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1.
Int J Biol Macromol ; 193(Pt B): 1845-1858, 2021 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-34762917

RESUMO

Microbial amino acid biosynthetic pathways are underexploited for the development of anti-bacterial agents. N-acetyl glutamate synthase (ArgA) catalyses the first committed step in L-arginine biosynthesis and is essential for M. tuberculosis growth. Here, we have purified and optimized assay conditions for the acetylation of l-glutamine by ArgA. Using the optimized conditions, high throughput screening was performed to identify ArgA inhibitors. We identified 2,5-Bis (2-chloro-4-guanidinophenyl) furan, a dicationic diaryl furan derivatives, as ArgA inhibitor, with a MIC99 values of 1.56 µM against M. tuberculosis. The diaryl furan derivative displayed bactericidal killing against both M. bovis BCG and M. tuberculosis. Inhibition of ArgA by the lead compound resulted in transcriptional reprogramming and accumulation of reactive oxygen species. The lead compound and its derivatives showed micromolar binding with ArgA as observed in surface plasmon resonance and tryptophan quenching experiments. Computational and dynamic analysis revealed that these scaffolds share similar binding site residues with L-arginine, however, with slight variations in their interaction pattern. Partial restoration of growth upon supplementation of liquid cultures with either L-arginine or N-acetyl cysteine suggests a multi-target killing mechanism for the lead compound. Taken together, we have identified small molecule inhibitors against ArgA enzyme from M. tuberculosis.


Assuntos
Aminoácido N-Acetiltransferase , Antituberculosos/química , Proteínas de Bactérias , Inibidores Enzimáticos/química , Mycobacterium tuberculosis/enzimologia , Aminoácido N-Acetiltransferase/antagonistas & inibidores , Aminoácido N-Acetiltransferase/química , Antituberculosos/uso terapêutico , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , Inibidores Enzimáticos/uso terapêutico , Furanos , Mycobacterium bovis/enzimologia
2.
Int J Mol Sci ; 22(21)2021 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-34769421

RESUMO

Haloalkane dehalogenases (EC 3.8.1.5) play an important role in hydrolytic degradation of halogenated compounds, resulting in a halide ion, a proton, and an alcohol. They are used in biocatalysis, bioremediation, and biosensing of environmental pollutants and also for molecular tagging in cell biology. The method of ancestral sequence reconstruction leads to prediction of sequences of ancestral enzymes allowing their experimental characterization. Based on the sequences of modern haloalkane dehalogenases from the subfamily II, the most common ancestor of thoroughly characterized enzymes LinB from Sphingobium japonicum UT26 and DmbA from Mycobacterium bovis 5033/66 was in silico predicted, recombinantly produced and structurally characterized. The ancestral enzyme AncLinB-DmbA was crystallized using the sitting-drop vapor-diffusion method, yielding rod-like crystals that diffracted X-rays to 1.5 Å resolution. Structural comparison of AncLinB-DmbA with their closely related descendants LinB and DmbA revealed some differences in overall structure and tunnel architecture. Newly prepared AncLinB-DmbA has the highest active site cavity volume and the biggest entrance radius on the main tunnel in comparison to descendant enzymes. Ancestral sequence reconstruction is a powerful technique to study molecular evolution and design robust proteins for enzyme technologies.


Assuntos
Hidrolases/química , Mycobacterium bovis/enzimologia , Sphingomonadaceae/enzimologia , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X/métodos , Evolução Molecular , Hidrolases/metabolismo , Hidrólise , Modelos Moleculares , Engenharia de Proteínas/métodos , Análise de Sequência de Proteína/métodos
3.
Acta Crystallogr D Struct Biol ; 77(Pt 5): 618-627, 2021 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-33950018

RESUMO

Peptidoglycan comprises repeating units of N-acetylmuramic acid, N-acetylglucosamine and short cross-linking peptides. After the conversion of UDP-N-acetylglucosamine (UNAG) to UDP-N-acetylmuramic acid (UNAM) by the MurA and MurB enzymes, an amino acid is added to UNAM by UDP-N-acetylmuramic acid L-alanine ligase (MurC). As peptidoglycan is an essential component of the bacterial cell wall, the enzymes involved in its biosynthesis represent promising targets for the development of novel antibacterial drugs. Here, the crystal structure of Mycobacterium bovis MurC (MbMurC) is reported, which exhibits a three-domain architecture for the binding of UNAM, ATP and an amino acid as substrates, with a nickel ion at the domain interface. The ATP-binding loop adopts a conformation that is not seen in other MurCs. In the UNAG-bound structure of MbMurC, the substrate mimic interacts with the UDP-binding domain of MbMurC, which does not invoke rearrangement of the three domains. Interestingly, the glycine-rich loop of the UDP-binding domain of MbMurC interacts through hydrogen bonds with the glucose moiety of the ligand, but not with the pyrophosphate moiety. These findings suggest that UNAG analogs might serve as potential candidates for neutralizing the catalytic activity of bacterial MurC.


