RESUMEN
Protein N-glycosylation is a widespread post-translational modification. The first committed step in this process is catalysed by dolichyl-phosphate N-acetylglucosamine-phosphotransferase DPAGT1 (GPT/E.C. 2.7.8.15). Missense DPAGT1 variants cause congenital myasthenic syndrome and disorders of glycosylation. In addition, naturally-occurring bactericidal nucleoside analogues such as tunicamycin are toxic to eukaryotes due to DPAGT1 inhibition, preventing their clinical use. Our structures of DPAGT1 with the substrate UDP-GlcNAc and tunicamycin reveal substrate binding modes, suggest a mechanism of catalysis, provide an understanding of how mutations modulate activity (thus causing disease) and allow design of non-toxic "lipid-altered" tunicamycins. The structure-tuned activity of these analogues against several bacterial targets allowed the design of potent antibiotics for Mycobacterium tuberculosis, enabling treatment in vitro, in cellulo and in vivo, providing a promising new class of antimicrobial drug.
Asunto(s)
Antibióticos Antituberculosos/farmacología , Trastornos Congénitos de Glicosilación/metabolismo , Inhibidores Enzimáticos/farmacología , N-Acetilglucosaminiltransferasas/química , Animales , Antibióticos Antituberculosos/química , Sitios de Unión , Trastornos Congénitos de Glicosilación/genética , Inhibidores Enzimáticos/química , Femenino , Células HEK293 , Células Hep G2 , Humanos , Metabolismo de los Lípidos , Ratones , Simulación del Acoplamiento Molecular , Mutación , N-Acetilglucosaminiltransferasas/antagonistas & inhibidores , N-Acetilglucosaminiltransferasas/genética , N-Acetilglucosaminiltransferasas/metabolismo , Unión Proteica , Células Sf9 , Spodoptera , Tunicamicina/química , Tunicamicina/farmacología , Uridina Difosfato Ácido Glucurónico/química , Uridina Difosfato Ácido Glucurónico/metabolismoRESUMEN
Resistance of bacterial pathogens against antibiotics is declared by WHO as a major global health threat. As novel antibacterial agents are urgently needed, we re-assessed the broad-spectrum myxobacterial antibiotic myxovalargin and found it to be extremely potent against Mycobacterium tuberculosis. To ensure compound supply for further development, we studied myxovalargin biosynthesis in detail enabling production via fermentation of a native producer. Feeding experiments as well as functional genomics analysis suggested a structural revision, which was eventually corroborated by the development of a concise total synthesis. The ribosome was identified as the molecular target based on resistant mutant sequencing, and a cryo-EM structure revealed that myxovalargin binds within and completely occludes the exit tunnel, consistent with a mode of action to arrest translation during a late stage of translation initiation. These studies open avenues for structure-based scaffold improvement toward development as an antibacterial agent.
Asunto(s)
Mycobacterium tuberculosis , Myxococcales , Antibacterianos/química , Ribosomas/metabolismo , Biosíntesis de ProteínasRESUMEN
Mycobacterium tuberculosis (Mtb) was responsible for approximately 1.6 million deaths in 2021. With the emergence of extensive drug resistance, novel therapeutic agents are urgently needed, and continued drug discovery efforts required. Host-derived lipids such as cholesterol not only support Mtb growth, but are also suspected to function in immunomodulation, with links to persistence and immune evasion. Mtb cytochrome P450 (CYP) enzymes facilitate key steps in lipid catabolism and thus present potential targets for inhibition. Here we present a series of compounds based on an ethyl 5-(pyridin-4-yl)-1H-indole-2-carboxylate pharmacophore which bind strongly to both Mtb cholesterol oxidases CYP125 and CYP142. Using a structure-guided approach, combined with biophysical characterization, compounds with micromolar range in-cell activity against clinically relevant drug-resistant isolates were obtained. These will incite further development of much-needed additional treatment options and provide routes to probe the role of CYP125 and CYP142 in Mtb pathogenesis.
