RESUMO
Diverse aerobic bacteria use atmospheric H2 as an energy source for growth and survival1. This globally significant process regulates the composition of the atmosphere, enhances soil biodiversity and drives primary production in extreme environments2,3. Atmospheric H2 oxidation is attributed to uncharacterized members of the [NiFe] hydrogenase superfamily4,5. However, it remains unresolved how these enzymes overcome the extraordinary catalytic challenge of oxidizing picomolar levels of H2 amid ambient levels of the catalytic poison O2 and how the derived electrons are transferred to the respiratory chain1. Here we determined the cryo-electron microscopy structure of the Mycobacterium smegmatis hydrogenase Huc and investigated its mechanism. Huc is a highly efficient oxygen-insensitive enzyme that couples oxidation of atmospheric H2 to the hydrogenation of the respiratory electron carrier menaquinone. Huc uses narrow hydrophobic gas channels to selectively bind atmospheric H2 at the expense of O2, and 3 [3Fe-4S] clusters modulate the properties of the enzyme so that atmospheric H2 oxidation is energetically feasible. The Huc catalytic subunits form an octameric 833 kDa complex around a membrane-associated stalk, which transports and reduces menaquinone 94 Å from the membrane. These findings provide a mechanistic basis for the biogeochemically and ecologically important process of atmospheric H2 oxidation, uncover a mode of energy coupling dependent on long-range quinone transport, and pave the way for the development of catalysts that oxidize H2 in ambient air.
Assuntos
Atmosfera , Hidrogênio , Hidrogenase , Mycobacterium smegmatis , Microscopia Crioeletrônica , Hidrogênio/química , Hidrogênio/metabolismo , Hidrogenase/química , Hidrogenase/metabolismo , Hidrogenase/ultraestrutura , Oxirredução , Oxigênio , Vitamina K 2/metabolismo , Atmosfera/química , Mycobacterium smegmatis/enzimologia , Mycobacterium smegmatis/metabolismo , HidrogenaçãoAssuntos
Trifosfato de Adenosina , Antituberculosos , Desenho de Fármacos , Mycobacterium tuberculosis , Tuberculose , Humanos , Trifosfato de Adenosina/metabolismo , Antituberculosos/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/metabolismo , Tuberculose/tratamento farmacológico , Tuberculose/microbiologia , AnimaisRESUMO
Antimicrobial tolerance is the ability of a microbial population to survive, but not proliferate, during antimicrobial exposure. Significantly, it has been shown to precede the development of bona fide antimicrobial resistance. We have previously identified the two-component system CroRS as a critical regulator of tolerance to antimicrobials like teixobactin in the bacterial pathogen Enterococcus faecalis. To understand the molecular mechanism of this tolerance, we have carried out RNA-seq analyses in the E. faecalis wild-type and isogenic ∆ croRS mutant to determine the teixobactin-induced CroRS regulon. We identified a 132 gene CroRS regulon and demonstrate that CroRS upregulates biosynthesis of all major components of the enterococcal cell envelope in response to teixobactin. This suggests a coordinating role of this regulatory system in maintaining integrity of the multiple layers of the enterococcal envelope during antimicrobial stress, likely contributing to bacterial survival. Using experimental evolution, we observed that truncation of HppS, a key enzyme in the synthesis of the quinone electron carrier demethylmenaquinone, was sufficient to rescue tolerance in the croRS deletion strain. This highlights a key role for isoprenoid biosynthesis in antimicrobial tolerance in E. faecalis. Here, we propose a model of CroRS acting as a master regulator of cell envelope biogenesis and a gate-keeper between isoprenoid biosynthesis and respiration to ensure tolerance against antimicrobial challenge.
Assuntos
Antibacterianos , Anti-Infecciosos , Antibacterianos/farmacologia , Enterococcus faecalis/genética , Proteínas de Bactérias/genética , Homeostase , Terpenos , Testes de Sensibilidade MicrobianaRESUMO
Testing Plasmodium vivax antimicrobial sensitivity is limited to ex vivo schizont maturation assays, which preclude determining the IC50s of delayed action antimalarials such as doxycycline. Using Plasmodium cynomolgi as a model for P. vivax, we determined the physiologically significant delayed death effect induced by doxycycline [IC50(96 h), 1,401 ± 607 nM]. As expected, IC50(96 h) to chloroquine (20.4 nM), piperaquine (12.6 µM), and tafenoquine (1,424 nM) were not affected by extended exposure.
