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AIMS: The incidence of lung infections is increasing worldwide in individuals suffering from cystic fibrosis and chronic obstructive pulmonary diseases. Mycobacterium abscessus is associated to chronic lung deterioration in these populations. The intrinsic resistance of M. abscessus to most conventional antibiotics jeopardizes treatment success rates. To date no single drug has been developed targeting specifically M. abscessus. Our objective was to characterize the pyrithione-core drug-like small molecule named VOMG as a new compound active against M. abscessus and other pathogens. METHODS: We used a multidisciplinary approach including microbiological, chemical, biochemical and transcriptomics procedures to validate VOMG as a promising anti-M. abscessus drug candidate. RESULTS: We report for the first time the in vitro and in vivo bactericidal activity of VOMG against M. abscessus and other pathogens. Besides being active against M. abscessus biofilm, the compound showed a favourable pharmacology (ADME-Tox) profile. Frequency of resistance studies were unable to isolate resistant mutants. VOMG inhibits cell division, particularly the FtsZ enzyme. CONCLUSIONS: VOMG is a new drug-like molecule discovered against M. abscessus inhibiting cell division with broad spectrum activity against other microbial pathogens.
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Tuberculosis is a serious public health problem worldwide. The search for new antibiotics has become a priority, especially with the emergence of resistant strains. A new family of imidazoquinoline derivatives, structurally analogous to triazolophthalazines, which had previously shown good antituberculosis activity, were designed to inhibit InhA, an essential enzyme for Mycobacterium tuberculosis survival. Over twenty molecules were synthesized and the results showed modest inhibitory efficacy against the protein. Docking experiments were carried out to show how these molecules could interact with the protein's substrate binding site. Disappointingly, unlike triazolophthlazines, these imidazoquinoline derivatives showed an absence of inhibition on mycobacterial growth.
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Antituberculosos , Proteínas Bacterianas , Simulación del Acoplamiento Molecular , Mycobacterium tuberculosis , Oxidorreductasas , Quinolinas , Mycobacterium tuberculosis/enzimología , Mycobacterium tuberculosis/efectos de los fármacos , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/química , Oxidorreductasas/antagonistas & inhibidores , Oxidorreductasas/metabolismo , Antituberculosos/farmacología , Antituberculosos/química , Antituberculosos/síntesis química , Quinolinas/química , Quinolinas/farmacología , Imidazoles/química , Imidazoles/farmacología , Imidazoles/síntesis química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/síntesis química , Relación Estructura-Actividad , Pruebas de Sensibilidad Microbiana , Sitios de Unión , Estructura MolecularRESUMEN
Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a worldwide scourge with more than 10 million people affected yearly. Among the proteins essential for the survival of Mtb, InhA has been and is still clinically validated as a therapeutic target. A new family of direct diaryl ether inhibitors, not requiring prior activation by the catalase peroxidase enzyme KatG, has been designed with the ambition of fully occupying the InhA substrate-binding site. Thus, eleven compounds, featuring three pharmacophores within the same molecule, were synthesized. One of them, 5-(((4-(2-hydroxyphenoxy)benzyl)(octyl)amino)methyl)-2-phenoxyphenol (compound 21), showed good inhibitory activity against InhA with IC50 of 0.70 µM. The crystal structure of compound 21 in complex with InhA/NAD+ showed how the molecule fills the substrate-binding site as well as the minor portal of InhA. This study represents a further step towards the design of new inhibitors of InhA.
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Antituberculosos , Imidazoles , Mycobacterium tuberculosis , Sulfonamidas , Tiofenos , Humanos , Antituberculosos/farmacología , Antituberculosos/química , Éter , Éteres , Sitios de Unión , Éteres de Etila , Proteínas Bacterianas/metabolismoRESUMEN
Vγ9Vδ2 T lymphocytes are programmed for broad antimicrobial responses with rapid production of Th1 cytokines even before birth, and thus thought to play key roles against pathogens in infants. The process regulating Vδ2 cell acquisition of cytotoxic potential shortly after birth remains understudied. We observed that perforin production in cord blood Vδ2 cells correlates with phenotypes defined by the concomitant assessment of PD-1 and CD56. Bulk RNA sequencing of sorted Vδ2 cell fractions indicated that transcripts related to cytotoxic activity and NK function are enriched in the subset with the highest proportion of perforin+ cells. Among differentially expressed transcripts, IRF8, previously linked to CD8 T cell effector differentiation and NK maturation, has the potential to mediate Vδ2 cell differentiation towards cytotoxic effectors. Our current and past results support the hypothesis that distinct mechanisms regulate Vδ2 cell cytotoxic function before and after birth, possibly linked to different levels of microbial exposure.