Asunto(s)
Mycobacterium tuberculosis , Sistema Enzimático del Citocromo P-450/metabolismo , Colesterol/química , Descubrimiento de Drogas , Antituberculosos/farmacología , Antituberculosos/químicaRESUMEN
The emergence of drug resistant Mycobacterium tuberculosis, the causative agent of tuberculosis, demands the development of new drugs and new drug targets. We have recently reported that the d-phenylalanine benzoxazole Q112 has potent antibacterial activity against this pathogen with a distinct mechanism of action from other antimycobacterial agents. Q112 and previously reported derivatives were unstable in plasma and no free compound could be observed. Here we expand the structure-activity relationship for antimycobacterial activity and find nonhydrolyzable derivatives with decreased plasma binding. We also show that there is no correlation between antibacterial activity and inhibition of PanG, a putative target for these compounds.
Asunto(s)
Mycobacterium tuberculosis , Tuberculosis Resistente a Múltiples Medicamentos , Tuberculosis , Humanos , Benzoxazoles/farmacología , Antituberculosos/farmacología , Antituberculosos/química , Relación Estructura-Actividad , Pruebas de Sensibilidad MicrobianaRESUMEN
Described here is a series of spiropyrimidinetrione (SPT) compounds with activity against Mycobacterium tuberculosis through inhibition of DNA gyrase. The SPT class operates via a novel mode of inhibition, which involves Mg2+-independent stabilization of the DNA cleavage complex with DNA gyrase and is thereby not cross-resistant with other DNA gyrase-inhibiting antibacterials, including fluoroquinolones. Compound 22 from the series was profiled broadly and showed in vitro cidality as well as intracellular activity against M. tuberculosis in macrophages. Evidence for the DNA gyrase mode of action was supported by inhibition of the target in a DNA supercoiling assay and elicitation of an SOS response seen in a recA reporter strain of M. tuberculosis. Pharmacokinetic properties of 22 supported evaluation of efficacy in an acute model of M. tuberculosis infection, where modest reduction in CFU numbers was seen. This work offers promise for deriving a novel drug class of tuberculosis agent without preexisting clinical resistance.
Asunto(s)
Mycobacterium tuberculosis , Tuberculosis , Girasa de ADN/genética , Fluoroquinolonas/farmacología , Fluoroquinolonas/uso terapéutico , Humanos , Inhibidores de Topoisomerasa II/farmacología , Tuberculosis/tratamiento farmacológicoRESUMEN
As a result of a high-throughput compound screening campaign using Mycobacterium tuberculosis-infected macrophages, a new drug candidate for the treatment of tuberculosis has been identified. GSK2556286 inhibits growth within human macrophages (50% inhibitory concentration [IC50] = 0.07 µM), is active against extracellular bacteria in cholesterol-containing culture medium, and exhibits no cross-resistance with known antitubercular drugs. In addition, it has shown efficacy in different mouse models of tuberculosis (TB) and has an adequate safety profile in two preclinical species. These features indicate a compound with a novel mode of action, although still not fully defined, that is effective against both multidrug-resistant (MDR) or extensively drug-resistant (XDR) and drug-sensitive (DS) M. tuberculosis with the potential to shorten the duration of treatment in novel combination drug regimens. (This study has been registered at ClinicalTrials.gov under identifier NCT04472897).
Asunto(s)
Mycobacterium tuberculosis , Tuberculosis Resistente a Múltiples Medicamentos , Tuberculosis , Animales , Antituberculosos/farmacología , Antituberculosos/uso terapéutico , Macrófagos , Ratones , Pruebas de Sensibilidad Microbiana , Tuberculosis/tratamiento farmacológico , Tuberculosis Resistente a Múltiples Medicamentos/tratamiento farmacológicoRESUMEN
Translational frameshift errors are often deleterious to the synthesis of functional proteins and could therefore be promoted therapeutically to kill bacteria. TrmD (tRNA-(N(1)G37) methyltransferase) is an essential tRNA modification enzyme in bacteria that prevents +1 errors in the reading frame during protein translation and represents an attractive potential target for the development of new antibiotics. Here, we describe the application of a structure-guided fragment-based drug discovery approach to the design of a new class of inhibitors against TrmD in Mycobacterium abscessus. Fragment library screening, followed by structure-guided chemical elaboration of hits, led to the rapid development of drug-like molecules with potent in vitro TrmD inhibitory activity. Several of these compounds exhibit activity against planktonic M. abscessus and M. tuberculosis as well as against intracellular M. abscessus and M. leprae, indicating their potential as the basis for a novel class of broad-spectrum mycobacterial drugs.