Assuntos
Aminoquinolinas , Antimaláricos , Doxiciclina , Piperazinas , Plasmodium cynomolgi , Plasmodium vivax , Doxiciclina/farmacologia , Antimaláricos/farmacologia , Aminoquinolinas/farmacologia , Plasmodium vivax/efeitos dos fármacos , Plasmodium cynomolgi/efeitos dos fármacos , Cloroquina/farmacologia , Animais , Malária Vivax/tratamento farmacológico , Malária Vivax/parasitologia , Quinolinas/farmacologia , Concentração Inibidora 50 , Humanos , Testes de Sensibilidade ParasitáriaRESUMO
Synergistic interactions between chemical inhibitors, whilst informative, can be difficult to interpret, as chemical inhibitors can often have multiple targets, many of which can be unknown. Here, using multiplexed transcriptional repression, we have validated that the simultaneous repression of glutamate racemase and alanine racemase has a synergistic interaction in Mycobacterium tuberculosis. This confirms prior observations from chemical interaction studies and highlights the potential of targeting multiple enzymes involved in mycobacterial cell wall synthesis.
Assuntos
Alanina Racemase , Isomerases de Aminoácido , Mycobacterium tuberculosis , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/efeitos dos fármacos , Isomerases de Aminoácido/genética , Isomerases de Aminoácido/metabolismo , Alanina Racemase/genética , Alanina Racemase/metabolismo , Regulação Bacteriana da Expressão Gênica , Transcrição Gênica , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Inibidores Enzimáticos/farmacologia , Parede Celular/metabolismo , Parede Celular/genéticaRESUMO
Discovered in the 1920s, cytochrome bd is a terminal oxidase that has received renewed attention as a drug target since its atomic structure was first determined in 2016. Only found in prokaryotes, we study it here as a drug target for Mycobacterium tuberculosis (Mtb). Most previous drug discovery efforts toward cytochrome bd have involved analogues of the canonical substrate quinone, known as Aurachin D. Here, we report six new cytochrome bd inhibitor scaffolds determined from a computational screen and confirmed on target activity through in vitro testing. These scaffolds provide new avenues for lead optimization toward Mtb therapeutics.
Assuntos
Antituberculosos , Inibidores Enzimáticos , Mycobacterium tuberculosis , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/efeitos dos fármacos , Antituberculosos/farmacologia , Antituberculosos/química , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/química , Tuberculose/tratamento farmacológico , Oxirredutases/antagonistas & inibidores , Oxirredutases/metabolismo , Oxirredutases/química , Modelos Moleculares , Simulação de Acoplamento MolecularRESUMO
The brevicidines represent a novel class of nonribosomal antimicrobial peptides that possess remarkable potency and selectivity toward highly problematic and resistant Gram-negative pathogenic bacteria. A recently discovered member of the brevicidine family, coined brevicidine B (2), comprises a single amino acid substitution (from d-Tyr2 to d-Phe2) in the amino acid sequence of the linear moiety of brevicidine (1) and was reported to exhibit broader antimicrobial activity against both Gram-negative (MIC = 2-4 µgmL-1) and Gram-positive (MIC = 2-8 µgmL-1) pathogens. Encouraged by this, we herein report the first total synthesis of the proposed structure of brevicidine B (2), building on our previously reported synthetic strategy to access brevicidine (1). In agreement with the original isolation paper, pleasingly, synthetic 2 demonstrated antimicrobial activity toward Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae (MIC = 4-8 µgmL-1). Interestingly, however, synthetic 2 was inactive toward all of the tested Gram-positive pathogens, including methicillin-resistant Staphylococcus aureus strains. Substitution of d-Phe2 with its enantiomer, and other hydrophobic residues, yields analogues that were either inactive or only exhibited activity toward Gram-negative strains. The striking difference in the biological activity of our synthetic 2 compared to the reported natural compound warrants the re-evaluation of the original natural product for purity or possible differences in relative configuration. Finally, the evaluation of synthetic 1 and 2 in a human kidney organoid model of nephrotoxicity revealed substantial toxicity of both compounds, although 1 was less toxic than 2 and polymyxin B. These results indicate that modification to position 2 may afford a strategy to mitigate the nephrotoxicity of brevicidine.
