged version of FPPS. Also, the present data demonstrate that the recombinant protein is inhibited by risedronate. CONCLUSIONS: The rPfFPPS is a bifunctional FPPS/GGPPS enzyme and the structure of products FOH and GGOH were confirmed mass spectrometry. Plasmodial FPPS represents a potential target for the rational design of chemotherapeutic agents to treat malaria.
Assuntos
Farnesiltranstransferase/genética , Farnesiltranstransferase/metabolismo , Plasmodium falciparum/enzimologia , Cromatografia Líquida , Clonagem Molecular , Farnesiltranstransferase/química , Plasmodium falciparum/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem , Terpenos/metabolismoRESUMO
Mycobacterium tuberculosis InhA (MtInhA) is an attractive enzyme to drug discovery efforts due to its validation as an effective biological target for tuberculosis therapy. In this work, two different virtual-ligand-screening approaches were applied in order to identify new InhA inhibitors' candidates from a library of ligands selected from the ZINC database. First, a 3-D pharmacophore model was built based on 36 available MtInhA crystal structures. By combining structure-based and ligand-based information, four pharmacophoric points were designed to select molecules able to satisfy the binding features of MtInhA substrate-binding cavity. The second approach consisted of using four well established docking programs, with different search algorithms, to compare the binding mode and score of the selected molecules from the aforementioned library. After detailed analyses of the results, six ligands were selected for in vitro analysis. Three of these molecules presented a satisfactory inhibitory activity with IC50 values ranging from 24 (±2) µM to 83 (±5) µM. The best compound presented an uncompetitive inhibition mode to NADH and 2-trans-dodecenoyl-CoA substrates, with Ki values of 24 (±3) µM and 20 (±2) µM, respectively. These molecules were not yet described as antituberculars or as InhA inhibitors, making its novelty interesting to start efforts on ligand optimization in order to identify new effective drugs against tuberculosis having InhA as a target. More studies are underway to dissect the discovered uncompetitive inhibitor interactions with MtInhA.
Assuntos
Proteínas de Bactérias/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Simulação de Acoplamento Molecular , Mycobacterium tuberculosis/enzimologia , Oxirredutases/antagonistas & inibidores , Interface Usuário-Computador , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Ligantes , Oxirredutases/química , Oxirredutases/metabolismo , Conformação ProteicaRESUMO
There has been a dramatic increase in the identification of non-conical translation and a significant expansion of the protein-coding genome and proteome. Among the strategies used to identify novel small ORFs (smORFs), Ribosome profiling (Ribo-Seq) is the gold standard for the annotation of novel coding sequences by reporting on smORF translation. In Ribo-Seq, ribosome-protected footprints (RPFs) that map to multiple sites in the genome are computationally removed since they cannot unambiguously be assigned to a specific genomic location, or to a specific transcript in the case of multiple isoforms. Furthermore, RPFs necessarily result in short (25-34 nucleotides) reads, increasing the chance of ambiguous and multi-mapping alignments, such that smORFs that reside in these regions cannot be identified by Ribo-Seq. Here, we show that the inclusion of proteogenomics to create a Ribosome Profiling and Proteogenomics Pipeline (RP3) bypasses this limitation to identify a group of microprotein-encoding smORFs that are missed by current Ribo-Seq pipelines. Moreover, we show that the microproteins identified by RP3 have different sequence compositions from the ones identified by Ribo-Seq-only pipelines, which can affect proteomics identification. In aggregate, the development of RP3 maximizes the detection and confidence of protein-encoding smORFs and microproteins.
RESUMO
Administration of the current tuberculosis (TB) vaccine to newborns is not a reliable route for preventing TB in adults. The conversion of XMP to GMP is catalyzed by guaA-encoded GMP synthetase (GMPS), and deletions in the Shiguella flexneri guaBA operon led to an attenuated auxotrophic strain. Here we present the cloning, expression, and purification of recombinant guaA-encoded GMPS from Mycobacterium tuberculosis (MtGMPS). Mass spectrometry data, oligomeric state determination, steady-state kinetics, isothermal titration calorimetry (ITC), and multiple sequence alignment are also presented. The homodimeric MtGMPS catalyzes the conversion of XMP, MgATP, and glutamine into GMP, ADP, PP(i), and glutamate. XMP, NH(4)(+), and Mg(2+) displayed positive homotropic cooperativity, whereas ATP and glutamine displayed hyperbolic saturation curves. The activity of ATP pyrophosphatase domain is independent of glutamine amidotransferase domain, whereas the latter cannot catalyze hydrolysis of glutamine to NH(3) and glutamate in the absence of substrates. ITC data suggest random order of binding of substrates, and PP(i) is the last product released. Sequence comparison analysis showed conservation of both Cys-His-Glu catalytic triad of N-terminal Class I amidotransferase and of amino acid residues of the P-loop of the N-type ATP pyrophosphatase family.