Asunto(s)
Antibacterianos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , ARN de Transferencia/metabolismo , ARNt Metiltransferasas/antagonistas & inhibidores , Antibacterianos/química , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Sitios de Unión , Descubrimiento de Drogas/métodos , Inhibidores Enzimáticos/química , Simulación del Acoplamiento Molecular , Mycobacterium abscessus/efectos de los fármacos , Mycobacterium abscessus/enzimología , Mycobacterium leprae/efectos de los fármacos , Mycobacterium leprae/enzimología , Unión Proteica , ARNt Metiltransferasas/química , ARNt Metiltransferasas/metabolismoRESUMEN
Combination chemotherapy can increase treatment efficacy and suppress drug resistance. Knowledge of how to engineer rational, mechanism-based drug combinations, however, remains lacking. Although studies of drug activity have historically focused on the primary drug-target interaction, growing evidence has emphasized the importance of the subsequent consequences of this interaction. Bedaquiline (BDQ) is the first new drug for tuberculosis (TB) approved in more than 40 y, and a species-selective inhibitor of the Mycobacterium tuberculosis (Mtb) ATP synthase. Curiously, BDQ-mediated killing of Mtb lags significantly behind its inhibition of ATP synthase, indicating a mode of action more complex than the isolated reduction of ATP pools. Here, we report that BDQ-mediated inhibition of Mtb's ATP synthase triggers a complex metabolic response indicative of a specific hierarchy of ATP-dependent reactions. We identify glutamine synthetase (GS) as an enzyme whose activity is most responsive to changes in ATP levels. Chemical supplementation with exogenous glutamine failed to affect BDQ's antimycobacterial activity. However, further inhibition of Mtb's GS synergized with and accelerated the onset of BDQ-mediated killing, identifying Mtb's glutamine synthetase as a collateral, rather than directly antimycobacterial, metabolic vulnerability of BDQ. These findings reveal a previously unappreciated physiologic specificity of ATP and a facet of mode-of-action biology we term collateral vulnerability, knowledge of which has the potential to inform the development of rational, mechanism-based drug combinations.
Asunto(s)
Diarilquinolinas/farmacología , Glutamato-Amoníaco Ligasa/efectos de los fármacos , Mycobacterium tuberculosis/efectos de los fármacos , Antituberculosos/farmacología , Proteínas Bacterianas/metabolismo , Diarilquinolinas/metabolismo , Glutamato-Amoníaco Ligasa/metabolismo , Pruebas de Sensibilidad Microbiana/métodos , Mycobacterium tuberculosis/metabolismo , Tuberculosis/microbiologíaRESUMEN
Candida auris is an emerging fatal fungal infection that has resulted in several outbreaks in hospitals and care facilities. Current treatment options are limited by the development of drug resistance. Identification of new pharmaceuticals to combat these drug-resistant infections will thus be required to overcome this unmet medical need. We have established a bioluminescent ATP-based assay to identify new compounds and potential drug combinations showing effective growth inhibition against multiple strains of multidrug-resistant Candida auris The assay is robust and suitable for assessing large compound collections by high-throughput screening (HTS). Utilizing this assay, we conducted a screen of 4,314 approved drugs and pharmacologically active compounds that yielded 25 compounds, including 6 novel anti-Candida auris compounds and 13 sets of potential two-drug combinations. Among the drug combinations, the serine palmitoyltransferase inhibitor myriocin demonstrated a combinational effect with flucytosine against all tested isolates during screening. This combinational effect was confirmed in 13 clinical isolates of Candida auris.