Assuntos
Antibacterianos , Testes de Sensibilidade Microbiana , Relação Estrutura-Atividade , Antibacterianos/farmacologia , Antibacterianos/síntese química , Antibacterianos/química , Estrutura Molecular , Pseudomonas aeruginosa/efeitos dos fármacos , Humanos , Depsipeptídeos/farmacologia , Depsipeptídeos/química , Depsipeptídeos/síntese química , Klebsiella pneumoniae/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Peptídeos Cíclicos/farmacologia , Peptídeos Cíclicos/síntese química , Peptídeos Cíclicos/químicaRESUMO
Lipopeptides are an important class of biomolecules for drug development. Compared with conventional acylation, a chemoselective lipidation strategy offers a more efficient strategy for late-stage structural derivatisation of a peptide scaffold. It provides access to chemically diverse compounds possessing intriguing and non-native moieties. Utilising an allenamide, we report the first semisynthesis of antimicrobial lipopeptides leveraging a highly efficient thia-Michael addition of chemically diverse lipophilic thiols. Using chemoenzymatically prepared polymyxin B nonapeptide (PMBN) as a model scaffold, an optimised allenamide-mediated thia-Michael addition effected rapid and near quantitative lipidation, affording vinyl sulfide-linked lipopeptide derivatives. Harnessing the utility of this new methodology, 22â lipophilic thiols of unprecedented chemical diversity were introduced to the PMBN framework. These included alkyl thiols, substituted aromatic thiols, heterocyclic thiols and those bearing additional functional groups (e.g., amines), ultimately yielding analogues with potent Gram-negative antimicrobial activity and substantially attenuated nephrotoxicity. Furthermore, we report facile routes to transform the allenamide into a ß-keto amide on unprotected peptides, offering a powerful "jack-of-all-trades" synthetic intermediate to enable further peptide modification.
RESUMO
Oxidation of malate to oxaloacetate, catalyzed by either malate dehydrogenase (Mdh) or malate quinone oxidoreductase (Mqo), is a critical step of the tricarboxylic acid cycle. Both Mqo and Mdh are found in most bacterial genomes, but the level of functional redundancy between these enzymes remains unclear. A bioinformatic survey revealed that Mqo was not as widespread as Mdh in bacteria but that it was highly conserved in mycobacteria. We therefore used mycobacteria as a model genera to study the functional role(s) of Mqo and its redundancy with Mdh. We deleted mqo from the environmental saprophyte Mycobacterium smegmatis, which lacks Mdh, and found that Mqo was essential for growth on nonfermentable carbon sources. On fermentable carbon sources, the Δmqo mutant exhibited delayed growth and lowered oxygen consumption and secreted malate and fumarate as terminal end products. Furthermore, heterologous expression of Mdh from the pathogenic species Mycobacterium tuberculosis shortened the delayed growth on fermentable carbon sources and restored growth on nonfermentable carbon sources at a reduced growth rate. In M. tuberculosis, CRISPR interference of either mdh or mqo expression resulted in a slower growth rate compared to controls, which was further inhibited when both genes were knocked down simultaneously. These data reveal that exergonic Mqo activity powers mycobacterial growth under nonenergy limiting conditions and that endergonic Mdh activity complements Mqo activity, but at an energetic cost for mycobacterial growth. We propose Mdh is maintained in slow-growing mycobacterial pathogens for use under conditions such as hypoxia that require reductive tricarboxylic acid cycle activity.