Assuntos
Acetilglucosamina/metabolismo , Proteínas de Bactérias/química , Ligases/química , Mycobacterium bovis/enzimologia , Ligação Proteica , Conformação Proteica , Especificidade por Substrato
4.
Org Biomol Chem ; 19(8): 1818-1826, 2021 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-33565547

RESUMO

An in situ screening assay for UDP-galactopyranose mutase (UGM, an essential enzyme of M. tuberculosis cell wall biosynthesis) has been developed to discover novel UGM inhibitors. The approach is based on the amide-forming reaction of an amino acid core with various cinnamic acids, followed by a direct fluorescence polarization assay to identify the best UGM binders without isolation and purification of the screened ligands. This assay allows us to perform one-pot high-throughput synthesis and screening of enzyme inhibitors in a 384-well plate format. UGM ligands were successfully identified by this technology and their inhibition levels were established from pure synthetic compounds in vitro and in a whole cell antibacterial assay. This study provides a blueprint for designing enamide structures as new UGM inhibitors and anti-mycobacterial agents.


Assuntos
Aminoácidos/farmacologia , Antituberculosos/farmacologia , Cinamatos/farmacologia , Inibidores Enzimáticos/farmacologia , Transferases Intramoleculares/antagonistas & inibidores , Aminoácidos/síntese química , Aminoácidos/metabolismo , Antituberculosos/síntese química , Antituberculosos/metabolismo , Cinamatos/síntese química , Cinamatos/metabolismo , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/metabolismo , Transferases Intramoleculares/química , Transferases Intramoleculares/metabolismo , Cinética , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Mycobacterium bovis/efeitos dos fármacos , Mycobacterium bovis/enzimologia , Mycobacterium tuberculosis/enzimologia , Ligação Proteica
5.
Tuberculosis (Edinb) ; 123: 101956, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32741533

RESUMO

The ability to perform genetic manipulation of mycobacteria is important for characterization of gene function. Homologous recombination-based protocols are frequently used for reverse genetics studies with mycobacteria. It is known that Mycobacteriumbovis BCG Russia, closely related to M. bovis BCG Moreau, is a natural recA deficient strain and is non-permissive to homologous recombination assays. In this work we show that M. bovis BCG Moreau is also deficient in homologous recombination, shown by a specialized transduction assay, but this phenotype can be reverted by complementation with heterologous recombinases, using a recombineering protocol. Sequence analysis of the genes known to be involved in homologous recombination annotated in the genome of BCG Moreau detected no differences compared to the genome of BCG Pasteur. Further studies are needed in order to determine the exact mechanism underlying this deficiency in BCG Moreau.


Assuntos
Proteínas de Bactérias/genética , Recombinação Homóloga , Mycobacterium bovis/genética , Recombinases Rec A/genética , Proteínas de Bactérias/metabolismo , Genótipo , Mycobacterium bovis/enzimologia , Fenótipo , Recombinases Rec A/metabolismo
6.
J Microbiol Biotechnol ; 30(6): 811-821, 2020 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-32238759

RESUMO

Mycobacterium tuberculosis produces mycolic acids which are relevant for persistence, recalcitrance to antibiotics and defiance to host immunity. c-di-GMP is a second messenger involved in transition from planktonic cells to biofilms, whose levels are controlled by diguanylate cyclases (DGC) and phosphodiesterases (PDE). The transcriptional regulator dosR, is involved in response to low oxygen, a condition likely happening to a subset of cells within biofilms. Here, we found that in M. bovis BCG, expression of both BCG1416c and BCG1419c genes, which code for a DGC and a PDE, respectively, decreased in both stationary phase and during biofilm production. The kasA, kasB, and fas genes, which are involved in mycolic acid biosynthesis, were induced in biofilm cultures, as was dosR, therefore suggesting an inverse correlation in their expression compared with that of genes involved in c-di-GMP metabolism. The relative abundance within trehalose dimycolate (TDM) of α-mycolates decreased during biofilm maturation, with methoxy mycolates increasing over time, and keto species remaining practically stable. Moreover, addition of synthetic c-di-GMP to mid-log phase BCG cultures reduced methoxy mycolates, increased keto species and practically did not affect α-mycolates, showing a differential effect of c-di-GMP on keto- and methoxy-mycolic acid metabolism.