Asunto(s)
Candida , Preparaciones Farmacéuticas , Antifúngicos/farmacología , Antifúngicos/uso terapéutico , Reposicionamiento de Medicamentos , Pruebas de Sensibilidad MicrobianaRESUMEN
Diphenyl ether derivatives inhibit mycobacterial cell wall synthesis by inhibiting an enzyme, enoyl-acyl carrier protein reductase (InhA), which catalyses the last step in the fatty acid synthesis cycle of genus Mycobacterium. To select and validate a protein crystal structure of enoyl-acyl carrier protein reductase of Mycobacterium tuberculosis for designing inhibitors using molecular modelling, a cross-docking and correlation study was performed. A series of novel 1-(3-(3-hydroxy-4-phenoxyphenyl)-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl) ethan-1-ones were synthesized from this model and screened for their antitubercular activity against M. tuberculosis H37Rv. Compound PYN-8 showed good antitubercular activity on M. tuberculosis H37Rv (MIC = 4-7 µM) and Mycobacterium bovis (% inhibition at 10 µM = 95.91%). Cytotoxicity of all the synthesized derivatives was assessed using various cell lines, and they were found to be safe. Structure of PYN-8 was also confirmed by single-crystal X-ray diffraction. The molecular modelling studies also corroborated the biological activity of the compounds. Further, in silico findings revealed that all these tested compounds exhibited good ADME properties and drug likeness and thus may be considered as potential candidates for further drug development.
Asunto(s)
Antituberculosos/síntesis química , Antituberculosos/farmacología , Éteres Fenílicos/síntesis química , Éteres Fenílicos/farmacología , Tuberculosis/tratamiento farmacológico , Proteínas Bacterianas/metabolismo , Línea Celular , Línea Celular Tumoral , Simulación por Computador , Cristalografía por Rayos X , Diseño de Fármacos , Células HEK293 , Células HeLa , Humanos , Células MCF-7 , Pruebas de Sensibilidad Microbiana/métodos , Simulación del Acoplamiento Molecular , Mycobacterium tuberculosis/efectos de los fármacos , Células PC-3 , Relación Estructura-Actividad , Tuberculosis/metabolismoRESUMEN
A new series of novel triclosan (2,4,4'-trichloro-2'-hydroxydiphenylether) analogues were designed, synthesized, and screened for their in vitro antimycobacterial and antibacterial activities. Most of the compounds showed significant activity against Mycobacterium tuberculosis H37Rv strain with minimum inhibitory concentration (MIC) values in 20-40 µM range in GAST/Fe medium when compared with triclosan (43 µM) in the first week of assay, and after additional incubation, seven compounds, that is, 2a, 2c, 2g, 2h, 2i, 2j, and 2m, exhibited MIC values at the concentration of 20-40 µM. The compounds also showed more significant activity against Bacillus subtilis and Staphylococcus aureus. The synthesized compounds showed druggable properties, and the predicted ADME (absorption, distribution, metabolism, and excretion) properties were within the acceptable limits. The in silico studies predicted better interactions of compounds with target protein residues and a higher dock score in comparison with triclosan. Molecular dynamics simulation study of the most active compound 2i was performed in order to further explore the stability of the protein-ligand complex and the protein-ligand interaction in detail.
Asunto(s)
Antituberculosos/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Triclosán/farmacología , Aminas/síntesis química , Aminas/química , Aminas/farmacología , Antibacterianos/síntesis química , Antibacterianos/química , Antibacterianos/farmacología , Antituberculosos/síntesis química , Antituberculosos/química , Bacillus subtilis/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Staphylococcus aureus/efectos de los fármacos , Relación Estructura-Actividad , Triclosán/análogos & derivados , Triclosán/químicaRESUMEN
A series of Mycobacterium tuberculosis TMPK (MtbTMPK) inhibitors based on a reported compound 3 were synthesized and evaluated for their capacity to inhibit MtbTMPK catalytic activity and the growth of a virulent M. tuberculosis strain (H37Rv). Modifications of the scaffold of 3 failed to afford substantial improvements in MtbTMPK inhibitory activity and antimycobacterial activity. Optimization of the substitution pattern of the D ring of 3 resulted in compound 21j with improved MtbTMPK inhibitory potency (three-fold) and H37Rv growth inhibitory activity (two-fold). Moving the 3-chloro substituent of 21j to the para-position afforded isomer 21h, which, despite a 10-fold increase in IC50-value, displayed promising whole cell activity (minimum inhibitory concentration (MIC) = 12.5 µM).