Assuntos
Malato Desidrogenase , Malatos , Oxirredutases , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Carbono/metabolismo , Ciclo do Ácido Cítrico , Malato Desidrogenase/genética , Malato Desidrogenase/metabolismo , Malatos/metabolismo , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/metabolismo , Ácido Oxaloacético/metabolismo , Oxirredutases/genética , Oxirredutases/metabolismoRESUMO
Neutrophils are often the major leukocyte at sites of mycobacterial infection, yet little is known about their ability to kill mycobacteria. In this study we have investigated whether the potent antibacterial oxidant hypochlorous acid (HOCl) contributes to killing of Mycobacterium smegmatis when this bacterium is phagocytosed by human neutrophils. We found that M. smegmatis were ingested by neutrophils into intracellular phagosomes but were killed slowly. We measured a t 1/2 of 30 min for the survival of M. smegmatis inside neutrophils, which is 5 times longer than that reported for Staphylococcus aureus and 15 times longer than Escherichia coli Live-cell imaging indicated that neutrophils generated HOCl in phagosomes containing M. smegmatis; however, inhibition of HOCl production did not alter the rate of bacterial killing. Also, the doses of HOCl that are likely to be produced inside phagosomes failed to kill isolated bacteria. Lethal doses of reagent HOCl caused oxidation of mycothiol, the main low-m.w. thiol in this bacterium. In contrast, phagocytosed M. smegmatis maintained their original level of reduced mycothiol. Collectively, these findings suggest that M. smegmatis can cope with the HOCl that is produced inside neutrophil phagosomes. A mycothiol-deficient mutant was killed by neutrophils at the same rate as wild-type bacteria, indicating that mycothiol itself is not the main driver of M. smegmatis resistance. Understanding how M. smegmatis avoids killing by phagosomal HOCl could provide new opportunities to sensitize pathogenic mycobacteria to destruction by the innate immune system.
Assuntos
Antibacterianos/metabolismo , Ácido Hipocloroso/metabolismo , Infecções por Mycobacterium não Tuberculosas/imunologia , Mycobacterium smegmatis/fisiologia , Neutrófilos/metabolismo , Fagossomos/metabolismo , Células Cultivadas , Cisteína/metabolismo , Glicopeptídeos/metabolismo , Humanos , Evasão da Resposta Imune , Imunidade Inata , Inositol/metabolismo , Infecções por Mycobacterium não Tuberculosas/microbiologia , Neutrófilos/imunologia , FagocitoseRESUMO
Mycobacterium tuberculosis remains a leading cause of death for which new drugs are needed. The identification of drug targets has been advanced by high-throughput and targeted genetic deletion strategies. Each though has limitations including the inability to distinguish between levels of vulnerability, lethality, and scalability as a molecular tool. Using mycobacterial CRISPR interference in combination with phenotypic screening, we have overcome these individual issues to investigate essentiality, vulnerability and lethality for 94 target genes from a diverse array of cellular pathways, many of which are potential antibiotic targets. Essential genes involved in cell wall synthesis and central cellular functions were equally vulnerable and often had bactericidal consequences. Conversely, essential genes involved in metabolism, oxidative phosphorylation, or amino acid synthesis were less vulnerable to inhibition and frequently bacteriostatic. In conclusion, this study provides novel insights into mycobacterial genetics and biology that will help to prioritize potential drug targets.
Assuntos
Parede Celular/genética , Genes Essenciais , Ensaios de Triagem em Larga Escala/métodos , Redes e Vias Metabólicas , Mycobacterium tuberculosis/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Edição de Genes/métodos , Genes Bacterianos , Humanos , Mycobacterium tuberculosis/metabolismo , Fenótipo , RNA Bacteriano , Tuberculose/microbiologiaRESUMO
Multidrug-resistant (MDR) tuberculosis (TB) is defined by the resistance of Mycobacterium tuberculosis, the causative organism, to the first-line antibiotics rifampicin and isoniazid. Mitigating or reversing resistance to these drugs offers a means of preserving and extending their use in TB treatment. R-loops are RNA/DNA hybrids that are formed in the genome during transcription, and they can be lethal to the cell if not resolved. RNase HI is an enzyme that removes R-loops, and this activity is essential in M. tuberculosis: knockouts of rnhC, the gene encoding RNase HI, are nonviable. This essentiality makes it a candidate target for the development of new antibiotics. In the model organism Mycolicibacterium smegmatis, RNase HI activity is provided by two enzymes, RnhA and RnhC. We show that the partial depletion of RNase HI activity in M. smegmatis, by knocking out either of the genes encoding RnhA or RnhC, led to the accumulation of R-loops. The sensitivity of the knockout strains to the antibiotics moxifloxacin, streptomycin, and rifampicin was increased, the latter by a striking near 100-fold. We also show that R-loop accumulation accompanies partial transcriptional inhibition, suggesting a mechanistic basis for the synergy between RNase HI depletion and rifampicin. A model of how transcriptional inhibition can potentiate R-loop accumulation is presented. Finally, we identified four small molecules that inhibit recombinant RnhC activity and that also potentiated rifampicin activity in whole-cell assays against M. tuberculosis, supporting an on-target mode of action and providing the first step in developing a new class of antimycobacterial drug.