Assuntos
Proteínas de Bactérias/metabolismo , Biofilmes , GMP Cíclico/análogos & derivados , Mycobacterium bovis/enzimologia , Ácidos Micólicos/metabolismo , Proteínas de Bactérias/genética , GMP Cíclico/metabolismo , GMP Cíclico/farmacologia , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Mycobacterium bovis/genética , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/metabolismo , Fósforo-Oxigênio Liases/genética , Fósforo-Oxigênio Liases/metabolismo
7.
Tuberculosis (Edinb) ; 121: 101890, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32279869

RESUMO

CpG motifs in DNA sequences are recognized by Toll-like receptor 9 and activate immune cells. Bacterial genomic DNA (gDNA) has modified cytosine bases (5-methylcytosine [5 mC]) and modified adenine bases (6-methyladenine [6 mA]). 5 mC inhibits immune activation by CpG DNA; however, it is unclear whether 6 mA inhibits immune activation by CpG DNA. Mycobacterium bovis BCG (BCG) has three adenine methyltransferases (MTases) that act on specific target sequences. In this study, we examined whether the 6 mA at the target sites of adenine MTases affected the immunostimulatory activity of CpG DNA. Our results showed that only 6 mA located at the target sequence of mamA, an adenine MTase from BCG, enhanced interleukin (IL)-12p40 production from murine bone marrow-derived macrophages (BMDMs) stimulated with CpG DNA. Enhancement of IL-12p40 production in BMDMs was also observed when BMDMs were stimulated with CpG DNA ligated to oligodeoxynucleotides (ODNs) harboring 6 mA. Accordingly, we then evaluated whether gDNA from adenine MTase-deficient BCG was less efficient with regard to stimulation of BMDMs. Indeed, gDNA from a mamA-deficient BCG had less ability to activate BMDMs than that from wild-type BCG. We concluded from these results that adenine methylation on ODNs and bacterial gDNA may enhance immune activity induced by CpG DNA.


Assuntos
Adenina/análogos & derivados , Adjuvantes Imunológicos/farmacologia , DNA Bacteriano/imunologia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Metiltransferases/imunologia , Mycobacterium bovis/imunologia , Oligodesoxirribonucleotídeos/farmacologia , Receptor Toll-Like 9/agonistas , Adenina/imunologia , Animais , Células Cultivadas , DNA Bacteriano/genética , Interações Hospedeiro-Patógeno , Subunidade p40 da Interleucina-12/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Metiltransferases/deficiência , Metiltransferases/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mycobacterium bovis/enzimologia , Mycobacterium bovis/genética , Transdução de Sinais , Receptor Toll-Like 9/genética , Receptor Toll-Like 9/metabolismo
8.
Tuberculosis (Edinb) ; 120: 101891, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31778929

RESUMO

Fluoroquinolone (FQ) resistance in Mycobacterium tuberculosis (Mtb), caused by amino acid substitutions in DNA gyrase, has been increasingly reported worldwide. WQ-3810 is a newly developed FQ that is highly active against FQ-resistant pathogens; however, its activity against Mtb has not been evaluated. Herein we examined the efficacy of WQ-3810 against Mtb through the use of recombinant Mtb DNA gyrases. In addition, in vitro antimycobacterial activity of WQ-3810 was evaluated against recombinant Mtb var. bovis Bacille Calmette-Guérin strains in which gyrase-coding genes were replaced with Mtb variants containing resistance-conferring mutations. WQ-3810 showed a higher inhibitory activity than levofloxacin against most recombinant DNA gyrases with FQ-resistance mutations. Furthermore, WQ-3810 showed inhibition even against a DNA gyrase variant harboring a G88C mutation which is thought to confer the highest resistance against FQs in clinical Mtb isolates. In contrast, the FQ susceptibility test showed that WQ-3810 had relatively weak mycobactericidal activity compared with moxifloxacin. However, the combination of WQ-3810 and ethambutol showed the greatest degree of synergistic activity against recombinant strains. Since FQs and ethambutol have been used in multi-drug therapy for tuberculosis, WQ-3810 might represent a new, potent anti-tuberculosis drug that can be effective even against FQ-resistant Mtb strains.


