RESUMEN
Tubercular Mycobacteria and Legionella pneumophila are the causative agents of potentially fatal respiratory diseases due to their intrinsic pathogenesis but also due to the emergence of antibiotic resistance that limits treatment options. The aim of our study was to explore the antimicrobial activity of a small ligand-based chemical library of 1255 structurally diverse compounds. These compounds were screened in a combination of three assays, two monitoring the intracellular growth of the pathogenic bacteria, Mycobacterium marinum and L. pneumophila, and one assessing virulence of M. marinum. We set up these assays using two amoeba strains, the genetically tractable social amoeba Dictyostelium discoideum and the free-living amoeba Acanthamoeba castellanii. In summary, 64 (5.1%) compounds showed anti-infective/anti-virulence activity in at least one of the three assays. The intracellular assays hit rate varied between 1.7% (n = 22) for M. marinum and 2.8% (n = 35) for L. pneumophila with seven compounds in common for both pathogens. In parallel, 1.2% (n = 15) of the tested compounds were able to restore D. discoideum growth in the presence of M. marinum spiked in a lawn of food bacteria. We also validated the generality of the hits identified in the A. castellanii-M. marinum anti-infective screen using the D. discoideum-M. marinum host-pathogen model. The characterization of anti-infective and antibacterial hits in the latter infection model revealed compounds able to reduce intracellular growth more than 50% at 30 µM. Moreover, the chemical space and physico-chemical properties of the anti-M. marinum hits were compared to standard and candidate Mycobacterium tuberculosis (Mtb) drugs using ChemGPS-NP. A principle component analysis identified separate clusters for anti-M. marinum and anti-L. pneumophila hits unveiling the potentially new physico-chemical properties of these hits compared to standard and candidate M. tuberculosis drugs. Our studies underscore the relevance of using a combination of low-cost and low-complexity assays with full 3R compliance in concert with a rationalized focused library of compounds to identify new chemical scaffolds and to dissect some of their properties prior to taking further steps toward compound development.
RESUMEN
Tuberculosis remains a serious threat to human health world-wide, and improved efficiency of medical treatment requires a better understanding of the pathogenesis and the discovery of new drugs. In the present study, we performed a whole-cell based screen in order to complete the characterization of 168 compounds from the GlaxoSmithKline TB-set. We have established and utilized novel previously unexplored host-model systems to characterize the GSK compounds, i.e. the amoeboid organisms D. discoideum and A. castellanii, as well as a microglial phagocytic cell line, BV2. We infected these host cells with Mycobacterium marinum to monitor and characterize the anti-infective activity of the compounds with quantitative fluorescence measurements and high-content microscopy. In summary, 88.1% of the compounds were confirmed as antibiotics against M. marinum, 11.3% and 4.8% displayed strong anti-infective activity in, respectively, the mammalian and protozoan infection models. Additionally, in the two systems, 13-14% of the compounds displayed pro-infective activity. Our studies underline the relevance of using evolutionarily distant pathogen and host models in order to reveal conserved mechanisms of virulence and defence, respectively, which are potential "universal" targets for intervention. Subsequent mechanism of action studies based on generation of over-expresser M. bovis BCG strains, generation of spontaneous resistant mutants and whole genome sequencing revealed four new molecular targets, including FbpA, MurC, MmpL3 and GlpK.