Asunto(s)
Antituberculosos , Proteínas Bacterianas/antagonistas & inhibidores , Inhibidores Enzimáticos , Mycobacterium tuberculosis/enzimología , Nucleósido-Fosfato Quinasa/antagonistas & inhibidores , Timina , Antituberculosos/síntesis química , Antituberculosos/química , Antituberculosos/farmacología , Proteínas Bacterianas/metabolismo , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Humanos , Modelos Moleculares , Estructura Molecular , Nucleósido-Fosfato Quinasa/metabolismo , Relación Estructura-Actividad , Timina/análogos & derivados , Timina/síntesis química , Timina/química , Timina/farmacologíaRESUMEN
Mechanisms of magnesium homeostasis in Mycobacterium tuberculosis are poorly understood. Here, we describe the characterization of a pyrimidinetrione amide scaffold that disrupts magnesium homeostasis in the pathogen by direct binding to the CorA Mg2+/Co2+ transporter. Mutations in domains of CorA that are predicted to regulate the pore opening in response to Mg2+ ions conferred resistance to this scaffold. The pyrimidinetrione amides were cidal against the pathogen under both actively replicating and nonreplicating conditions in vitro and were efficacious against the organism during macrophage infection. However, the compound lacked efficacy in infected mice, possibly due to limited exposure. Our results indicate that inhibition of Mg2+ homeostasis by CorA is an attractive target for tuberculosis drug discovery and encourage identification of improved CorA inhibitors.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas de Transporte de Catión/metabolismo , Magnesio/metabolismo , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/metabolismo , Antibacterianos/química , Antibacterianos/farmacocinética , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Proteínas de Transporte de Catión/genética , Homeostasis/efectos de los fármacos , Pirimidinas/química , Pirimidinas/farmacocinética , Pirimidinas/farmacología , Relación Estructura-ActividadRESUMEN
C4-phenylthio ß-lactams are a new family of antibacterial agents that have activity against two phylogenetically distant bacteria - Mycobacterium tuberculosis (Mtb) and Moraxella catarrhalis (M. cat). These compounds are effective against ß-lactamase producing Mtb and M. cat unlike the clinically relevant ß-lactam antibiotics. The structure-activity relationship for the C4 phenylthio ß-lactams has not yet been completely defined. Earlier efforts in our laboratories established that the C4-phenylthio substituent is essential for antimicrobial activity, while the N1 carbamyl substituent plays a more subtle role. In this present study, we investigated the role that the stereochemistry at C4 plays in these compounds' antibacterial activity. This was achieved by synthesizing and testing the antimicrobial activity of diastereomers with a chiral carbamyl group at N1. Our findings indicate that a strict stereochemistry for the C4-phenylthio ß-lactams is not required to obtain optimal anti-Mtb and anti-M. cat activity. Furthermore, the structure-bioactivity profiles more closely relate to the electronic requirement of the phenylthiogroup. In addition, the MICs of Mtb are sensitive to growth medium composition. Select compounds showed activity against non-replicating and multi-drug resistant Mtb.
Asunto(s)
Antibacterianos/farmacología , Moraxella catarrhalis/efectos de los fármacos , Mycobacterium tuberculosis/efectos de los fármacos , Compuestos de Sulfhidrilo/farmacología , beta-Lactamas/farmacología , Animales , Antibacterianos/síntesis química , Antibacterianos/química , Supervivencia Celular/efectos de los fármacos , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Farmacorresistencia Bacteriana/efectos de los fármacos , Macrófagos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Estructura Molecular , Moraxella catarrhalis/crecimiento & desarrollo , Mycobacterium tuberculosis/crecimiento & desarrollo , Relación Estructura-Actividad , Compuestos de Sulfhidrilo/síntesis química , Compuestos de Sulfhidrilo/química , beta-Lactamas/síntesis química , beta-Lactamas/químicaRESUMEN
Enzymes in essential metabolic pathways are attractive targets for the treatment of bacterial diseases, but in many cases, the presence of homologous human enzymes makes them impractical candidates for drug development. Fumarate hydratase, an essential enzyme in the tricarboxylic acid (TCA) cycle, has been identified as one such potential therapeutic target in tuberculosis. We report the discovery of the first small molecule inhibitor, to our knowledge, of the Mycobacterium tuberculosis fumarate hydratase. A crystal structure at 2.0-Å resolution of the compound in complex with the protein establishes the existence of a previously unidentified allosteric regulatory site. This allosteric site allows for selective inhibition with respect to the homologous human enzyme. We observe a unique binding mode in which two inhibitor molecules interact within the allosteric site, driving significant conformational changes that preclude simultaneous substrate and inhibitor binding. Our results demonstrate the selective inhibition of a highly conserved metabolic enzyme that contains identical active site residues in both the host and the pathogen.