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Antígeno CD56 , Linfocitos T CD8-positivos , Citotoxicidad Inmunológica , Receptor de Muerte Celular Programada 1 , Receptores de Antígenos de Linfocitos T gamma-delta , Subgrupos de Linfocitos T , Humanos , Linfocitos T CD8-positivos/inmunología , Citocinas/metabolismo , Sangre Fetal , Perforina/genética , Perforina/metabolismo , Receptor de Muerte Celular Programada 1/metabolismo , Receptores de Antígenos de Linfocitos T gamma-delta/metabolismo , Subgrupos de Linfocitos T/inmunología , Antígeno CD56/metabolismoRESUMEN
Mycobacterium abscessus has recently emerged as the cause of an increasing number of human infections worldwide. Unfortunately, it is highly resistant to existing drugs, and new specific agents to combat M. abscessus have not yet been found. The discovery of antibiotics that are effective not only against replicating but also against dormant and often recalcitrant cells is a daunting challenge. In this study, we developed a model of non-replicating M. abscessus, which represents a valuable screening tool for antibacterial agents. Thus, we demonstrated that, under a deficiency of potassium ions in the growth media and prolonged incubation, M. abscessus entered a 'non-culturable' state with a significant loss of colony-forming ability, but it retained viability, as confirmed using the most-probable-number (MPN) assay. The 'non-culturable' mycobacteria possessed decelerated cellular metabolism and noticeable differences in cell morphology from actively growing mycobacteria. 'Non-culturable' cells were used in a comprehensive screening of the efficacy of antibiotics, along with actively growing cells. Both CFU and MPN tests confirmed the prominent bactericidal effect of moxifloxacin on actively growing and 'non-culturable' M. abscessus, as proven by less than 0.01% of cells surviving after antibiotic treatment and prolonged storage. Bedaquiline exhibited a comparable bactericidal effect only on metabolically inactive non-culturable cells aged for 44 days. There were reductions ranging from 1000 to 10,000-fold in CFU and MPN, but it was not so efficient with respect to active cells, resulting in a bacteriostatic effect. The demonstrated specificity of bedaquiline in relation to inert non-replicating M. abscessus offers a new and unexpected result. Based on the findings of this research, moxifloxacin and bedaquiline can be regarded as potential treatments for infections caused by M. abscessus. In addition, a key outcome is the proposal to include the combination of viability assays for comprehensive testing of drug candidates. Relying on CFU-based assays alone resulted in overestimates of antibacterial efficacy, as demonstrated in our experiments.
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Hydrazone compounds represent an important area of research that includes, among others, synthetic approaches and biological studies. A series of 17 hydrazones have been synthesized by mechanochemical means. The fragments chosen were phenolic and furanyl aldehydes coupled with 12 heterocyclic hydrazines or hydrazinamides. All compounds can be obtained quantitatively when operating on a planetary ball mill and a maximum reaction time of 180 min (6 cycles of 30 min each). Complete spectroscopic analyses of hydrazones revealed eight compounds (3-5, 8-11, 16) present in one geometric form, six compounds (1, 2, 13-15) present in two isomeric forms, and three compounds (6, 7, 12) where one rotation is restricted giving rise to two different forms. The single crystal X-ray structure of one of the hydrazones bearing the isoniazid fragment (8) indicates a crystal lattice consisting of two symmetry-independent molecules with different geometries. All compounds obtained were tested for anti-infectious and antibacterial activities. Four compounds (1, 3, 5 and 8) showed good activity against Mycobacterium tuberculosis, and one (7) was very potent against Staphylococcus aureus. Most interesting, this series of compounds displayed very promising antileishmanial activity. Among all, compound 9 exhibited an IC50 value of 0.3 µM on the Leishmania donovani intramacrophage amastigote in vitro model and a good selectivity index, better than miltefosine, making it worth evaluating in vivo.