Assuntos
Infecções por Mycobacterium , Mycobacterium tuberculosis , Tuberculose Resistente a Múltiplos Medicamentos , Humanos , Rifampina/farmacologia , Isoniazida/farmacologia , Moxifloxacina , Mycobacterium tuberculosis/genética , Antibacterianos/farmacologia , Estreptomicina , RNA , Morte Celular , Antituberculosos/farmacologiaRESUMO
OBJECTIVES: There is an urgent need for novel drugs that target unique cellular pathways to combat infections caused by Mycobacterium tuberculosis. CRISPR interference (CRISPRi)-mediated transcriptional repression has recently been developed for use in mycobacteria as a genetic tool for identifying and validating essential genes as novel drug targets. Whilst CRISPRi has been applied to extracellular bacteria, no studies to date have determined whether CRISPRi can be used in M. tuberculosis infection models. METHODS: Using the human monocytic macrophage-like THP-1 cell line as a model for M. tuberculosis infection we investigated if CRISPRi can be activated within intracellular M. tuberculosis. RESULTS: The transcriptional repression of two candidate M. tuberculosis genes, i.e. mmpL3 and qcrB, leads to a reduction in viable M. tuberculosis within infected THP-1 cells. The reduction in viable colonies is dependent on both the level of CRISPRi-mediated repression and the duration of repression. CONCLUSIONS: These results highlight the utility of CRISPRi in exploring mycobacterial gene function and essentiality under a variety of conditions pertinent to host infection.
Assuntos
Mycobacterium tuberculosis , Tuberculose , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Expressão Gênica , Humanos , Macrófagos , Mycobacterium tuberculosis/genética , Tuberculose/microbiologiaRESUMO
Our inability to predict which mutations could result in antibiotic resistance has made it difficult to rapidly identify the emergence of resistance, identify pre-existing resistant populations, and manage our use of antibiotics to effectively treat patients and prevent or slow the spread of resistance. Here we investigated the potential for resistance against the new antitubercular nitroimidazole prodrugs pretomanid and delamanid to emerge in Mycobacterium tuberculosis, the causative agent of tuberculosis (TB). Deazaflavin-dependent nitroreductase (Ddn) is the only identified enzyme within M. tuberculosis that activates these prodrugs, via an F420H2-dependent reaction. We show that the native menaquinone-reductase activity of Ddn is essential for emergence from hypoxia, which suggests that for resistance to spread and pose a threat to human health, the native activity of Ddn must be at least partially retained. We tested 75 unique mutations, including all known sequence polymorphisms identified among ~15,000 sequenced M. tuberculosis genomes. Several mutations abolished pretomanid and delamanid activation in vitro, without causing complete loss of the native activity. We confirmed that a transmissible M. tuberculosis isolate from the hypervirulent Beijing family already possesses one such mutation and is resistant to pretomanid, before being exposed to the drug. Notably, delamanid was still effective against this strain, which is consistent with structural analysis that indicates delamanid and pretomanid bind to Ddn differently. We suggest that the mutations identified in this work be monitored for informed use of delamanid and pretomanid treatment and to slow the emergence of resistance.