Asunto(s)
Fumarato Hidratasa/metabolismo , Mycobacterium tuberculosis/enzimología , Regulación Alostérica , Cristalografía por Rayos X , Fluorescencia , Fumarato Hidratasa/antagonistas & inhibidoresRESUMEN
Elucidating the target or mechanism of action of potential drugs in the discovery pipeline is an integral component of most programs. For antibacterial compounds, generation of resistant mutants followed by whole genome sequencing has often been successful in uncovering the proteins involved in regulating compound activation, uptake, efflux and importantly, target processes. When this process succeeds, we are quick to declare a target. In a study reported by Sing and Dhar et al. (in press), the combination of resistant mutant generation, whole genome sequencing and recombineering to identify the target of a Mycobacterium tuberculosis growth inhibitor, pointed to a mechanism involving a scaffolding protein, Wag31, involved in polar elongation of mycobacterial cells. Time-lapse microscopy and electron microscopy confirmed the view that this inhibitor resulted in interruption of nascent cell wall biosynthesis. However, co-expression as well as regulated titration of the putative Wag31 target demonstrated that the wild-type allele was dominant and showed no synergy with the inhibitor. The most plausible explanation from their results was that this inhibitor interfered with the interaction of Wag31 with one of its interacting partners in the elongation complex.
Asunto(s)
Proteínas Bacterianas/metabolismo , Descubrimiento de Drogas/métodos , Antibacterianos/farmacocinética , Antibacterianos/farmacología , Mycobacterium tuberculosis/genética , Peptidoglicano/metabolismo , Unión ProteicaRESUMEN
Tuberculosis (TB) recently became the leading infectious cause of death in adults, while attempts to shorten therapy have largely failed. Dormancy, persistence, and drug tolerance are among the factors driving the long therapy duration. Assays to measure in situ drug susceptibility of Mycobacterium tuberculosis bacteria in pulmonary lesions are needed if we are to discover new fast-acting regimens and address the global TB threat. Here we take a first step toward this goal and describe an ex vivo assay developed to measure the cidal activity of anti-TB drugs against M. tuberculosis bacilli present in cavity caseum obtained from rabbits with active TB. We show that caseum M. tuberculosis bacilli are largely nonreplicating, maintain viability over the course of the assay, and exhibit extreme tolerance to many first- and second-line TB drugs. Among the drugs tested, only the rifamycins fully sterilized caseum. A similar trend of phenotypic drug resistance was observed in the hypoxia- and starvation-induced nonreplicating models, but with notable qualitative and quantitative differences: (i) caseum M. tuberculosis exhibits higher drug tolerance than nonreplicating M. tuberculosis in the Wayne and Loebel models, and (ii) pyrazinamide is cidal in caseum but has no detectable activity in these classic nonreplicating assays. Thus, ex vivo caseum constitutes a unique tool to evaluate drug potency against slowly replicating or nonreplicating bacilli in their native caseous environment. Intracaseum cidal concentrations can now be related to the concentrations achieved in the necrotic foci of granulomas and cavities to establish correlations between clinical outcome and lesion-centered pharmacokinetics-pharmacodynamics (PK-PD) parameters.
Asunto(s)
Antituberculosos/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Tuberculosis/tratamiento farmacológico , Animales , Tolerancia a Medicamentos , Pirazinamida/farmacología , Conejos , Rifamicinas/farmacologíaRESUMEN
We deleted subunits I (cydA) and II (cydB) of the Mycobacterium tuberculosis cytochrome bd menaquinol oxidase. The resulting ΔcydA and ΔcydAB mutants were hypersusceptible to compounds targeting the mycobacterial bc1 menaquinol-cytochrome c oxidoreductase and exhibited bioenergetic profiles indistinguishable from strains deficient in the ABC-type transporter, CydDC, predicted to be essential for cytochrome bd assembly. These results confirm CydAB and CydDC as potential targets for drugs aimed at inhibiting a terminal respiratory oxidase implicated in pathogenesis.