Assuntos
Antibióticos Antituberculose/farmacologia , Azetidinas/farmacologia , Proteínas de Bactérias/metabolismo , DNA Girase/metabolismo , Farmacorresistência Bacteriana , Fluoroquinolonas/farmacologia , Mycobacterium bovis/efeitos dos fármacos , Mycobacterium tuberculosis/efeitos dos fármacos , Inibidores da Topoisomerase II/farmacologia , Proteínas de Bactérias/genética , DNA Girase/genética , Farmacorresistência Bacteriana/genética , Sinergismo Farmacológico , Quimioterapia Combinada , Etambutol/farmacologia , Testes de Sensibilidade Microbiana , Mutação , Mycobacterium bovis/enzimologia , Mycobacterium bovis/crescimento & desenvolvimento , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/genética
9.
Artigo em Inglês | MEDLINE | ID: mdl-31285226

RESUMO

Tuberculosis (TB) is a global health concern, and this situation has further worsened due to the emergence of drug-resistant strains and the failure of BCG vaccine to impart protection. There is an imperative need to develop highly sensitive, specific diagnostic tools, novel therapeutics, and vaccines for the eradication of TB. In the present study, a chemical screen of a pharmacologically active compound library was performed to identify antimycobacterial compounds. The phenotypic screen identified a few novel small-molecule inhibitors, including NU-6027, a known CDK-2 inhibitor. We demonstrate that NU-6027 inhibits Mycobacterium bovis BCG growth in vitro and also displayed cross-reactivity with Mycobacterium tuberculosis protein kinase D (PknD) and protein kinase G (PknG). Comparative structural and sequence analysis along with docking simulation suggest that the unique binding site stereochemistry of PknG and PknD accommodates NU-6027 more favorably than other M. tuberculosis Ser/Thr protein kinases. Further, we also show that NU-6027 treatment induces the expression of proapoptotic genes in macrophages. Finally, we demonstrate that NU-6027 inhibits M. tuberculosis growth in both macrophage and mouse tissues. Taken together, these results indicate that NU-6027 can be optimized further for the development of antimycobacterial agents.


Assuntos
Antituberculosos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Mycobacterium bovis/efeitos dos fármacos , Mycobacterium tuberculosis/efeitos dos fármacos , Compostos Nitrosos/farmacologia , Proteína Quinase C/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Pirimidinas/farmacologia , Antituberculosos/química , Proteínas Reguladoras de Apoptose/agonistas , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Regulação da Expressão Gênica , Ensaios de Triagem em Larga Escala , Interações Hospedeiro-Patógeno , Macrófagos/metabolismo , Macrófagos/microbiologia , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Mycobacterium bovis/enzimologia , Mycobacterium bovis/genética , Mycobacterium bovis/crescimento & desenvolvimento , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/crescimento & desenvolvimento , Compostos Nitrosos/química , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteína Quinase C/química , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Secundária de Proteína , Pirimidinas/química , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia
10.
Vaccine ; 37(27): 3539-3551, 2019 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-31122861

RESUMO

The Mycobacterium bovis Bacille Calmette Guérin (BCG) vaccine shows variable efficacy in protection against adult tuberculosis (TB). Earlier, we have described a BCG mutant vaccine with a transposon insertion in the gene coding for the secreted acid phosphatase SapM, which led to enhanced long-term survival of vaccinated mice challenged with TB infection. To facilitate development of this mutation as part of a future improved live attenuated TB vaccine, we have now characterized the genome and transcriptome of this sapM::Tn mutant versus parental BCG Pasteur. Furthermore, we show that the sapM::Tn mutant had an equal low pathogenicity as WT BCG upon intravenous administration to immunocompromised SCID mice, passing this important safety test. Subsequently, we investigated the clearance of this improved vaccine strain following vaccination and found a more effective innate immune control over the sapM::Tn vaccine bacteria as compared to WT BCG. This leads to a fast contraction of IFNγ producing Th1 and Tc1 cells after sapM::Tn BCG vaccination. These findings corroborate that a live attenuated vaccine that affords improved long-term survival upon TB infection can be obtained by a mutation that further attenuates BCG. These findings suggest that an analysis of the effectiveness of innate immune control of the vaccine bacteria could be instructive also for other live attenuated TB vaccines that are currently under development, and encourage further studies of SapM mutation as a strategy in developing a more protective live attenuated TB vaccine.