Assuntos
Antituberculosos/farmacologia , Proteínas de Bactérias , Farmacorresistência Bacteriana , Mutação , Mycobacterium tuberculosis , Nitroimidazóis/farmacologia , Nitrorredutases , Oxazóis/farmacologia , Engenharia de Proteínas , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Farmacorresistência Bacteriana/efeitos dos fármacos , Farmacorresistência Bacteriana/genética , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Nitrorredutases/genética , Nitrorredutases/metabolismo , Polimorfismo GenéticoRESUMO
The crystal structure of the F1-catalytic domain of the adenosine triphosphate (ATP) synthase has been determined from Mycobacterium smegmatis which hydrolyzes ATP very poorly. The structure of the α3ß3-component of the catalytic domain is similar to those in active F1-ATPases in Escherichia coli and Geobacillus stearothermophilus However, its ε-subunit differs from those in these two active bacterial F1-ATPases as an ATP molecule is not bound to the two α-helices forming its C-terminal domain, probably because they are shorter than those in active enzymes and they lack an amino acid that contributes to the ATP binding site in active enzymes. In E. coli and G. stearothermophilus, the α-helices adopt an "up" state where the α-helices enter the α3ß3-domain and prevent the rotor from turning. The mycobacterial F1-ATPase is most similar to the F1-ATPase from Caldalkalibacillus thermarum, which also hydrolyzes ATP poorly. The ßE-subunits in both enzymes are in the usual "open" conformation but appear to be occupied uniquely by the combination of an adenosine 5'-diphosphate molecule with no magnesium ion plus phosphate. This occupation is consistent with the finding that their rotors have been arrested at the same point in their rotary catalytic cycles. These bound hydrolytic products are probably the basis of the inhibition of ATP hydrolysis. It can be envisaged that specific as yet unidentified small molecules might bind to the F1 domain in Mycobacterium tuberculosis, prevent ATP synthesis, and inhibit the growth of the pathogen.
Assuntos
Complexos de ATP Sintetase/antagonistas & inibidores , Antituberculosos , Proteínas de Bactérias/antagonistas & inibidores , Diarilquinolinas/química , Farmacorresistência Bacteriana Múltipla , Mycobacterium smegmatis/enzimologia , Mycobacterium tuberculosis/enzimologia , Complexos de ATP Sintetase/química , Antituberculosos/química , Antituberculosos/farmacologia , Proteínas de Bactérias/química , Humanos , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológicoRESUMO
Multidrug resistance is a major threat to global elimination of tuberculosis (TB). We performed phenotypic drug-susceptibility testing and whole-genome sequencing for 309 isolates from 342 consecutive patients who were given a diagnosis of TB in Yangon, Myanmar, during July 2016âJune 2018. We identified isolates by using the GeneXpert platform to evaluate drug-resistance profiles. A total of 191 (62%) of 309 isolates had rifampin resistance; 168 (88%) of these rifampin-resistant isolates were not genomically related, indicating the repeated emergence of resistance in the population, rather than extensive local transmission. We did not detect resistance mutations to new oral drugs, including bedaquiline and pretomanid. The current GeneXpert MTB/RIF system needs to be modified by using the newly launched Xpert MTB/XDR cartridge or line-probe assay. Introducing new oral drugs to replace those currently used in treatment regimens for multidrug-resistant TB will also be useful for treating TB in Myanmar.
Assuntos
Mycobacterium tuberculosis , Tuberculose Resistente a Múltiplos Medicamentos , Farmacorresistência Bacteriana , Genômica , Humanos , Testes de Sensibilidade Microbiana , Mianmar/epidemiologia , Mycobacterium tuberculosis/genética , Rifampina , Tuberculose Resistente a Múltiplos Medicamentos/diagnóstico , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Tuberculose Resistente a Múltiplos Medicamentos/epidemiologiaRESUMO
Mycobacterium tuberculosis, the causative agent of human tuberculosis, harbors a branched electron transport chain, preventing the bactericidal action of cytochrome bc1 inhibitors (e.g., TB47). Here, we investigated, using luminescent mycobacterial strains, the in vitro combination activity of cytochrome bc1 inhibitors and nitric oxide (NO) donors including pretomanid (PMD) and explored the mechanisms of combination activity. The TB47 and PMD combination quickly abolished the light emission of luminescent bacilli, as was the case for the combination of TB47 and aurachin D, a putative cytochrome bd inhibitor. The TB47 and PMD combination inhibited M. tuberculosis oxygen consumption, decreased ATP levels, and had a delayed bactericidal effect. The NO scavenger carboxy-PTIO prevented the bactericidal activity of the drug combination, suggesting the requirement for NO. In addition, cytochrome bc1 inhibitors were largely bactericidal when administered with DETA NONOate, another NO donor. Proteomic analysis revealed that the cotreated bacilli had a compromised expression of the dormancy regulon proteins, PE/PPE proteins, and proteins required for the biosynthesis of several cofactors, including mycofactocin. Some of these proteomic changes, e.g., the impaired dormancy regulon induction, were attributed to PMD. In conclusion, combination of cytochrome bc1 inhibitors with PMD inhibited M. tuberculosis respiration and killed the bacilli. The activity of cytochrome bc1 inhibitors can be greatly enhanced by NO donors. Monitoring of luminescence may be further exploited to screen cytochrome bd inhibitors.
