RESUMEN
Results from clinical strains and knockouts of the H37Rv and CDC1551 laboratory strains demonstrated that ndh (Rv1854c) is not a resistance-conferring gene for isoniazid, ethionamide, delamanid, or pretomanid in Mycobacterium tuberculosis. This difference in the susceptibility to NAD-adduct-forming drugs compared with other mycobacteria may be driven by differences in the absolute intrabacterial NADH concentration.
Asunto(s)
Mycobacterium tuberculosis , Tuberculosis Resistente a Múltiples Medicamentos , Humanos , Isoniazida/farmacología , Etionamida/farmacología , Mycobacterium tuberculosis/genética , Antituberculosos/farmacología , Proteínas Bacterianas/genética , Mutación , Tuberculosis Resistente a Múltiples Medicamentos/microbiologíaRESUMEN
It is only recently that the abundant presence of circular RNAs (circRNAs) in all kingdoms of Life, including the hyperthermophilic archaeon Pyrococcus abyssi, has emerged. This led us to investigate the physiologic significance of a previously observed weak intramolecular ligation activity of Pab1020 RNA ligase. Here we demonstrate that this enzyme, despite sharing significant sequence similarity with DNA ligases, is indeed an RNA-specific polynucleotide ligase efficiently acting on physiologically significant substrates. Using a combination of RNA immunoprecipitation assays and RNA-seq, our genome-wide studies revealed 133 individual circRNA loci in P. abyssi. The large majority of these loci interacted with Pab1020 in cells and circularization of selected C/D Box and 5S rRNA transcripts was confirmed biochemically. Altogether these studies revealed that Pab1020 is required for RNA circularization. Our results further suggest the functional speciation of an ancestral NTase domain and/or DNA ligase toward RNA ligase activity and prompt for further characterization of the widespread functions of circular RNAs in prokaryotes. Detailed insight into the cellular substrates of Pab1020 may facilitate the development of new biotechnological applications e.g. in ligation of preadenylated adaptors to RNA molecules.
Asunto(s)
Empalme Alternativo , Proteínas Arqueales/genética , Genoma Arqueal , Pyrococcus abyssi/genética , ARN Ligasa (ATP)/genética , ARN de Archaea/genética , ARN/genética , Proteínas Arqueales/metabolismo , Biología Computacional , Inmunoprecipitación , Pyrococcus abyssi/enzimología , ARN/metabolismo , ARN Ligasa (ATP)/metabolismo , Estabilidad del ARN , ARN de Archaea/metabolismo , ARN Circular , ARN Ribosómico 5S/genética , ARN Ribosómico 5S/metabolismo , Análisis de Secuencia de ARN , Especificidad por SustratoRESUMEN
Thymidylate synthase ThyX, required for DNA synthesis in many pathogenic bacteria, is considered a promising antimicrobial target. It binds FAD and three substrates, producing dTMP (2'-deoxythymidine-5'-monophosphate) from dUMP (2'-deoxyuridine-5'-monophosphate). However, ThyX proteins also act as NADPH oxidase by reacting directly with O2. In the present study we investigated the dynamic interplay between the substrates and their role in competing with this wasteful and potentially harmful oxidase reaction in catalytically efficient ThyX from Paramecium bursaria Chlorella virus-1. dUMP binding accelerates the O2-insensitive half-reaction between NADPH and FAD by over four orders of magnitude to ~30 s-1. Thus, although dUMP does not have a direct role in FAD reduction, any turnover with molecular O2 requires its presence. Inversely, NADPH accommodation accelerates dUMP binding ~3-fold and apparently precedes dUMP binding under physiological conditions. In the oxidative half-reaction, excess CH2H4folate (N5,N10-methylene-5,6,7,8-tetrahydrofolate) was found to re-oxidize FADH2 within 1 ms, thus very efficiently competing with FADH2 oxidation by O2 (1.5 s-1 under aerobic conditions). The resulting reaction scheme points out how the interplay between the fast reactions with the native substrates, although not rate-limiting for overall catalysis, avoids NADPH oxidase activity in aerobic micro-organisms, including many pathogens. These observations also explain why ThyX proteins are also present in aerobic micro-organisms.