Assuntos
Fosfatase Ácida/genética , Vacina BCG/efeitos adversos , Avaliação Pré-Clínica de Medicamentos , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/patologia , Mutação , Mycobacterium bovis/patogenicidade , Fatores de Virulência/genética , Animais , Vacina BCG/genética , Feminino , Interferon gama/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos SCID , Mycobacterium bovis/enzimologia , Mycobacterium bovis/genética , Linfócitos T/imunologia
11.
Cells ; 8(5)2019 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-31035386

RESUMO

M. tuberculosis (Mtb), which causes tuberculosis disease, continues to be a major global health threat. Correct identification of valid drug targets is important for the development of novel therapeutics that would shorten the current 6-9 month treatment regimen and target resistant bacteria. Methionine aminopeptidases (MetAP), which remove the N-terminal methionine from newly synthesized proteins, are essential in all life forms (eukaryotes and prokaryotes). The MetAPs contribute to the cotranslational control of proteins as they determine their half life (N-terminal end rule) and facilitate further modifications such as acetylation and others. Mtb (and M. bovis) possess two MetAP isoforms, MetAP1a and MetAP1c, encoded by the mapA and mapB genes, respectively. Conflicting evidence was reported in the literature on which of the two variants is essential. To resolve this question, we performed a targeted genetic deletion of each of these two genes. We show that a deletion mutant of mapA is viable with only a weak growth defect. In contrast, we provide two lines of genetic evidence that mapB is indispensable. Furthermore, construction of double-deletion mutants as well as the introduction of point mutations into mapB resulting in proteins with partial activity showed partial, but not full, redundancy between mapB and mapA. We propose that it is MetAP1c (mapB) that is essentially required for mycobacteria and discuss potential reasons for its vitality.


Assuntos
Proteínas de Bactérias/genética , Isoenzimas/genética , Metionil Aminopeptidases/genética , Mycobacterium tuberculosis/enzimologia , Metionina/metabolismo , Mycobacterium bovis/enzimologia
12.
FEMS Microbiol Lett ; 365(23)2018 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-30418577

RESUMO

We developed a method involving recombinant Mycobacterium bovis bacillus Calmette-Guérin (BCG) and recombinant Mycobacterium smegmatis to determine which mutations in Mycobacterium tuberculosis (Mtb) gyrBA are associated with fluoroquinolone (FQ) resistance. The minimal inhibitory concentration (MIC) for FQ for recombinant strains with wild-type Mtb gyrBA was equivalent to that for strains with intrinsic gyrBA. Among 27 gyrBA mutations, the fold-changes in FQ MIC for M. smegmatis and M. bovis BCG backgrounds were comparable and were in part equivalent to those previously reported for recombinant Mtb strains. Mutations at position 90 or 94 of gyrA conferred strong and synergistic FQ resistance, which may be associated with the clinical observation that isolates carrying these mutations are the most or second most frequent. Sitafloxacin hydrate had the lowest MIC among the FQs tested in this study, which is similar to findings from a previous in vivo animal study. Most gyrBA mutations detected in clinical Mtb isolates could confer FQ resistance, but several mutations reduced bacterial growth rates. Overall, recombinant M. smegmatis appears to be a beneficial surrogate system to evaluate FQ susceptibility of virulent mycobacteria.


Assuntos
Antibacterianos/farmacologia , DNA Girase/metabolismo , Farmacorresistência Bacteriana , Fluoroquinolonas/farmacologia , Mutação , Mycobacterium bovis/crescimento & desenvolvimento , Mycobacterium smegmatis/crescimento & desenvolvimento , DNA Girase/genética , Testes de Sensibilidade Microbiana , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mycobacterium bovis/efeitos dos fármacos , Mycobacterium bovis/enzimologia , Mycobacterium bovis/genética , Mycobacterium smegmatis/efeitos dos fármacos , Mycobacterium smegmatis/enzimologia , Mycobacterium smegmatis/genética , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/isolamento & purificação , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tuberculose/microbiologia
13.
Sci Rep ; 8(1): 12664, 2018 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-30140040

