RESUMEN
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
The coenzyme A (CoA) biosynthesis pathway has attracted attention as a potential target for much-needed novel antimicrobial drugs, including for the treatment of tuberculosis (TB), the lethal disease caused by Mycobacterium tuberculosis (Mtb). Seeking to identify inhibitors of Mtb phosphopantetheine adenylyltransferase (MtbPPAT), the enzyme that catalyses the penultimate step in CoA biosynthesis, we performed a fragment screen. In doing so, we discovered three series of fragments that occupy distinct regions of the MtbPPAT active site, presenting a unique opportunity for fragment linking. Here we show how, guided by X-ray crystal structures, we could link weakly-binding fragments to produce an active site binder with a KD <20â µM and on-target anti-Mtb activity, as demonstrated using CRISPR interference. This study represents a big step toward validating MtbPPAT as a potential drug target and designing a MtbPPAT-targeting anti-TB drug.
Asunto(s)
Mycobacterium tuberculosis , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Nucleotidiltransferasas/metabolismo , Antituberculosos/farmacologíaRESUMEN
Tuberculosis (TB), caused by the bacillus Mycobacterium tuberculosis (Mtb), remains a leading cause of death by infectious disease, overshadowed only recently by the COVID-19 pandemic [...].
Asunto(s)
COVID-19 , Mycobacterium tuberculosis , Tuberculosis , Humanos , Pandemias , Tuberculosis/microbiologíaRESUMEN
Rifampicin is an effective drug for treating tuberculosis (TB) but is not used to treat Mycobacterium abscessus infections due to poor in vitro activity. While rifabutin, another rifamycin, has better anti-M. abscessus activity, its activity is far from the nanomolar potencies of rifamycins against Mycobacterium tuberculosis. Here, we asked (i) why is rifabutin more active against M. abscessus than rifampicin, and (ii) why is rifabutin's anti-M. abscessus activity poorer than its anti-TB activity? Comparative analysis of naphthoquinone- versus naphthohydroquinone-containing rifamycins suggested that the improved activity of rifabutin over rifampicin is linked to its less readily oxidizable naphthoquinone core. Although rifabutin is resistant to bacterial oxidation, metabolite and genetic analyses showed that this rifamycin is metabolized by the ADP-ribosyltransferase ArrMab like rifampicin, preventing it from achieving the nanomolar activity that it displays against M. tuberculosis. Based on the identified dual mechanism of intrinsic rifamycin resistance, we hypothesized that rifamycins more potent than rifabutin should contain the molecule's naphthoquinone core plus a modification that blocks ADP-ribosylation at its C-23. To test these predictions, we performed a blinded screen of a diverse collection of 189 rifamycins and identified two molecules more potent than rifabutin. As predicted, these compounds contained both a more oxidatively resistant naphthoquinone core and C-25 modifications that blocked ADP-ribosylation. Together, this work revealed dual bacterial metabolism as the mechanism of intrinsic resistance of M. abscessus to rifamycins and provides proof of concept for the repositioning of rifamycins for M. abscessus disease by developing derivatives that resist both bacterial oxidation and ADP-ribosylation.
Asunto(s)
Mycobacterium abscessus , Rifamicinas , ADP-Ribosilación , Pruebas de Sensibilidad Microbiana , Rifabutina/farmacología , Rifamicinas/farmacologíaRESUMEN
Mycobacterium tuberculosis, the causative agent of tuberculosis, remains a leading infectious killer globally, demanding the urgent development of faster-acting drugs with novel mechanisms of action. Riminophenazines such as clofazimine are clinically efficacious against both drug-susceptible and drug-resistant strains of M. tuberculosis We determined the in vitro anti-M. tuberculosis activities, absorption, distribution, metabolism, and excretion properties, and in vivo mouse pharmacokinetics of a series of structurally related phenoxazines. One of these, PhX1, displayed promising drug-like properties and potent in vitro efficacy, supporting its further investigation in an M. tuberculosis-infected animal model.