Assuntos
Mycobacterium tuberculosis , Citocromos , Transporte de Elétrons , Complexo III da Cadeia de Transporte de Elétrons , Humanos , Óxido Nítrico , Nitroimidazóis , ProteômicaRESUMO
TB47, a new drug candidate targeting QcrB in the electron transport chain, has shown a unique synergistic activity with clofazimine and forms a highly sterilizing combination. Here, we investigated the sterilizing effects of several all-oral regimens containing TB47 plus clofazimine and linezolid as a block and the roles of fluoroquinolones and pyrazinamide in them. All these regimens cured tuberculosis within 4 to 6 months in a well-established mouse model, and adding pyrazinamide showed a significant difference in bactericidal effects.
Assuntos
Mycobacterium tuberculosis , Tuberculose , Animais , Antituberculosos/uso terapêutico , Clofazimina/uso terapêutico , Modelos Animais de Doenças , Linezolida , Camundongos , Camundongos Endogâmicos BALB C , Tuberculose/tratamento farmacológicoRESUMO
With the emergence of multidrug-resistant strains of Mycobacterium tuberculosis (MDR-TB) and extensive drug-resistant strains (XDR-TB), there is an urgent need to develop novel drugs for the treatment of tuberculosis. Here, we designed and synthesized a series of 5-methylpyrimidopyridone analogues as potential antitubercular agents. The most potent compound 6q exhibited a MIC value of 4 µM in vitro against Mycobacterium tuberculosis. The antitubercular activities of the synthesized compounds were impacted by the amantadine and 2-chlorophenyl groups, and were enhanced by the presence of 3-methyl(4-dimethylamino)piperidinylphenyl. Molecular modeling and binding studies suggest that PknB is the potential molecular target of 5-methylpyrimidopyridone compounds. This study provides insights for the future development of new antimycobacterial agents with novel mechanisms of action.
Assuntos
Antituberculosos/farmacologia , Descoberta de Drogas , Mycobacterium tuberculosis/efeitos dos fármacos , Piridonas/farmacologia , Animais , Antituberculosos/síntese química , Antituberculosos/química , Sobrevivência Celular/efeitos dos fármacos , Chlorocebus aethiops , Relação Dose-Resposta a Droga , Testes de Sensibilidade Microbiana , Modelos Moleculares , Estrutura Molecular , Piridonas/síntese química , Piridonas/química , Relação Estrutura-Atividade , Células VeroRESUMO
The K+-sparing diuretic amiloride elicits anticancer activities in multiple animal models. During our recent medicinal chemistry campaign aiming to identify amiloride analogs with improved properties for potential use in cancer, we discovered novel 6-(hetero)aryl-substituted amiloride and 5-(N,N-hexamethylene)amiloride (HMA) analogs with up to 100-fold higher potencies than the parent compounds against urokinase plasminogen activator (uPA), one of amiloride's putative anticancer targets, and no diuretic or antikaliuretic effects. Here, we report the systematic evaluation of structure-property relationships (lipophilicity, aqueous solubility and in vitro metabolic stability in human and mouse liver microsomes) in twelve matched pair analogs selected from our 6-substituted amiloride and HMA libraries. Mouse plasma stability, plasma protein binding, Caco-2 cell permeability, cardiac ion channel activity and pharmacokinetics in mice (PO and IV) and rats (IV) are described alongside amiloride and HMA comparators for a subset of the four most promising matched-pair analogs. The findings combined with earlier uPA activity/selectivity and other data ultimately drove selection of two analogs (AA1-39 and AA1-41) that showed efficacy in separate mouse cancer metastasis studies.