Asunto(s)
Citocromos c/efectos de los fármacos , Complejo IV de Transporte de Electrones/efectos de los fármacos , Complejo IV de Transporte de Electrones/genética , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/genética , Antituberculosos/farmacología , Descubrimiento de Drogas , Genoma Bacteriano/genética , Pruebas de Sensibilidad Microbiana , Fosforilación Oxidativa/efectos de los fármacos , Oxígeno/metabolismo , Consumo de Oxígeno/genética , Eliminación de Secuencia/genéticaRESUMEN
Francisella tularensis is the causative agent of tularemia and a potential biowarfare agent. The virulence of F. tularensis is decreased by deletion of guaB, the gene encoding IMP dehydrogenase (IMPDH), suggesting that this enzyme is a target for antibacterial design. Here we report that F. tularensis growth is blocked by inhibitors of bacterial IMPDHs. Seventeen compounds from two different frameworks, designated the D and Q series, display antibacterial activities with MICs of <1 µM. These compounds are also active against intracellular infections. Surprisingly, antibacterial activity does not correlate with IMPDH inhibition. In addition, the presence of guanine does not affect the antibacterial activity of most compounds, nor does the deletion of guaB These observations suggest that antibacterial activity derives from inhibition of another target(s). Moreover, D compounds display antibacterial activity only against F. tularensis, suggesting the presence of a unique target or uptake mechanism. A ΔguaB mutant resistant to compound D73 contained a missense mutation (Gly45Cys) in nuoB, which encodes a subunit of bacterial complex I. Overexpression of the nuoB mutant conferred resistance to D73 in both wild-type and ΔguaB strains. This strain was not resistant to Q compounds, suggesting that a different off-target mechanism operates for these compounds. Several Q compounds are also effective against Mycobacterium tuberculosis, in which a second target has also been implicated, in addition to IMPDH. The fortuitous presence of multiple targets with overlapping structure-activity relationships presents an intriguing opportunity for the development of robust antibiotics that may avoid the emergence of resistance.
Asunto(s)
Antibacterianos/farmacología , Benzoxazoles/farmacología , Francisella tularensis/efectos de los fármacos , IMP Deshidrogenasa/antagonistas & inhibidores , Ftalazinas/farmacología , Animales , Línea Celular , Complejo I de Transporte de Electrón/genética , Humanos , IMP Deshidrogenasa/genética , Ratones , Pruebas de Sensibilidad Microbiana , Relación Estructura-Actividad , Tularemia/tratamiento farmacológico , Tularemia/microbiologíaRESUMEN
Nicotinamide adenine dinucleotide (NAD+) synthetase catalyzes the last step in NAD+ biosynthesis. Depletion of NAD+ is bactericidal for both active and dormant Mycobacterium tuberculosis (Mtb). By inhibiting NAD+ synthetase (NadE) from Mtb, we expect to eliminate NAD+ production which will result in cell death in both growing and nonreplicating Mtb. NadE inhibitors have been investigated against various pathogens, but few have been tested against Mtb. Here, we report on the expansion of a series of urea-sulfonamides, previously reported by Brouillette et al. Guided by docking studies, substituents on a terminal phenyl ring were varied to understand the structure-activity-relationships of substituents on this position. Compounds were tested as inhibitors of both recombinant Mtb NadE and Mtb whole cells. While the parent compound displayed very weak inhibition against Mtb NadE (IC50=1000µM), we observed up to a 10-fold enhancement in potency after optimization. Replacement of the 3,4-dichloro group on the phenyl ring of the parent compound with 4-nitro yielded 4f, the most potent compound of the series with an IC50 value of 90µM against Mtb NadE. Our modeling results show that these urea-sulfonamides potentially bind to the intramolecular ammonia tunnel, which transports ammonia from the glutaminase domain to the active site of the enzyme. This hypothesis is supported by data showing that, even when treated with potent inhibitors, NadE catalysis is restored when treated with exogenous ammonia. Most of these compounds also inhibited Mtb cell growth with MIC values of 19-100µg/mL. These results improve our understanding of the SAR of the urea-sulfonamides, their mechanism of binding to the enzyme, and of Mtb NadE as a potential antitubercular drug target.