Asunto(s)
Antituberculosos/farmacología , Antituberculosos/farmacocinética , Mycobacterium tuberculosis/efectos de los fármacos , Oxazinas/farmacología , Oxazinas/farmacocinética , Animales , Clofazimina/farmacocinética , Clofazimina/farmacología , Modelos Animales de Enfermedad , Ratones , Pruebas de Sensibilidad Microbiana/métodos , Tuberculosis/tratamiento farmacológico , Tuberculosis/microbiologíaRESUMEN
HIV significantly affects the immunological environment during tuberculosis coinfection, and therefore may influence the selective landscape upon which M. tuberculosis evolves. To test this hypothesis whole genome sequences were determined for 169 South African M. tuberculosis strains from HIV-1 coinfected and uninfected individuals and analyzed using two Bayesian codon-model based selection analysis approaches: FUBAR which was used to detect persistent positive and negative selection (selection respectively favoring and disfavoring nonsynonymous substitutions); and MEDS which was used to detect episodic directional selection specifically favoring nonsynonymous substitutions within HIV-1 infected individuals. Among the 25,251 polymorphic codon sites analyzed, FUBAR revealed that 189-fold more were detectably evolving under persistent negative selection than were evolving under persistent positive selection. Three specific codon sites within the genes celA2b, katG, and cyp138 were identified by MEDS as displaying significant evidence of evolving under directional selection influenced by HIV-1 coinfection. All three genes encode proteins that may indirectly interact with human proteins that, in turn, interact functionally with HIV proteins. Unexpectedly, epitope encoding regions were enriched for sites displaying weak evidence of directional selection influenced by HIV-1. Although the low degree of genetic diversity observed in our M. tuberculosis data set means that these results should be interpreted carefully, the effects of HIV-1 on epitope evolution in M. tuberculosis may have implications for the design of M. tuberculosis vaccines that are intended for use in populations with high HIV-1 infection rates.
Asunto(s)
Mycobacterium tuberculosis/metabolismo , Tuberculosis Pulmonar/metabolismo , Teorema de Bayes , Codón , Coinfección/metabolismo , Evolución Molecular , Variación Genética , Infecciones por VIH/complicaciones , Infecciones por VIH/genética , VIH-1/genética , Humanos , Modelos Genéticos , Mutación , Mycobacterium tuberculosis/efectos de los fármacos , Selección Genética , Tuberculosis Pulmonar/complicacionesRESUMEN
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
Tuberculosis is one of the deadliest infectious diseases in the world, and the increased number of multidrug-resistant and extensively drug-resistant strains is a reason for concern. We have previously reported a series of substituted 5-(2-aminothiazol-4-yl)isoxazole-3-carboxamides with growth inhibitory activity against Mycobacterium tuberculosis strains and low propensity to be substrate of efflux pumps. Encouraged by these preliminary results, we have undertaken a medicinal chemistry campaign to determine the metabolic fate of these compounds and to delineate a reliable body of Structure-Activity Relationships. Keeping intact the (thiazol-4-yl)isoxazole-3-carboxamide core, as it is deemed to be the pharmacophore of the molecule, we have extensively explored the structural modifications able to confer good activity and avoid rapid clearance. Also, a small set of analogues based on isostere manipulation of the 2-aminothiazole were prepared and tested, with the aim to disclose novel antitubercular chemotypes. These studies, combined, were instrumental in designing improved compounds such as 42g and 42l, escaping metabolic degradation by human liver microsomes and, at the same time, maintaining good antitubercular activity against both drug-susceptible and drug-resistant strains.
Asunto(s)
Isoxazoles , Mycobacterium tuberculosis , Humanos , Isoxazoles/farmacología , Antituberculosos/farmacología , Relación Estructura-Actividad , Química FarmacéuticaRESUMEN
The coenzyme A (CoA) biosynthesis pathway has attracted attention as a potential target for much-needed novel antimicrobial drugs, including for the treatment of tuberculosis (TB), the lethal disease caused by Mycobacterium tuberculosis (Mtb). Seeking to identify inhibitors of Mtb phosphopantetheine adenylyltransferase (MtbPPAT), the enzyme that catalyses the penultimate step in CoA biosynthesis, we performed a fragment screen. In doing so, we discovered three series of fragments that occupy distinct regions of the MtbPPAT active site, presenting a unique opportunity for fragment linking. Here we show how, guided by X-ray crystal structures, we could link weakly-binding fragments to produce an active site binder with a K D <20â µM and on-target anti-Mtb activity, as demonstrated using CRISPR interference. This study represents a big step toward validating MtbPPAT as a potential drug target and designing a MtbPPAT-targeting anti-TB drug.
RESUMEN
Coenzyme A (CoA) is a ubiquitous cofactor present in all living cells and estimated to be required for up to 9% of intracellular enzymatic reactions. Mycobacterium tuberculosis (Mtb) relies on its own ability to biosynthesize CoA to meet the needs of the myriad enzymatic reactions that depend on this cofactor for activity. As such, the pathway to CoA biosynthesis is recognized as a potential source of novel tuberculosis drug targets. In prior work, we genetically validated CoaBC as a bactericidal drug target in Mtb in vitro and in vivo. Here, we describe the identification of compound 1f, a small molecule inhibitor of the 4'-phosphopantothenoyl-l-cysteine synthetase (PPCS; CoaB) domain of the bifunctional Mtb CoaBC, and show that this compound displays on-target activity in Mtb. Compound 1f was found to inhibit CoaBC uncompetitively with respect to 4'-phosphopantothenate, the substrate for the CoaB-catalyzed reaction. Furthermore, metabolomic profiling of wild-type Mtb H37Rv following exposure to compound 1f produced a signature consistent with perturbations in pantothenate and CoA biosynthesis. As the first report of a direct small molecule inhibitor of Mtb CoaBC displaying target-selective whole-cell activity, this study confirms the druggability of CoaBC and chemically validates this target.