Asunto(s)
Proteínas Bacterianas/metabolismo , Simulación de Dinámica Molecular , Consumo de Oxígeno/fisiología , Timidilato Sintasa/metabolismo , Animales , Sitios de Unión/fisiología , Dominio Catalítico/fisiología , Bovinos , Unión Proteica/fisiología , Especificidad por Sustrato/fisiologíaRESUMEN
3,5-Dinitrobenzylsulfanyl tetrazoles and 1,3,4-oxadiazoles, previously identified as having high in vitro activities against both replicating and nonreplicating mycobacteria and favorable cytotoxicity and genotoxicity profiles were investigated. First we demonstrated that these compounds act in a deazaflavin-dependent nitroreduction pathway and thus require a nitro group for their activity. Second, we confirmed the necessity of both nitro groups for antimycobacterial activity through extensive structure-activity relationship studies using 32 structural types of analogues, each in a five-membered series. Only the analogues with shifted nitro groups, namely, 2,5-dinitrobenzylsulfanyl oxadiazoles and tetrazoles, maintained high antimycobacterial activity but in this case mainly as a result of DprE1 inhibition. However, these analogues also showed increased toxicity to the mammalian cell line. Thus, both nitro groups in 3,5-dinitrobenzylsulfanyl-containing antimycobacterial agents remain essential for their high efficacy, and further efforts should be directed at finding ways to address the possible toxicity and solubility issues, for example, by targeted delivery.
Asunto(s)
Mycobacterium tuberculosis , Animales , Oxadiazoles/farmacología , Oxadiazoles/química , Tetrazoles/farmacología , Tetrazoles/química , Pruebas de Sensibilidad Microbiana , Antituberculosos/farmacología , Antituberculosos/química , Relación Estructura-Actividad , Nitrorreductasas , MamíferosRESUMEN
Drug-resistant Mycobacterium tuberculosis (Mtb) remains a major public health concern requiring complementary approaches to standard anti-tuberculous regimens. Anti-virulence molecules or compounds that enhance the activity of antimicrobial prodrugs are promising alternatives to conventional antibiotics. Exploiting host cell-based drug discovery, we identified an oxadiazole compound (S3) that blocks the ESX-1 secretion system, a major virulence factor of Mtb. S3-treated mycobacteria showed impaired intracellular growth and a reduced ability to lyse macrophages. RNA sequencing experiments of drug-exposed bacteria revealed strong upregulation of a distinct set of genes including ethA, encoding a monooxygenase activating the anti-tuberculous prodrug ethionamide. Accordingly, we found a strong ethionamide boosting effect in S3-treated Mtb. Extensive structure-activity relationship experiments revealed that anti-virulence and ethionamide-boosting activity can be uncoupled by chemical modification of the primary hit molecule. To conclude, this series of dual-active oxadiazole compounds targets Mtb via two distinct mechanisms of action.
Asunto(s)
Mycobacterium tuberculosis , Tuberculosis , Sistemas de Secreción Tipo VII , Humanos , Etionamida/farmacología , Oxadiazoles/farmacología , Proteínas Bacterianas/genéticaRESUMEN
Mycobacterium tuberculosis, the pathogen that causes tuberculosis, is responsible for the death of 1.5 million people each year and the number of bacteria resistant to the standard regimen is constantly increasing. This highlights the need to discover molecules that act on new M. tuberculosis targets. Mycolic acids, which are very long-chain fatty acids essential for M. tuberculosis viability, are synthesized by two types of fatty acid synthase (FAS) systems. MabA (FabG1) is an essential enzyme belonging to the FAS-II cycle. We have recently reported the discovery of anthranilic acids as MabA inhibitors. Here, the structure-activity relationships around the anthranilic acid core, the binding of a fluorinated analog to MabA by NMR experiments, the physico-chemical properties and the antimycobacterial activity of these inhibitors were explored. Further investigation of the mechanism of action in bacterio showed that these compounds affect other targets than MabA in mycobacterial cells and that their antituberculous activity is due to the carboxylic acid moiety which induces intrabacterial acidification.
