RESUMEN
There is an urgent need to discover new anti-tubercular agents with novel mechanisms of action in order to tackle the scourge of drug-resistant tuberculosis. Here, we report the identification of such a molecule - an AminoPYrimidine-Sulfonamide (APYS1) that has potent, bactericidal activity against M. tuberculosis. Mutations in APYS1-resistant M. tuberculosis mapped exclusively to wag31, a gene that encodes a scaffolding protein thought to orchestrate cell elongation. Recombineering confirmed that a Gln201Arg mutation in Wag31 was sufficient to cause resistance to APYS1, however, neither overexpression nor conditional depletion of wag31 impacted M. tuberculosis susceptibility to this compound. In contrast, expression of the wildtype allele of wag31 in APYS1-resistant M. tuberculosis was dominant and restored susceptibility to APYS1 to wildtype levels. Time-lapse imaging and scanning electron microscopy revealed that APYS1 caused gross malformation of the old pole of M. tuberculosis, with eventual lysis. These effects resembled the morphological changes observed following transcriptional silencing of wag31 in M. tuberculosis. These data show that Wag31 is likely not the direct target of APYS1, but the striking phenotypic similarity between APYS1 exposure and genetic depletion of Wag31 in M. tuberculosis suggests that APYS1 might indirectly affect Wag31 through an as yet unknown mechanism.
Asunto(s)
Antituberculosos/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Pirimidinas/farmacocinética , Antibacterianos/farmacocinética , Aumento de la Célula , Descubrimiento de Drogas/métodos , Regulación Bacteriana de la Expresión Génica/genética , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/metabolismo , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Pirimidinas/química , Pirimidinas/metabolismo , Homología de Secuencia de Aminoácido , Sulfonamidas/metabolismo , Sulfonamidas/farmacocinética , Imagen de Lapso de TiempoRESUMEN
The unique cell wall of mycobacteria is essential to their viability and the target of many clinically used anti-tuberculosis drugs and inhibitors under development. Despite intensive efforts to identify the ligase(s) responsible for the covalent attachment of the two major heteropolysaccharides of the mycobacterial cell wall, arabinogalactan (AG) and peptidoglycan (PG), the enzyme or enzymes responsible have remained elusive. We here report on the identification of the two enzymes of Mycobacterium tuberculosis, CpsA1 (Rv3267) and CpsA2 (Rv3484), responsible for this function. CpsA1 and CpsA2 belong to the widespread LytR-Cps2A-Psr (LCP) family of enzymes that has been shown to catalyze a variety of glycopolymer transfer reactions in Gram-positive bacteria, including the attachment of wall teichoic acids to PG. Although individual cpsA1 and cpsA2 knock-outs of M. tuberculosis were readily obtained, the combined inactivation of both genes appears to be lethal. In the closely related microorganism Corynebacterium glutamicum, the ortholog of cpsA1 is the only gene involved in this function, and its conditional knockdown leads to dramatic changes in the cell wall composition and morphology of the bacteria due to extensive shedding of cell wall material in the culture medium as a result of defective attachment of AG to PG. This work marks an important step in our understanding of the biogenesis of the unique cell envelope of mycobacteria and opens new opportunities for drug development.