Asunto(s)
Mycobacterium tuberculosis , Péptido Sintasas/antagonistas & inhibidores , Coenzima A , Cisteína/análogos & derivados , Mycobacterium tuberculosis/enzimología , Mycobacterium tuberculosis/genética , Ácido Pantoténico/análogos & derivados , Péptido Sintasas/genéticaRESUMEN
Coenzyme A (CoA) is a fundamental co-factor for all life, involved in numerous metabolic pathways and cellular processes, and its biosynthetic pathway has raised substantial interest as a drug target against multiple pathogens including Mycobacterium tuberculosis. The biosynthesis of CoA is performed in five steps, with the second and third steps being catalysed in the vast majority of prokaryotes, including M. tuberculosis, by a single bifunctional protein, CoaBC. Depletion of CoaBC was found to be bactericidal in M. tuberculosis. Here we report the first structure of a full-length CoaBC, from the model organism Mycobacterium smegmatis, describe how it is organised as a dodecamer and regulated by CoA thioesters. A high-throughput biochemical screen focusing on CoaB identified two inhibitors with different chemical scaffolds. Hit expansion led to the discovery of potent and selective inhibitors of M. tuberculosis CoaB, which we show to bind to a cryptic allosteric site within CoaB.
Asunto(s)
Antituberculosos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Carboxiliasas/antagonistas & inhibidores , Mycobacterium smegmatis/enzimología , Mycobacterium tuberculosis/efectos de los fármacos , Péptido Sintasas/antagonistas & inhibidores , Regulación Alostérica/efectos de los fármacos , Sitio Alostérico/efectos de los fármacos , Antituberculosos/uso terapéutico , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/ultraestructura , Carboxiliasas/genética , Carboxiliasas/metabolismo , Carboxiliasas/ultraestructura , Coenzima A/biosíntesis , Cristalografía por Rayos X , Pruebas de Enzimas , Técnicas de Silenciamiento del Gen , Ensayos Analíticos de Alto Rendimiento , Humanos , Pruebas de Sensibilidad Microbiana , Mycobacterium tuberculosis/enzimología , Mycobacterium tuberculosis/genética , Péptido Sintasas/genética , Péptido Sintasas/metabolismo , Péptido Sintasas/ultraestructura , Tuberculosis/tratamiento farmacológico , Tuberculosis/microbiologíaRESUMEN
Claiming close to two million lives each year, tuberculosis is now the leading cause of death from an infectious disease. The rise in number of Mycobacterium tuberculosis (Mtb) strains resistant to existing TB drugs has underscored the urgent need to develop new antimycobacterials with novel mechanisms of action. To meet this need, a drug pipeline has been established that is populated with new and repurposed drugs. Recent advances in identifying molecules with inhibitory activity against Mtb under conditions modelled on those encountered during infection, and in elucidating their mechanisms of action, have primed the pipeline with promising drug/target couples, hit compounds and new targets. In this review, we highlight recent advances and emerging areas of opportunity in this field.
Asunto(s)
Antituberculosos/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Tuberculosis/tratamiento farmacológico , Animales , Diseño de Fármacos , Farmacorresistencia Bacteriana , Humanos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/fisiología , Tuberculosis/microbiologíaRESUMEN
Mycobacterium tuberculosis relies on its own ability to biosynthesize coenzyme A to meet the needs of the myriad enzymatic reactions that depend on this cofactor for activity. As such, the essential pantothenate and coenzyme A biosynthesis pathways have attracted attention as targets for tuberculosis drug development. To identify the optimal step for coenzyme A pathway disruption in M. tuberculosis, we constructed and characterized a panel of conditional knockdown mutants in coenzyme A pathway genes. Here, we report that silencing of coaBC was bactericidal in vitro, whereas silencing of panB, panC, or coaE was bacteriostatic over the same time course. Silencing of coaBC was likewise bactericidal in vivo, whether initiated at infection or during either the acute or chronic stages of infection, confirming that CoaBC is required for M. tuberculosis to grow and persist in mice and arguing against significant CoaBC bypass via transport and assimilation of host-derived pantetheine in this animal model. These results provide convincing genetic validation of CoaBC as a new bactericidal drug target.