RESUMEN
The restrictions posed by the COVID-19 pandemic obliged the French Society for Medicinal Chemistry (Société de chimie thérapeutique) and the French Microbiology Society (Société Française de Microbiologie) to organize their joint autumn symposium (entitled "On the hunt for next-generation antimicrobial agents") online on 9-10 December 2021. The meeting attracted more than 200 researchers from France and abroad with interests in drug discovery, antimicrobial resistance, medicinal chemistry, and related disciplines. This review summarizes the 13 invited keynote lectures. The symposium generated high-level scientific dialogue on the most recent advances in combating antimicrobial resistance. The University of Lille, the Institut Pasteur de Lille, the journal Pharmaceuticals, Oxeltis, and INCATE, sponsored the event.
RESUMEN
The sensitivity of Mycobacterium tuberculosis, the pathogen that causes tuberculosis (TB), to antibiotic prodrugs is dependent on the efficacy of the activation process that transforms the prodrugs into their active antibacterial moieties. Various oxidases of M. tuberculosis have the potential to activate the prodrug ethionamide. Here, we used medicinal chemistry coupled with a phenotypic assay to select the N-acylated 4-phenylpiperidine compound series. The lead compound, SMARt751, interacted with the transcriptional regulator VirS of M. tuberculosis, which regulates the mymA operon encoding a monooxygenase that activates ethionamide. SMARt751 boosted the efficacy of ethionamide in vitro and in mouse models of acute and chronic TB. SMARt751 also restored full efficacy of ethionamide in mice infected with M. tuberculosis strains carrying mutations in the ethA gene, which cause ethionamide resistance in the clinic. SMARt751 was shown to be safe in tests conducted in vitro and in vivo. A model extrapolating animal pharmacokinetic and pharmacodynamic parameters to humans predicted that as little as 25 mg of SMARt751 daily would allow a fourfold reduction in the dose of ethionamide administered while retaining the same efficacy and reducing side effects.
Asunto(s)
Mycobacterium tuberculosis , Profármacos , Tuberculosis , Animales , Antituberculosos/farmacología , Antituberculosos/uso terapéutico , Etionamida/química , Etionamida/farmacología , Etionamida/uso terapéutico , Ratones , Profármacos/farmacología , Profármacos/uso terapéutico , Tuberculosis/tratamiento farmacológicoRESUMEN
Mycobacterium tuberculosis (M.tb), the etiologic agent of tuberculosis, remains the leading cause of death from a single infectious agent worldwide. The emergence of drug-resistant M.tb strains stresses the need for drugs acting on new targets. Mycolic acids are very long chain fatty acids playing an essential role in the architecture and permeability of the mycobacterial cell wall. Their biosynthesis involves two fatty acid synthase (FAS) systems. Among the four enzymes (MabA, HadAB/BC, InhA and KasA/B) of the FAS-II cycle, MabA (FabG1) remains the only one for which specific inhibitors have not been reported yet. The development of a new LC-MS/MS based enzymatic assay allowed the screening of a 1280 fragment-library and led to the discovery of the first small molecules that inhibit MabA activity. A fragment from the anthranilic acid series was optimized into more potent inhibitors and their binding to MabA was confirmed by 19F ligand-observed NMR experiments.
Asunto(s)
Proteínas Bacterianas/antagonistas & inhibidores , Descubrimiento de Drogas , Inhibidores Enzimáticos/farmacología , Ácido Graso Sintasas/antagonistas & inhibidores , Mycobacterium tuberculosis/enzimología , ortoaminobenzoatos/farmacología , Proteínas Bacterianas/metabolismo , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/química , Ácido Graso Sintasas/metabolismo , Estructura Molecular , Relación Estructura-Actividad , ortoaminobenzoatos/químicaRESUMEN
Antibiotic resistance is one of the biggest threats to human health globally. Alarmingly, multidrug-resistant and extensively drug-resistant Mycobacterium tuberculosis have now spread worldwide. Some key antituberculosis antibiotics are prodrugs, for which resistance mechanisms are mainly driven by mutations in the bacterial enzymatic pathway required for their bioactivation. We have developed drug-like molecules that activate a cryptic alternative bioactivation pathway of ethionamide in M. tuberculosis, circumventing the classic activation pathway in which resistance mutations have now been observed. The first-of-its-kind molecule, named SMARt-420 (Small Molecule Aborting Resistance), not only fully reverses ethionamide-acquired resistance and clears ethionamide-resistant infection in mice, it also increases the basal sensitivity of bacteria to ethionamide.