Asunto(s)
Proteínas Bacterianas/genética , Pared Celular/metabolismo , Galactanos/metabolismo , Mycobacterium tuberculosis/metabolismo , Peptidoglicano/metabolismo , Ácidos Teicoicos/metabolismo , Proteínas Bacterianas/metabolismo , Pared Celular/genética , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , Galactanos/genética , Mycobacterium tuberculosis/genética , Peptidoglicano/genética , Ácidos Teicoicos/genéticaRESUMEN
Apart from the SARS-CoV-2 virus, tuberculosis remains the leading cause of death from a single infectious agent according to the World Health Organization. As part of our long-term research, we prepared a series of hybrid compounds combining pyrazinamide, a first-line antitubercular agent, and 4-aminosalicylic acid (PAS), a second-line agent. Compound 11 was found to be the most potent, with a broad spectrum of antimycobacterial activity and selectivity toward mycobacterial strains over other pathogens. It also retained its in vitro activity against multiple-drug-resistant mycobacterial strains. Several structural modifications were attempted to improve the in vitro antimycobacterial activity. The δ-lactone form of compound 11 (11') had more potent in vitro antimycobacterial activity against Mycobacterium tuberculosis H37Rv. Compound 11 was advanced for in vivo studies, where it was proved to be nontoxic in Galleria mellonella and zebrafish models, and it reduced the number of colony-forming units in spleens in the murine model of tuberculosis. Biochemical studies showed that compound 11 targets mycobacterial dihydrofolate reductases (DHFR). An in silico docking study combined with molecular dynamics identified a viable binding mode of compound 11 in mycobacterial DHFR. The lactone 11' opens in human plasma to its parent compound 11 (t1/2 = 21.4 min). Compound 11 was metabolized by human liver fraction by slow hydrolysis of the amidic bond (t1/2 = 187 min) to yield PAS and its starting 6-chloropyrazinoic acid. The long t1/2 of compound 11 overcomes the main drawback of PAS (short t1/2 necessitating frequent administration of high doses of PAS).
Asunto(s)
Ácido Aminosalicílico , COVID-19 , Mycobacterium tuberculosis , Tuberculosis , Humanos , Animales , Ratones , Pirazinamida/farmacología , Ácido Aminosalicílico/farmacología , Pez Cebra , SARS-CoV-2 , Antituberculosos/química , Tuberculosis/tratamiento farmacológico , LactonasRESUMEN
Tuberculosis is the number one killer of infectious diseases caused by a single microbe, namely Mycobacterium tuberculosis (Mtb). The success rate of curing this infection is decreasing due to emerging antimicrobial resistance. Therefore, novel treatments are urgently needed. As an attempt to develop new antituberculars effective against both drugs-sensitive and drug-resistant Mtb, we report the synthesis of a novel series inspired by combining fragments from the first-line agents isoniazid and pyrazinamide (series I) and isoniazid with the second-line agent 4-aminosalicylic acid (series II). We identified compound 10c from series II with selective, potent in vitro antimycobacterial activity against both drug-sensitive and drug-resistant Mtb H37Rv strains with no in vitro or in vivo cytotoxicity. In the murine model of tuberculosis, compound 10c caused a statistically significant decrease in colony-forming units (CFU) in spleen. Despite having a 4-aminosalicylic acid fragment in its structure, biochemical studies showed that compound 10c does not directly affect the folate pathway but rather methionine metabolism. In silico simulations indicated the possibility of binding to mycobacterial methionine-tRNA synthetase. Metabolic study in human liver microsomes revealed that compound 10c does not have any known toxic metabolites and has a half-life of 630 min, overcoming the main drawbacks of isoniazid (toxic metabolites) and 4-aminosalicylic acid (short half-life).
Asunto(s)
Ácido Aminosalicílico , Mycobacterium tuberculosis , Tuberculosis , Humanos , Animales , Ratones , Isoniazida/farmacología , Ácido Aminosalicílico/farmacología , Antituberculosos/química , Tuberculosis/tratamiento farmacológico , Tuberculosis/microbiología , Metionina , Pruebas de Sensibilidad MicrobianaRESUMEN
Latent Mycobacterium tuberculosis infection presents one of the largest challenges for tuberculosis control and novel antimycobacterial drug development. A series of pyrano[3,2-b]indolone-based compounds was designed and synthesized via an original eight-step scheme. The synthesized compounds were evaluated for their in vitro activity against M. tuberculosis strains H37Rv and streptomycin-starved 18b (SS18b), representing models for replicating and nonreplicating mycobacteria, respectively. Compound 10a exhibited good activity with MIC99 values of 0.3 and 0.4 µg/mL against H37Rv and SS18b, respectively, as well as low toxicity, acceptable intracellular activity, and satisfactory metabolic stability and was selected as the lead compound for further studies. An analysis of 10a-resistant M. bovis mutants disclosed a cross-resistance with pretomanid and altered relative amounts of different forms of cofactor F420 in these strains. Complementation experiments showed that F420-dependent glucose-6-phosphate dehydrogenase and the synthesis of mature F420 were important for 10a activity. Overall these studies revealed 10a to be a prodrug that is activated by an unknown F420-dependent enzyme in mycobacteria.