Asunto(s)
Proteínas Bacterianas/antagonistas & inhibidores , Coenzima A/metabolismo , Inhibidores Enzimáticos/administración & dosificación , Mycobacterium tuberculosis/efectos de los fármacos , Péptido Sintasas/antagonistas & inhibidores , Tuberculosis/microbiología , Animales , Antituberculosos/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Inhibidores Enzimáticos/química , Femenino , Humanos , Ratones , Ratones Endogámicos C57BL , Mycobacterium tuberculosis/enzimología , Mycobacterium tuberculosis/genética , Péptido Sintasas/genética , Péptido Sintasas/metabolismo , Tuberculosis/tratamiento farmacológicoRESUMEN
Although efforts to identify novel therapies for the treatment of tuberculosis have led to the identification of several promising drug candidates, the identification of high-quality hits from conventional whole-cell screens remains disappointingly low. The elucidation of the genome sequence of Mycobacterium tuberculosis (Mtb) facilitated a shift to target-based approaches to drug design but these efforts have proven largely unsuccessful. More recently, regulated gene expression systems that enable dose-dependent modulation of gene expression have been applied in target validation to evaluate the requirement of individual genes for the growth of Mtb both in vitro and in vivo. Notably, these systems can also provide a measure of the extent to which putative targets must be depleted in order to manifest a growth inhibitory phenotype. Additionally, the successful implementation of Mtb strains engineered to under-express specific molecular targets in whole-cell screens has enabled the simultaneous identification of cell-permeant inhibitors with defined mechanisms of action. Here, we review the application of tetracycline-regulated gene expression systems in the validation of novel drug targets in Mtb, highlighting both the strengths and limitations associated with this approach to target validation.
RESUMEN
A combination of chemical genetic and biochemical assays was applied to investigate the mechanism of action of the anticancer drug 5-fluorouracil (5-FU), against Mycobacterium tuberculosis (Mtb). 5-FU resistance was associated with mutations in upp or pyrR. Upp-catalyzed conversion of 5-FU to FUMP was shown to constitute the first step in the mechanism of action, and resistance conferred by nonsynonymous SNPs in pyrR shown to be due to derepression of the pyr operon and rescue from the toxic effects of FUMP and downstream antimetabolites through de novo production of UMP. 5-FU-derived metabolites identified in Mtb were consistent with the observed incorporation of 5-FU into RNA and DNA and the reduced amount of mycolyl arabinogalactan peptidoglycan in 5-FU-treated cells. Conditional depletion of the essential thymidylate synthase ThyX resulted in modest hypersensitivity to 5-FU, implicating inhibition of ThyX by fluorodeoxyuridylate as a further component of the mechanism of antimycobacterial action of this drug.
Asunto(s)
Antimetabolitos/metabolismo , Fluorouracilo/metabolismo , Antibacterianos/química , Antibacterianos/metabolismo , Antibacterianos/farmacología , Antimetabolitos/química , Antimetabolitos/farmacología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Radioisótopos de Carbono/química , Pared Celular/efectos de los fármacos , Pared Celular/metabolismo , Fluorouracilo/química , Fluorouracilo/farmacología , Marcaje Isotópico , Mutación , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/metabolismo , Operón , Pentosiltransferasa/genética , Pentosiltransferasa/metabolismo , ARN Mensajero/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Timidilato Sintasa/genética , Timidilato Sintasa/metabolismoRESUMEN
The development and application of a highly versatile suite of tools for mycobacterial genetics, coupled with widespread use of "omics" approaches to elucidate the structure, function, and regulation of mycobacterial proteins, has led to spectacular advances in our understanding of the metabolism and physiology of mycobacteria. In this article, we provide an update on nucleotide metabolism and DNA replication in mycobacteria, highlighting key findings from the past 10 to 15 years. In the first section, we focus on nucleotide metabolism, ranging from the biosynthesis, salvage, and interconversion of purine and pyrimidine ribonucleotides to the formation of deoxyribonucleotides. The second part of the article is devoted to DNA replication, with a focus on replication initiation and elongation, as well as DNA unwinding. We provide an overview of replication fidelity and mutation rates in mycobacteria and summarize evidence suggesting that DNA replication occurs during states of low metabolic activity, and conclude by suggesting directions for future research to address key outstanding questions. Although this article focuses primarily on observations from Mycobacterium tuberculosis, it is interspersed, where appropriate, with insights from, and comparisons with, other mycobacterial species as well as better characterized bacterial models such as Escherichia coli. Finally, a common theme underlying almost all studies of mycobacterial metabolism is the potential to identify and validate functions or pathways that can be exploited for tuberculosis drug discovery. In this context, we have specifically highlighted those processes in mycobacterial DNA replication that might satisfy this critical requirement.