Asunto(s)
Antituberculosos/farmacología , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Etionamida/metabolismo , Tuberculosis Extensivamente Resistente a Drogas/microbiología , Isoxazoles/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Compuestos de Espiro/farmacología , Animales , ADN/metabolismo , Etionamida/farmacología , Humanos , Ratones , Mutación , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Oxadiazoles/farmacología , Piperidinas/farmacología , Unión Proteica/efectos de los fármacos , Proteínas Represoras/antagonistas & inhibidores , Proteínas Represoras/metabolismoRESUMEN
There is an urgent need to identify new treatments for tuberculosis (TB), a major infectious disease caused by Mycobacterium tuberculosis (Mtb), which results in 1.5 million deaths each year. We have targeted two essential enzymes in this organism that are promising for antibacterial therapy and reported to be inhibited by naphthoquinones. ThyX is an essential thymidylate synthase that is mechanistically and structurally unrelated to the human enzyme. DNA gyrase is a DNA topoisomerase present in bacteria and plants but not animals. The current study set out to understand the structure-activity relationships of these targets in Mtb using a combination of cheminformatics and in vitro screening. Here, we report the identification of new Mtb ThyX inhibitors, 2-chloro-3-(4-methanesulfonylpiperazin-1-yl)-1,4-dihydronaphthalene-1,4-dione) and idebenone, which show modest whole-cell activity and appear to act, at least in part, by targeting ThyX in Mtb.
Asunto(s)
Proteínas Bacterianas/metabolismo , Modelos Moleculares , Mycobacterium tuberculosis/enzimología , Timidilato Sintasa/metabolismo , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/química , Teorema de Bayes , Girasa de ADN/metabolismo , Inhibidores Enzimáticos/análisis , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Aprendizaje Automático , Naftoquinonas/química , Naftoquinonas/farmacología , Timidilato Sintasa/antagonistas & inhibidores , Timidilato Sintasa/química , Ubiquinona/análogos & derivados , Ubiquinona/química , Ubiquinona/farmacología , Interfaz Usuario-ComputadorRESUMEN
ThyX is an essential thymidylate synthase that is mechanistically and structurally unrelated to the functionally analogous human enzyme, thus providing means for selective inhibition of bacterial growth. To identify novel compounds with anti-bacterial activity against the human pathogenic bacterium Helicobacter pylori, based on our earlier biochemical and structural analyses, we designed a series of eighteen 2-hydroxy-1,4-naphthoquinones (2-OH-1,4-NQs) that target HpThyX. Our lead-like molecules markedly inhibited the NADPH oxidation and 2'-deoxythymidine-5'-monophosphate-forming activities of HpThyX enzyme in vitro, with inhibitory constants in the low nanomolar range. The identification of non-cytotoxic and non-mitotoxic 2-OH-1,4-NQ inhibitors permitted testing their in vivo efficacy in a mouse model for H. pylori infections. Despite the widely assumed toxicity of naphthoquinones (NQs), we identified tight-binding ThyX inhibitors that were tolerated in mice and can be associated with a modest effect in reducing the number of colonizing bacteria. Our results thus provide proof-of-concept that targeting ThyX enzymes is a highly feasible strategy for the development of therapies against H. pylori and a high number of other ThyX-dependent pathogenic bacteria. We also demonstrate that chemical reactivity of NQs does not prevent their exploitation as anti-microbial compounds, particularly when mitotoxicity screening is used to prioritize these compounds for further experimentation.