RESUMO

Mycobacterium tuberculosis, the causative agent of tuberculosis, has surpassed HIV as the leading cause of death due to an infectious disease worldwide, being responsible for more than 1.5 million deaths in low-income countries. In response to a pandemic threat by drug resistant strains, the tuberculosis research community is searching for new chemical entities with novel mechanisms of action to avoid drug resistance and shorten treatment regimens using combinatorial chemotherapy. Herein, we have identified several novel chemical scaffolds, GSK97C (spiro-oxazolidin-2-one), GSK93A (2-amino-1,3-thiazole, GSK85A and GSK92A (enamides), which target M. tuberculosis aspartyl-tRNA synthetase (Mt-AspRS), an essential component of the protein synthesis machinery of tuberculosis, using a whole-cell target-based screening strategy against a genetically modified Mycobacterium bovis BCG strain. We also provide further evidence of protein inhibition and inhibitor profiling through a classical aminoacylation reaction and a tRNA-independent assay, respectively. Altogether, our results have identified a number of hit new molecules with novel mechanism of action for further development through medicinal chemistry as hits and leads.


Assuntos
Antituberculosos/farmacologia , Aspartato-tRNA Ligase/metabolismo , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/enzimologia , Aspartato-tRNA Ligase/antagonistas & inibidores , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/farmacologia , Testes de Sensibilidade Microbiana , Mycobacterium bovis/efeitos dos fármacos , Mycobacterium bovis/enzimologia
14.
Pathog Dis ; 76(2)2018 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-29394352

RESUMO

Cyclic di-AMP (c-di-AMP) has been shown to play an important role in bacterial physiology and pathogen-host interactions. We previously reported that deletion of the sole c-di-AMP phosphodiesterase-encoding gene (cnpB) in Mycobacterium tuberculosis (Mtb) led to significant virulence attenuation. In this study, we found that ΔcnpB of M. bovisbacillus Calmette-Guerin (BCG) was unable to secrete c-di-AMP, which differs from Mtb ΔcnpB. We infected bone marrow-derived macrophages (BMDMs) with c-di-AMP-associated mutants generated from both Mtb and BCG. Our results showed that upon infection with Mtb ΔcnpB, BMDMs of wildtype mice secreted a large amount of interferon-ß (IFN-ß) post-infection similarly as we reported previously. In contrast, the response was less pronounced with BMDMs isolated from cGAS-/- mice and was nearly abolished with BMDMs prepared from STING-/- mice. Deletion of the region of difference 1 (RD1) locus in Mtb ΔcnpB did not alter the c-di-AMP secretion of ΔcnpB but eliminated the IFN-ß production in the infected cells. In contrast, neither BCG ΔcnpB nor a recombinant BCG ΔcnpB with a pRD1 cosmid induced a type I interferon response. Interestingly, multiple studies have demonstrated that type I IFN enhances BCG's immunity. Thus, amending BCG based on our findings might improve BCG vaccination.


Assuntos
3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Vacina BCG/imunologia , Fosfatos de Dinucleosídeos/metabolismo , Macrófagos/imunologia , Mycobacterium bovis/imunologia , Tuberculose/prevenção & controle , Vacinação/métodos , 3',5'-AMP Cíclico Fosfodiesterases/deficiência , Animais , Células Cultivadas , Interferon beta/metabolismo , Macrófagos/microbiologia , Camundongos , Camundongos Knockout , Mycobacterium bovis/enzimologia , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/imunologia
15.
Sci Rep ; 7(1): 15161, 2017 11 09.
Artigo em Inglês | MEDLINE | ID: mdl-29123253

RESUMO

Mycobacterium tuberculosis is a major health threat, necessitating novel drug targets. Protein synthesis in bacteria uses initiator tRNAi charged with formylated methionine residue. Deletion of the formylase gene, tRNAfMet-formyl transferase (fmt), causes severe growth-retardation in E. coli and in S. pneumoniae, but not in P. aeruginosa or S. aureus. fmt was predicted to be essential in M. tuberculosis by transposon library analysis, but this was never formally tested in any mycobacteria. We performed a targeted deletion of fmt in M. smegmatis as well as Mtb-complex (M. bovis). In both cases, we created a mero-diploid strain, deleted the native gene by two-step allelic exchange or specialized-phage transduction, and then removed the complementing gene to create full deletion mutants. In M. smegmatis a full deletion strain could be easily created. In contrast, in M. bovis-BCG, a full deletion strain could only be created after incubation of 6 weeks, with a generation time ~2 times longer than for wt bacteria. Our results confirm the importance of this gene in pathogenic mycobacteria, but as the deletion mutant is viable, validity of fmt as a drug target remains unclear. Our results also refute the previous reports that fmt is essential in M. tuberculosis-complex.