Asunto(s)
Tuberculosis Latente , Mycobacterium tuberculosis , Tuberculosis , Antituberculosos/farmacología , Humanos , Mycobacterium tuberculosis/genéticaRESUMEN
With the emerging primary resistance of Mycobacterium tuberculosis to current drugs and wide distribution of latent tuberculosis infection, the need for new compounds with a novel mode of action is growing. Copper-mediated innate immunity and its antibacterial toxicity pose novel strategies for tuberculosis drug discovery and development. Transcriptome response to 1-hydroxy-5-R-pyridine-2(1H)-thiones, which were found to be highly active in vitro against actively growing and dormant nonculturable M. tuberculosis, revealed signs of copper toxicity. 1-Hydroxy-5-R-pyridine-2(1H)-thiones were found to form stable charged lipophilic complexes with Cu2+ ions that could transport into mycobacterial cells. Copper accumulated inside treated bacilli as subsequent metabolic destruction of the complex led to chemical transformation of 1-hydroxy-5-R-pyridine-2(1H)-thiones and release of free Cu2+ into the cytoplasm. 1-Hydroxy-5-R-pyridine-2(1H)-thiones are a potent class of Cu-dependent inhibitors of M. tuberculosis, and may control infection by impairment of copper homeostasis.
Asunto(s)
Antituberculosos/química , Antituberculosos/farmacología , Cobre/toxicidad , Mycobacterium tuberculosis/efectos de los fármacos , Piridinas/química , Tionas/química , Tuberculosis/tratamiento farmacológico , Células Cultivadas , Humanos , Pruebas de Sensibilidad Microbiana , Oligoelementos/toxicidad , Tuberculosis/microbiologíaRESUMEN
The search for compounds with biological activity for many diseases is turning increasingly to drug repurposing. In this study, we have focused on the European Union-approved antimalarial pyronaridine which was found to have in vitro activity against Mycobacterium tuberculosis (MIC 5⯵g/mL). In macromolecular synthesis assays, pyronaridine resulted in a severe decrease in incorporation of 14C-uracil and 14C-leucine similar to the effect of rifampicin, a known inhibitor of M. tuberculosis RNA polymerase. Surprisingly, the co-administration of pyronaridine (2.5⯵g/ml) and rifampicin resulted in in vitro synergy with an MIC 0.0019-0.0009⯵g/mL. This was mirrored in a THP-1 macrophage infection model, with a 16-fold MIC reduction for rifampicin when the two compounds were co-administered versus rifampicin alone. Docking pyronaridine in M. tuberculosis RNA polymerase suggested the potential for it to bind outside of the RNA polymerase rifampicin binding pocket. Pyronaridine was also found to have activity against a M. tuberculosis clinical isolate resistant to rifampicin, and when combined with rifampicin (10% MIC) was able to inhibit M. tuberculosis RNA polymerase in vitro. All these findings, and in particular the synergistic behavior with the antitubercular rifampicin, inhibition of RNA polymerase in combination in vitro and its current use as a treatment for malaria, may suggest that pyronaridine could also be used as an adjunct for treatment against M. tuberculosis infection. Future studies will test potential for in vivo synergy, clinical utility and attempt to develop pyronaridine analogs with improved potency against M. tuberculosis RNA polymerase when combined with rifampicin.