Assuntos
Carbono-Nitrogênio Ligases/metabolismo , Mycobacterium tuberculosis/enzimologia , Tuberculose/microbiologia , Adenosina Trifosfatases/metabolismo , Sequência de Aminoácidos , Carbono-Nitrogênio Ligases/química , Carbono-Nitrogênio Ligases/genética , Carbono-Nitrogênio Ligases/isolamento & purificação , Clonagem Molecular , Glutaminase/metabolismo , Humanos , Cinética , Ligantes , Magnésio/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/genética , Ligação Proteica , Multimerização Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , TitulometriaRESUMO
Enoyl-ACP reductases (ENRs) are enzymes that catalyze the last step of the elongation cycle during fatty acid synthesis. In recent years, new bacterial ENR types were discovered, some of them with structures and mechanisms that differ from the canonical bacterial FabI enzymes. Here, we briefly review the diversity of structural and catalytic properties of the canonical FabI and the new FabK, FabV, FabL, and novel ENRs identified in a soil metagenome study. We also highlight recent efforts to use the newly discovered Fabs as targets for drug development and consider the complex evolutionary history of this diverse set of bacterial ENRs.
RESUMO
Tuberculosis (TB) remains one of the leading causes of death due to a single pathogen. The emergence and proliferation of multidrug-resistant (MDR-TB) and extensively drug-resistant strains (XDR-TB) represent compelling reasons to invest in the pursuit of new anti-TB agents. The shikimate pathway, responsible for chorismate biosynthesis, which is a precursor of important aromatic compounds, is required for Mycobacterium tuberculosis growth. The enzyme 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (MtbDAHPS) catalyzes the first step in the shikimate pathway and it is an attractive target for anti-tubercular agents. Here, we used a CRISPRi system to evaluate the DAHPS as a vulnerable target in M. tuberculosis. The silencing of aroG significantly reduces the M. tuberculosis growth in both rich medium and, especially, in infected murine macrophages. The supplementation with amino acids was only able to partially rescue the growth of bacilli, whereas the Aro supplement (aromix) was enough to sustain the bacterial growth at lower rates. This study shows that MtbDAHPS protein is vulnerable and, therefore, an attractive target to develop new anti-TB agents. In addition, the study contributes to a better understanding of the biosynthesis of aromatic compounds and the bacillus physiology. IMPORTANCE Determining the vulnerability of a potential target allows us to assess whether its partial inhibition will impact bacterial growth. Here, we evaluated the vulnerability of the enzyme 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAHPS) from M. tuberculosis by silencing the DAHPS-coding aroG gene in different contexts. These results could lead to the development of novel and potent anti-tubercular agents in the near future.
Assuntos
3-Desoxi-7-Fosfo-Heptulonato Sintase , Mycobacterium tuberculosis , 3-Desoxi-7-Fosfo-Heptulonato Sintase/química , 3-Desoxi-7-Fosfo-Heptulonato Sintase/genética , 3-Desoxi-7-Fosfo-Heptulonato Sintase/metabolismo , Animais , Antituberculosos/farmacologia , Camundongos , Mycobacterium tuberculosis/metabolismo , FosfatosRESUMO
BACKGROUND: Gut microbiota-derived short-chain fatty-acid (SFCA) acetate protects mice against RSV A2 strain infection by increasing interferon-ß production and expression of interferon-stimulated genes (ISGs). However, the role of SFCA in RSV infection using strains isolated from patients is unknown. METHODS: We first used RSV clinical strains isolated from infants hospitalized with RSV bronchiolitis to investigate the effects of in vitro SCFA-acetate treatment of human pulmonary epithelial cells. We next examined whether SCFA-acetate treatment is beneficial in a mouse model of RSV infection using clinical isolates. We sought to investigate the relationship of gut microbiota and fecal acetate with disease severity among infants hospitalized with RSV bronchiolitis, and whether treating their respiratory epithelial cells with SCFA-acetate ex-vivo impacts viral load and ISG expression. We further treated epithelial cells from SARS-CoV-2 infected patients with SCFA-acetate. FINDINGS: In vitro pre-treatment of A549 cells with SCFA-acetate reduced RSV infection with clinical isolates and increased the expression of RIG-I and ISG15. Animals treated with SCFA-acetate intranasally recovered significantly faster, with reduction in the RSV clinical isolates viral load, and increased lung expression of IFNB1 and the RIG-I. Experiments in RIG-I knockout A549 cells demonstrated that the protection relies on RIG-I presence. Gut microbial profile was associated with bronchiolitis severity and with acetate in stool. Increased SCFA-acetate levels were associated with increasing oxygen saturation at admission, and shorter duration of fever. Ex-vivo treatment of patients' respiratory cells with SCFA-acetate reduced RSV load and increased expression of ISGs OAS1 and ISG15, and virus recognition receptors MAVS and RIG-I, but not IFNB1. These SCFA-acetate effects were not found on cells from SARS-CoV-2 infected patients. INTERPRETATION: SCFA-acetate reduces the severity of RSV infection and RSV viral load through modulation of RIG-I expression. FUNDING: FAPERGS (FAPERGS/MS/CNPq/SESRS no. 03/2017 - PPSUS 17/2551-0001380-8 and COVID-19 20/2551-0000258-6); CNPq 312504/2017-9; CAPES) - Finance Code 001.