Assuntos
Genes Essenciais , Hidroximetil e Formil Transferases/metabolismo , Mycobacterium bovis/enzimologia , Mycobacterium bovis/crescimento & desenvolvimento , Deleção de Genes , Hidroximetil e Formil Transferases/genética , Viabilidade Microbiana , Mycobacterium bovis/genética , Mycobacterium smegmatis/enzimologia , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/crescimento & desenvolvimento
16.
Nat Commun ; 8(1): 244, 2017 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-28811474

RESUMO

Mycobacterium tuberculosis PtpA is a secreted effector protein that dephosphorylates several proteins in the host cell cytoplasm, such as p-JNK, p-p38, and p-VPS33B, leading to suppression of host innate immunity. Here we show that, in addition, PtpA enters the nucleus of host cells and regulates the expression of host genes, some of which are known to be involved in host innate immunity or in cell proliferation and migration (such as GADD45A). PtpA can bind directly to the promoter region of GADD45A in vitro. Both phosphatase activity and DNA-binding ability of PtpA are important in suppressing host innate immune responses. Furthermore, PtpA-expressing Mycobacterium bovis BCG promotes proliferation and migration of human lung adenoma A549 cells in vitro and in a mouse xenograft model. Further research is needed to test whether mycobacteria, via PtpA, might affect cell proliferation or migration in humans. Mycobacterium tuberculosis secretes a protein, PtpA, that dephosphorylates proteins in the host cell cytoplasm, weakening immune responses. Here, the authors show that PtpA also enters the nucleus, affects the expression of several host genes, and promotes proliferation and migration of a cancer cell line.


Assuntos
Proteínas de Bactérias/metabolismo , Mycobacterium bovis/enzimologia , Mycobacterium tuberculosis/enzimologia , Proteínas Tirosina Fosfatases/metabolismo , Tuberculose/genética , Tuberculose/fisiopatologia , Animais , Proteínas de Bactérias/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Movimento Celular , Proliferação de Células , Feminino , Interações Hospedeiro-Patógeno , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mycobacterium bovis/genética , Mycobacterium bovis/fisiologia , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/fisiologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Tirosina Fosfatases/genética , Tuberculose/metabolismo
17.
J Microbiol Methods ; 136: 34-39, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28249794

RESUMO

Signal transduction in bacteria is generally mediated via two-component systems. These systems depend on the transfer of a phosphate molecule from a donor to an acceptor by histidine kinases, thereby activating the acceptor to allow downstream signaling/activation. Several bacterial genomes, including the genome of M. tuberculosis, were shown to encode eukaryotic-like kinases. To better understand the function of these kinases and the regulatory networks within which they operate, identification of downstream targets is essential. We here present a straightforward approach for the identification of bacterial Ser/Thr-kinase substrates. This approach is based on the KESTREL (Kinase Tracking and Substrate Elucidation) procedure combined with reversed-phase chromatography and two-dimensional gel electrophoresis. Using this method, GarA was identified as one potential substrate for the mycobacterial Ser/Thr-protein kinase G (PknG). These results show that the modified KESTREL approach can be successfully employed for the identification of substrates for bacterial Ser/Thr-kinases.


Assuntos
Proteínas Quinases Dependentes de GMP Cíclico/isolamento & purificação , Mycobacterium tuberculosis/enzimologia , Proteínas Serina-Treonina Quinases/isolamento & purificação , Proteoma , Especificidade por Substrato , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Cromatografia/métodos , Proteínas Quinases Dependentes de GMP Cíclico/química , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Eletroforese em Gel Bidimensional/métodos , Ensaios Enzimáticos , Escherichia coli/genética , Genoma Bacteriano , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Mycobacterium bovis/enzimologia , Mycobacterium bovis/genética , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/genética , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Proteômica/métodos , Transdução de Sinais
18.
Tuberculosis (Edinb) ; 103: 37-43, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28237032