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Antiprotozoarios , Hidrazonas , Hidrazonas/farmacología , Hidrazonas/química , Aldehídos , Amidas , Hidrazinas , Antibacterianos/farmacología , Antiprotozoarios/farmacología , Antiprotozoarios/química , Relación Estructura-ActividadRESUMEN
Treatment against tuberculosis can lead to the selection of drug-resistant Mycobacterium tuberculosis strains. To tackle this serious threat, new targets from M. tuberculosis are needed to develop novel effective drugs. In this work, we aimed to provide a possible workflow to validate new targets and inhibitors by combining genetic, in silico, and enzymological approaches. CanB is one of the three M. tuberculosis ß-carbonic anhydrases that catalyze the reversible reaction of CO2 hydration to form HCO3- and H+. To this end, we precisely demonstrated that CanB is essential for the survival of the pathogen in vitro by constructing conditional mutants. In addition, to search for CanB inhibitors, conditional canB mutants were also constructed using the Pip-ON system. By molecular docking and minimum inhibitory concentration assays, we selected three molecules that inhibit the growth in vitro of M. tuberculosis wild-type strain and canB conditional mutants, thus implementing a target-to-drug approach. The lead compound also showed a bactericidal activity by the time-killing assay. We further studied the interactions of these molecules with CanB using enzymatic assays and differential scanning fluorimetry thermal shift analysis. In conclusion, the compounds identified by the in silico screening proved to have a high affinity as CanB ligands endowed with antitubercular activity.
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Tuberculosis (TB) is the historical leading cause of death by a single infectious agent. The European Regimen Accelerator for Tuberculosis (ERA4TB) is a public-private partnership of 30+ institutions with the objective to progress new anti-TB regimens into the clinic. Thus, robust and replicable results across independent laboratories are essential for reliable interpretation of treatment efficacy. A standardization workgroup unified in vitro protocols and data reporting templates. Time-kill assays provide essential input data for pharmacometric model-informed translation of single agents and regimens activity from in vitro to in vivo and the clinic. Five conditions were assessed by time-kill assays in six independent laboratories using four bacterial plating methods. Baseline bacterial burden varied between laboratories but variability was limited in net drug effect, confirming 2.5 µL equally robust as 100 µL plating. This exercise establishes the foundations of collaborative data generation, reporting, and integration within the overarching Antimicrobial Resistance Accelerator program.
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Mycobacterium abscessus is an opportunistic pathogen that mainly colonizes and infects cystic fibrosis patients' lungs. M. abscessus is naturally resistant to many antibiotics such as rifamycin, tetracyclines and ß-lactams. The current therapeutic regimens are not very effective and are mostly based on repurposed drugs used against Mycobacterium tuberculosis infections. Thus, new approaches and novel strategies are urgently needed. This review aims to provide an overview of the latest ongoing findings to fight M. abscessus infections by analyzing emerging and alternative treatments, novel drug delivery strategies, and innovative molecules.
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Fibrosis Quística , Infecciones por Mycobacterium no Tuberculosas , Mycobacterium abscessus , Humanos , Fibrosis Quística/tratamiento farmacológico , Antibacterianos/farmacología , Infecciones por Mycobacterium no Tuberculosas/tratamiento farmacológico , beta-Lactamas/farmacología , Pruebas de Sensibilidad MicrobianaRESUMEN
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.
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Isoxazoles , Mycobacterium tuberculosis , Humanos , Isoxazoles/farmacología , Antituberculosos/farmacología , Relación Estructura-Actividad , Química FarmacéuticaRESUMEN
Mycobacterium tuberculosis (Mtb) is the etiological agent of tuberculosis (TB), one of the most life-threatening communicable diseases, which causes 10 million new cases each year and results in an estimated 1 [...].
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Enfermedades Transmisibles , Mycobacterium tuberculosis , Tuberculosis , Humanos , Tuberculosis/tratamiento farmacológico , Tuberculosis/microbiologíaRESUMEN
Tuberculosis (TB) still poses a global menace as one of the deadliest infectious diseases. A quarter of the human population is indeed latently infected with Mycobacterium tuberculosis. People with latent infection have a 5 to 10% lifetime risk of becoming ill with TB, representing a reservoir for TB active infection. This is a worrisome problem to overcome in the case of relapse; unfortunately, few drugs are effective against nonreplicating M. tuberculosis cells. Novel strategies to combat TB, including its latent form, are urgently needed. In response to the lack of new effective drugs and after screening about 500 original chemical molecules, we selected a compound, 11726172, that is endowed with potent antitubercular activity against M. tuberculosis both in vitro and in vivo and importantly also against dormant nonculturable bacilli. We also investigated the mechanism of action of 11726172 by applying a multidisciplinary approach, including transcriptomic, labeled metabolomic, biochemical, and microbiological procedures. Our results represent an important step forward in the development of a new antitubercular compound with a novel mechanism of action active against latent bacilli. IMPORTANCE The discontinuation of TB services due to COVID-19 causes concern about a future resurgence of TB, also considering that latent infection affects a high number of people worldwide. To combat this situation, the identification of antitubercular compounds targeting Mycobacterium tuberculosis through novel mechanisms of action is necessary. These compounds should be active against not only replicating bacteria cells but also nonreplicating cells to limit the reservoir of latently infected people on which the bacterium can rely to spread after reactivation.