Assuntos
Bronquiolite , COVID-19 , Infecções por Vírus Respiratório Sincicial , Vírus Sincicial Respiratório Humano , Acetatos/metabolismo , Acetatos/farmacologia , Animais , Antivirais/metabolismo , Antivirais/farmacologia , Antivirais/uso terapêutico , Bronquiolite/tratamento farmacológico , Bronquiolite/metabolismo , Ácidos Graxos Voláteis/metabolismo , Humanos , Lactente , Pulmão/metabolismo , Camundongos , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Infecções por Vírus Respiratório Sincicial/genética , Vírus Sincicial Respiratório Humano/fisiologia , SARS-CoV-2RESUMO
The pyrH-encoded uridine 5'-monophosphate kinase (UMPK) is involved in both de novo and salvage synthesis of DNA and RNA precursors. Here we describe Mycobacterium tuberculosis UMPK (MtUMPK) cloning and expression in Escherichia coli. N-terminal amino acid sequencing and electrospray ionization mass spectrometry analyses confirmed the identity of homogeneous MtUMPK. MtUMPK catalyzed the phosphorylation of UMP to UDP, using ATP-Mg²(+) as phosphate donor. Size exclusion chromatography showed that the protein is a homotetramer. Kinetic studies revealed that MtUMPK exhibits cooperative kinetics towards ATP and undergoes allosteric regulation. GTP and UTP are, respectively, positive and negative effectors, maintaining the balance of purine versus pyrimidine synthesis. Initial velocity studies and substrate(s) binding measured by isothermal titration calorimetry suggested that catalysis proceeds by a sequential ordered mechanism, in which ATP binds first followed by UMP binding, and release of products is random. As MtUMPK does not resemble its eukaryotic counterparts, specific inhibitors could be designed to be tested as antitubercular agents.
Assuntos
Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Mycobacterium tuberculosis/enzimologia , Pirimidinas/metabolismo , Transferases/genética , Transferases/metabolismo , Trifosfato de Adenosina/metabolismo , Regulação Alostérica , Sequência de Aminoácidos , Clonagem Molecular , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/isolamento & purificação , Genes Supressores , Guanosina Trifosfato/metabolismo , Cinética , Ligantes , Dados de Sequência Molecular , Peso Molecular , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Reação em Cadeia da Polimerase , Alinhamento de Sequência , Análise de Sequência de DNA , Espectrometria de Massas por Ionização por Electrospray , Transferases/química , Transferases/isolamento & purificação , Uridina Trifosfato/metabolismoRESUMO
Cytidine deaminase (CDA) is a key enzyme in the pyrimidine salvage pathway. It is involved in the hydrolytic deamination of cytidine or 2'-deoxycytidine to uridine or 2'-deoxyuridine, respectively. Here we report the crystal structures of Mycobacterium tuberculosis CDA (MtCDA) in complex with uridine (2.4 Å resolution) and deoxyuridine (1.9 Å resolution). Molecular dynamics (MD) simulation was performed to analyze the physically relevant motions involved in the protein-ligand recognition process, showing that structural flexibility of some protein regions are important to product binding. In addition, MD simulations allowed the analysis of the stability of tetrameric MtCDA structure. These findings open-up the possibility to use MtCDA as a target in future studies aiming to the rational design of new inhibitor of MtCDA-catalyzed chemical reaction with potential anti-proliferative activity on cell growth of M. tuberculosis, the major causative agent of tuberculosis.
Assuntos
Citidina Desaminase/metabolismo , Desoxiuridina/metabolismo , Mycobacterium tuberculosis/enzimologia , Uridina/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Citidina Desaminase/química , Simulação de Dinâmica Molecular , Mycobacterium tuberculosis/química , Ligação ProteicaRESUMO
The emergence of drug-resistant strains of Mycobacterium tuberculosis, the major causative agent of tuberculosis (TB), and the deadly HIV-TB co-infection have led to an urgent need for the development of new anti-TB drugs. The histidine biosynthetic pathway is present in bacteria, archaebacteria, lower eukaryotes and plants, but is absent in mammals. Disruption of the hisD gene has been shown to be essential for M. tuberculosis survival. Here we present cloning, expression and purification of recombinant hisD-encoded histidinol dehydrogenase (MtHisD). N-terminal amino acid sequencing and electrospray ionization mass spectrometry analyses confirmed the identity of homogeneous MtHisD. Analytical gel filtration, metal requirement analysis, steady-state kinetics and isothermal titration calorimetry data showed that homodimeric MtHisD is a metalloprotein that follows a Bi Uni Uni Bi Ping-Pong mechanism. pH-rate profiles and a three-dimensional model of MtHisD allowed proposal of amino acid residues involved in either catalysis or substrate(s) binding.