RESUMO

As a eukaryotic-like Ser/Thr protein kinase, Mycobacterium tuberculosis virulent effector protein kinase G (PknG) mediates mycobacterial survival by regulating bacterial cell metabolic processes and preventing phagosome-lysosome fusion in host macrophages. Targeting PknG is an effective strategy for development of anti-tuberculosis (TB) drugs. In the study, we found that sclerotiorin, derived from marine fungi from the South China Sea, exhibited moderately strong inhibitory effects on recombinant PknG, with an IC50 value of 76.5 µM, and acted as a non-competitive inhibitor. The dissociation constant (KD) of sclerotiorin determined by MST was 11.4 µM, demonstrating a moderate binding strength between them. Sclerotiorin could substantially impair the mycobacterial survival in infected macrophages while the macrophage viability remained unaffected, though it did not inhibit the mycobacterial growth in culture. When sclerotiorin was used in combination with rifampicin, intracellular mycobacterial growth decreased as sclerotiorin concentration increased. Docking analysis suggested a binding mechanism of inhibition with performing interactions with the P-loop and catalytic loop of PknG. In summary, we reported that sclerotiorin had moderately strong PknG inhibitory activity, but no cytotoxicity, and it could substantially decrease the mycobacterial growth inside macrophages, suggesting that sclerotiorin has potential to supplement antibiotic therapy for TB.


Assuntos
Antituberculosos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Benzopiranos/farmacologia , Macrófagos/efeitos dos fármacos , Mycobacterium tuberculosis/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Tuberculose/tratamento farmacológico , Animais , Antituberculosos/química , Antituberculosos/metabolismo , Carga Bacteriana , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Benzopiranos/química , Benzopiranos/metabolismo , Sítios de Ligação , Relação Dose-Resposta a Droga , Células HeLa , Humanos , Células MCF-7 , Macrófagos/microbiologia , Camundongos , Simulação de Acoplamento Molecular , Terapia de Alvo Molecular , Mycobacterium bovis/efeitos dos fármacos , Mycobacterium bovis/enzimologia , Mycobacterium bovis/crescimento & desenvolvimento , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/crescimento & desenvolvimento , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Tuberculose/microbiologia
19.
Methods Mol Biol ; 1491: 75-85, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-27778282

RESUMO

This chapter provides a step-by-step protocol using activity-based protein profiling (ABPP) as a chemical-proteomic tool to survey the antibiotic properties of a small molecule. Here, we investigate the molecular mechanism behind the bactericidal activity of tetrahydrolipstatin (THL). ABPP relies on small molecule probes that target the active site of specific enzymes in complex proteomes. These probes in turn are equipped with a reporter tag that allows capturing, visualization, enrichment, identification, and quantification of its targets either in vitro or in situ. THL possesses bactericidal activities, but its precise spectrum of molecular targets is poorly characterized. Here, we used THL analogs functionalized to enable Huisgen-base cycloaddition, commonly known as "click chemistry," to identify target proteins after enrichment from mycobacterial cell lysates obtained from different physiological conditions.


Assuntos
Esterases/metabolismo , Lactonas/farmacologia , Metabolismo dos Lipídeos , Mycobacterium bovis/enzimologia , Proteômica , Antibacterianos/farmacologia , Química Click , Reação de Cicloadição , Inibidores Enzimáticos/farmacologia , Orlistate
20.
Sci Rep ; 6: 38986, 2016 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-27982051

RESUMO

High-throughput phenotypic screens have re-emerged as screening tools in antibiotic discovery. The advent of such technologies has rapidly accelerated the identification of 'hit' compounds. A pre-requisite to medicinal chemistry optimisation programmes required to improve the drug-like properties of a 'hit' molecule is identification of its mode of action. Herein, we have combined phenotypic screening with a biased target-specific screen. The inosine monophosphate dehydrogenase (IMPDH) protein GuaB2 has been identified as a drugable target in Mycobacterium tuberculosis, however previously identified compounds lack the desired characteristics necessary for further development into lead-like molecules. This study has identified 7 new chemical series from a high-throughput resistance-based phenotypic screen using Mycobacterium bovis BCG over-expressing GuaB2. Hit compounds were identified in a single shot high-throughput screen, validated by dose response and subjected to further biochemical analysis. The compounds were also assessed using molecular docking experiments, providing a platform for their further optimisation using medicinal chemistry. This work demonstrates the versatility and potential of GuaB2 as an anti-tubercular drug target.


Assuntos
Antituberculosos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , IMP Desidrogenase/antagonistas & inibidores , Mycobacterium tuberculosis/enzimologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Avaliação Pré-Clínica de Medicamentos , IMP Desidrogenase/genética , IMP Desidrogenase/metabolismo , Mycobacterium bovis/enzimologia , Mycobacterium bovis/genética , Mycobacterium tuberculosis/genética
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