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COVID-19 , Tuberculosis Latente , Mycobacterium tuberculosis , Tuberculosis , Humanos , Antituberculosos/farmacología , Tuberculosis/tratamiento farmacológico , Tuberculosis/microbiologíaRESUMEN
The first effective synthetic approach to naphthofuroquinones via a reaction involving lawsone, various aldehydes, and three isocyanides under microwave irradiation afforded derivatives in moderate to good yields. In addition, for less-reactive aldehydes, two naphtho-enaminodione quinones were obtained for the first time, as result of condensation between lawsone and isocyanides. X-ray structure determination for 9 and 2D-NMR spectra of 28 confirmed the obtained structures. All compounds were evaluated for their anti-infectious activities against Plasmodium falciparum, Leishmania donovani, and Mycobacterium tuberculosis. Among the naphthofuroquinone series, 17 exhibited comparatively the best activity against P. falciparum (IC50 = 2.5 µM) and M. tuberculosis (MIC = 9 µM) with better (P. falciparum) or equivalent (M. tuberculosis) values to already-known naphthofuroquinone compounds. Among the two naphtho-enaminodione quinones, 28 exhibited a moderate activity against P. falciparum with a good selectivity index (SI > 36) while also a very high potency against L. donovani (IC50 = 3.5 µM and SI > 28), rendering it very competitive to the reference drug miltefosine. All compounds were studied through molecular modeling on their potential targets for P. falciparum, Pfbc1, and PfDHODH, where 17 showed the most favorable interactions.
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Tuberculosis (TB) is one of the leading causes of death worldwide, due to a single pathogen, Mycobacterium tuberculosis. To eradicate TB, management of drug-resistant strains is fundamental, therefore, the identification and characterization of drug targets is pivotal. In this work we aim at describing the relationships with the well-known drug target DprE1 and DprE2, working in association for the biosynthesis of the arabinogalactan precursor, essential component of mycobacterial cell wall. We demonstrated that the enzymes behave as a stable heterodimeric complex, once co-expressed into the same system. This complex showed improved catalytic properties, compared to the singularly expressed enzymes, demonstrating that co-expression is fundamental to achieve the proper folding of the active sites. Our results represent an important step forward in deciphering the functional properties of these enzymes, and lay the foundations for structural studies, useful for development of more specific inhibitors helpful to contrast the spreading of drug-resistant strains.
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Mycobacterium tuberculosis , Tuberculosis , Antituberculosos/química , Antituberculosos/farmacología , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Dominio Catalítico , Humanos , Racemasas y Epimerasas , Tuberculosis/tratamiento farmacológico , Tuberculosis/microbiologíaRESUMEN
Avermectins are macrocyclic lactones with anthelmintic activity. Recently, they were found to be effective against Mycobacterium tuberculosis, which accounts for one third of the worldwide deaths from antimicrobial resistance. However, their anti-mycobacterial mode of action remains to be elucidated. The activity of selamectin was determined against a panel of M. tuberculosis mutants. Two strains carrying mutations in DprE1, the decaprenylphosphoryl-ß-D-ribose oxidase involved in the synthesis of mycobacterial arabinogalactan, were more susceptible to selamectin. Biochemical assays against the Mycobacterium smegmatis DprE1 protein confirmed this finding, and docking studies predicted a binding site in a loop that included Leu275. Sequence alignment revealed variants in this position among mycobacterial species, with the size and hydrophobicity of the residue correlating with their MIC values; M. smegmatis DprE1 variants carrying these point mutations validated the docking predictions. However, the correlation was not confirmed when M. smegmatis mutant strains were constructed and MIC phenotypic assays performed. Likewise, metabolic labeling of selamectin-treated M. smegmatis and M. tuberculosis cells with 14C-labeled acetate did not reveal the expected lipid profile associated with DprE1 inhibition. Together, our results confirm the in vitro interactions of selamectin and DprE1 but suggest that selamectin could be a multi-target anti-mycobacterial compound.