Asunto(s)
Antibióticos Antituberculosos/farmacología , Antimaláricos/farmacología , Antituberculosos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , ARN Polimerasas Dirigidas por ADN/antagonistas & inhibidores , Mycobacterium tuberculosis/efectos de los fármacos , Naftiridinas/farmacología , Rifampin/farmacología , Antimaláricos/química , Antituberculosos/química , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Sitios de Unión , ARN Polimerasas Dirigidas por ADN/química , ARN Polimerasas Dirigidas por ADN/metabolismo , Reposicionamiento de Medicamentos , Farmacorresistencia Bacteriana , Sinergismo Farmacológico , Quimioterapia Combinada , Humanos , Pruebas de Sensibilidad Microbiana , Simulación del Acoplamiento Molecular , Mycobacterium tuberculosis/enzimología , Mycobacterium tuberculosis/crecimiento & desarrollo , Naftiridinas/química , Unión Proteica , Conformación Proteica , Relación Estructura-Actividad , Células THP-1RESUMEN
Mycobacterium tuberculosis, the etiological agent of the infectious disease tuberculosis, kills approximately 1.5 million people annually, while the spread of multidrug-resistant strains is of great global concern. Thus, continuous efforts to identify new antitubercular drugs as well as novel targets are crucial. Recently, two prodrugs activated by the monooxygenase EthA, 7947882 and 7904688, which target the CTP synthetase PyrG, were identified and characterized. In this work, microbiological, biochemical, and in silico methodologies were used to demonstrate that both prodrugs possess a second target, the pantothenate kinase PanK. This enzyme is involved in coenzyme A biosynthesis, an essential pathway for M. tuberculosis growth. Moreover, compound 11426026, the active metabolite of 7947882, was demonstrated to directly inhibit PanK, as well. In an independent screen of a compound library against PyrG, two additional inhibitors were also found to be active against PanK. In conclusion, these direct PyrG and PanK inhibitors can be considered as leads for multitarget antitubercular drugs and these two enzymes could be employed as a "double-tool" in order to find additional hit compounds.
Asunto(s)
Ligasas de Carbono-Nitrógeno/efectos de los fármacos , Descubrimiento de Drogas/métodos , Fosfotransferasas (Aceptor de Grupo Alcohol)/efectos de los fármacos , Antituberculosos/química , Antituberculosos/metabolismo , Antituberculosos/farmacología , Proteínas Bacterianas/metabolismo , Simulación por Computador , Humanos , Modelos Moleculares , Mycobacterium tuberculosis/enzimología , Tuberculosis/tratamiento farmacológicoRESUMEN
Despite its great potential, the target-based approach has been mostly unsuccessful in tuberculosis drug discovery, while whole cell phenotypic screening has delivered several active compounds. However, for many of these hits, the cellular target has not yet been identified, thus preventing further target-based optimization of the compounds. In this context, the newly validated drug target CTP synthetase PyrG was exploited to assess a target-based approach of already known, but untargeted, antimycobacterial compounds. To this purpose the publically available GlaxoSmithKline antimycobacterial compound set was assayed, uncovering a series of 4-(pyridin-2-yl)thiazole derivatives which efficiently inhibit the Mycobacterium tuberculosis PyrG enzyme activity, one of them showing low activity against the human CTP synthetase. The three best compounds were ATP binding site competitive inhibitors, with Ki values ranging from 3 to 20 µM, but did not show any activity against a small panel of different prokaryotic and eukaryotic kinases, thus demonstrating specificity for the CTP synthetases. Metabolic labeling experiments demonstrated that the compounds directly interfere not only with CTP biosynthesis, but also with other CTP dependent biochemical pathways, such as lipid biosynthesis. Moreover, using a M. tuberculosis pyrG conditional knock-down strain, it was shown that the activity of two compounds is dependent on the intracellular concentration of the CTP synthetase. All these results strongly suggest a role of PyrG as a target of these compounds, thus strengthening the value of this kind of approach for the identification of new scaffolds for drug development.