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Oxidorreductasas de Alcohol/antagonistas & inhibidores , Antiparasitarios/farmacología , Antituberculosos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Ivermectina/análogos & derivados , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/enzimología , Oxidorreductasas de Alcohol/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Sitios de Unión , Relación Dosis-Respuesta a Droga , Descubrimiento de Drogas , Ivermectina/farmacología , Pruebas de Sensibilidad Microbiana , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Mutación , Relación Estructura-ActividadRESUMEN
Some nontuberculous mycobacteria (NTM) are considered opportunistic pathogens. Nevertheless, NTM infections are increasing worldwide, becoming a major public health threat. Furthermore, there is no current specific drugs to treat these infections, and the recommended regimens generally lack efficacy, emphasizing the need for novel antibacterial compounds. In this paper, we focused on the essential mycolic acids transporter MmpL3, which is a well-characterized target of several antimycobacterial agents, to identify new compounds active against Mycobacterium abscessus (Mab). From the crystal structure of MmpL3 in complex with known inhibitors, through an in silico approach, we developed a pharmacophore that was used as a three-dimensional filter to identify new putative MmpL3 ligands within databases of known drugs. Among the prioritized compounds, mefloquine showed appreciable activity against Mab (MIC = 16 µg/mL). The compound was confirmed to interfere with mycolic acids biosynthesis, and proved to also be active against other NTMs, including drug-resistant clinical isolates. Importantly, mefloquine is a well-known antimalarial agent, opening the possibility of repurposing an already approved drug, which is a useful strategy to reduce the time and cost of disclosing novel drug candidates.
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Antibacterianos/farmacología , Antimaláricos/farmacología , Mefloquina/farmacología , Mycobacterium abscessus/metabolismo , Ácidos Micólicos/metabolismoRESUMEN
A novel coumarin-based molecule, designed as a fluorescent surrogate of a thiacetazone-derived antitubercular agent, was quickly and easily synthesized from readily available starting materials. This small molecule, coined Coum-TAC, exhibited a combination of appropriate physicochemical and biological properties, including resistance toward hydrolysis and excellent antitubercular efficiency similar to that of well-known thiacetazone derivatives, as well as efficient covalent labeling of HadA, a relevant therapeutic target to combat Mycobacterium tuberculosis. More remarkably, Coum-TAC was successfully implemented as an imaging probe that is capable of labeling Mycobacterium tuberculosis in a selective manner, with an enrichment at the level of the poles, thus giving for the first time relevant insights about the polar localization of HadA in the mycobacteria.
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Lepidópteros , Mycobacterium tuberculosis , Tioacetazona , Animales , Antituberculosos/farmacología , CumarinasRESUMEN
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.
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Tuberculosis Latente , Mycobacterium tuberculosis , Tuberculosis , Antituberculosos/farmacología , Humanos , Mycobacterium tuberculosis/genéticaRESUMEN
The spread of acquired drug resistance and of microorganisms naturally resistant to antibiotics is a major threat to global health, leading to an urgent need for novel antimicrobial compounds. Exogenous nitric oxide (NO) represents an attractive and promising antimicrobial approach, showing both bactericidal and biofilm dispersal activities. Numerous studies have been performed to develop NO donor scaffolds, including small molecules, macromolecular compounds, nanoparticles (NPs), and polymeric materials. This approach has resulted in successful outcomes, with some NO-releasing compounds entering clinical practice. In this review, we highlight the importance of this strategy, with a focus on lung infections.
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Antibacterianos/farmacología , Óxido Nítrico/metabolismo , Infecciones del Sistema Respiratorio/tratamiento farmacológico , Animales , Biopelículas/efectos de los fármacos , Farmacorresistencia Bacteriana , Humanos , Enfermedades Pulmonares/tratamiento farmacológico , Enfermedades Pulmonares/microbiología , Nanopartículas , Polímeros/química , Infecciones del Sistema Respiratorio/microbiologíaRESUMEN
Human Vγ9Vδ2 T cells respond to several diverse pathogens by sensing microbial cholesterol intermediates. Unlike CD4 T cells, they are poised for rapid Th1-like responses even before birth, which allows them to play a key role in the first line of defense against pathogens in early life. However, their regulation and functional maturation during infancy (in particular the acquisition of cytotoxic potential) remain understudied. We thus characterized their responses to cholesterol intermediates and Bacille Calmette-Guérin in a cohort of African neonates and 12-month-old infants. Infant Vδ2 lymphocytes exhibited intermediate or adult-like expression of markers associated with differentiation or function, intermediate proliferative responses, and adult-like cytotoxic potential. The enhancement of Vδ2 cell cytotoxic potential coincided with decreasing PD-1 and increasing NKG2A expression. Our results are consistent with the hypothesis that switching from a PD-1+ to a NKG2A+ phenotype during infancy indicates a shift in mechanisms regulating Vδ2 T cell function.