Asunto(s)
Antituberculosos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Ligasas de Carbono-Nitrógeno/antagonistas & inhibidores , Mycobacterium tuberculosis/efectos de los fármacos , Piridinas/farmacología , Tiazoles/farmacología , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Antituberculosos/química , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Unión Competitiva , Ligasas de Carbono-Nitrógeno/química , Ligasas de Carbono-Nitrógeno/genética , Ligasas de Carbono-Nitrógeno/metabolismo , Descubrimiento de Drogas , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Expresión Génica , Ensayos Analíticos de Alto Rendimiento , Cinética , Lípidos/antagonistas & inhibidores , Lípidos/biosíntesis , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Simulación del Acoplamiento Molecular , Mycobacterium tuberculosis/enzimología , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/crecimiento & desarrollo , Unión Proteica , Piridinas/química , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Relación Estructura-Actividad , Tiazoles/químicaRESUMEN
Herein, we report the discovery and structure-activity relationships of 5-substituted-2-[(3,5-dinitrobenzyl)sulfanyl]-1,3,4-oxadiazoles and 1,3,4-thiadiazoles as a new class of antituberculosis agents. The majority of these compounds exhibited outstanding in vitro activity against Mycobacterium tuberculosis CNCTC My 331/88 and six multidrug-resistant clinically isolated strains of M. tuberculosis, with minimum inhibitory concentration values as low as 0.03 µM (0.011-0.026 µg/mL). The investigated compounds had a highly selective antimycobacterial effect because they showed no activity against the other bacteria or fungi tested in this study. Furthermore, the investigated compounds exhibited low in vitro toxicities in four proliferating mammalian cell lines and in isolated primary human hepatocytes. Several in vitro genotoxicity assays indicated that the selected compounds have no mutagenic activity. The oxadiazole and thiadiazole derivatives with the most favorable activity/toxicity profiles also showed potency comparable to that of rifampicin against the nonreplicating streptomycin-starved M. tuberculosis 18b-Lux strain, and therefore, these derivatives, are of particular interest.
Asunto(s)
Antituberculosos/síntesis química , Antituberculosos/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Oxazoles/síntesis química , Oxazoles/farmacología , Tiadiazoles/síntesis química , Tiadiazoles/farmacología , Animales , Antituberculosos/toxicidad , Bacterias/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Diseño de Fármacos , Farmacorresistencia Bacteriana Múltiple , Hongos/efectos de los fármacos , Humanos , Tuberculosis Latente/tratamiento farmacológico , Tuberculosis Latente/microbiología , Pruebas de Sensibilidad Microbiana , Microsomas/metabolismo , Mutágenos/toxicidad , Cultivo Primario de Células , Rifampin/farmacología , Relación Estructura-ActividadRESUMEN
A recently developed transformation system for the pathogenic yeast Candida parapsilosis opened a venue for studying the biological phenomena of this species at the molecular level. However, the standard chemical method yielded only about 1x10(3) transformants/microg of DNA, which is insufficient for certain types of experiment. With the aim of increasing the transformation efficiency, we employed two alternative methods for the introduction of plasmids into the recipient cells. Whereas biolistics resulted in about 5x10(2) transformants/microg of plasmid DNA, electroporation was an order of magnitude more efficient than the chemical method. Pretreatment of cells with 100 mM lithium acetate or 10 mM dithiothreitol resulted in a 5-fold (5x10(4)) or a 10-fold (1x10(5)) increase in transformation efficiency, respectively. This high-efficiency transformation method should be suitable for experiments such as the screening of DNA libraries.
Asunto(s)
Candida/genética , Transformación Genética/genética , Acetatos/farmacología , Biolística , Candida/metabolismo , Ditiotreitol/farmacología , Electroporación , Biblioteca de Genes , Pruebas Genéticas , Plásmidos/genética , Transformación Genética/fisiologíaRESUMEN
Candida parapsilosis is an important human pathogen, responsible for severe cases of systemic candidiasis and one of the leading causes of mortality in neonates. In this report, we describe the first system for genetic manipulation of C. parapsilosis. We isolated and subsequently determined DNA sequences of genes encoding galactokinase ( CpGAL1) and orotidine-5'-phosphate decarboxylase ( CpURA3) from a genomic DNA library of C. parapsilosis by functional complementation of corresponding mutations in Saccharomyces cerevisiae. The predicted protein products, Gal1p and Ura3p, displayed a high degree of homology with corresponding sequences of C. albicans and S. cerevisiae, respectively. A collection of galactokinase-deficient ( gal1) strains of C. parapsilosis was prepared using direct selection of mutagenized cells on media containing 2-deoxy-galactose. Additionally, we constructed a plasmid vector carrying CpGAL1 as a selection marker and a genomic DNA fragment with an autonomously replicating sequence activity that transforms the C. parapsilosis gal1 mutant strain with high efficiency. This system for genetic transformation of C. parapsilosis may significantly advance the study of this human pathogen, greatly improving our understanding of its biology and virulence, with implications for drug development.