Asunto(s)
Acanthamoeba castellanii/microbiología , Antituberculosos/farmacología , Dictyostelium/microbiología , Descubrimiento de Drogas/métodos , Mycobacterium marinum/efectos de los fármacos , Animales , Línea Celular , Farmacorresistencia Bacteriana/genética , Ratones , Pruebas de Sensibilidad Microbiana , Microglía/citología , Microglía/efectos de los fármacos , Mutación , Mycobacterium marinum/genética , Mycobacterium marinum/crecimiento & desarrolloRESUMEN
Tuberculosis (TB) is a global disease causing 1.8 million deaths each year. The appearance of drug-resistant strains raised the demand for new anti-mycobacterial drugs and therapies, because previously discovered antibiotics are shown to be inefficient. Moreover, the number of newly discovered drugs is not increasing in proportion to the emergence of drug resistance, which suggests that more optimized methodology and screening procedures are required including the incorporation of in vivo properties of TB infection. A way to improve efficacy of screening approaches is by introducing the use of different host-pathogen systems into primary screenings. These include whole cell-based screenings, zebrafish larvae-based screenings and the impact of artificial granuloma research on the drug discovery process. This review highlights current screening attempts and the identified molecular targets and summarizes findings of alternative, not fully explored host-pathogen systems for the characterization of anti-mycobacterial compounds.
Asunto(s)
Antituberculosos , Descubrimiento de Drogas/métodos , Modelos Biológicos , Mycobacterium tuberculosis/efectos de los fármacos , Tuberculosis , Animales , Antituberculosos/farmacología , Antituberculosos/uso terapéutico , Línea Celular , Interacciones Huésped-Patógeno , Humanos , Ratones , Tuberculosis/tratamiento farmacológico , Tuberculosis/microbiologíaRESUMEN
lonophore-based ion-selective optical nanosensors have been explored for a number of years. Voltage sensitive dyes (VSDs) have been introduced into this type of sensors only very recently, forming a new class of analytical tools. Here, K(+)-sensitive nanospheres incorporating a lipophilic VSD were successfully fabricated and characterized. The nanosensors were readily delivered into the social amoeba Dictyostelium discoideum in a non-invasive manner, forming a promising new platform for intracellular ion quantification and imaging.
Asunto(s)
Técnicas Biosensibles , Colorantes/química , Técnicas Electroquímicas/instrumentación , Nanotecnología/instrumentación , Fenómenos Ópticos , Potasio/química , Algoritmos , Técnicas Electroquímicas/métodos , Aprendizaje Automático , Nanotecnología/métodosRESUMEN
Ionophore-based ion selective optical nanosensors that operate independently of the sample pH are developed here by the use of electrically charged solvatochromic dyes as signal transducers. A series of dye molecules with a D-π-A structure was synthesized and characterized in various solvents and incorporated into ion selective nanospheres for K(+), Na(+), and H(+). Since dye leakage was greatly suppressed when the solvatochromic dyes were encapsulated in the nanosphere core, ion sensing nanospheres were explored for cellular ion imaging in Dictyostelium discoideum live cells but spontaneous dye loss resulted in undesired staining of cells. The in vitro analysis of potassium in human plasma was successfully demonstrated with this approach. A theoretical model was developed for the response of the ion selective nanosensors containing charged solvatochromic dyes. The nanosensors exhibited a tunable response range, high sensitivity, and good stability.
Asunto(s)
Colorimetría/métodos , Colorantes/química , Hidrógeno/análisis , Potasio/análisis , Sodio/análisis , Cationes/análisis , Dictyostelium/química , Dictyostelium/citología , Fluorescencia , Humanos , Concentración de Iones de Hidrógeno , Nanosferas/química , Nanotecnología/métodos , Imagen Óptica , Plasma/química , Potasio/sangre , Espectrometría de Fluorescencia/métodosRESUMEN
The causative agent of Legionnaires' disease, Legionella pneumophila, grows in environmental amoebae and mammalian macrophages within a distinct compartment, the 'Legionella-containing vacuole' (LCV). Intracellular bacteria are protected from many antibiotics, and thus are notoriously difficult to eradicate. To identify novel compounds that restrict intracellular bacterial replication, we previously developed an assay based on a coculture of amoebae and GFP-producing L. pneumophila. This assay was used to screen a pathway-based, highly diverse chemical library, referred to as the Sinergia library. In this work, we chose to focus on a group of 11 hit compounds, the majority of which originated from the query molecule CN585, a compound that targets the protein phosphatase calcineurin. Further studies on 78 related compound variants revealed crucial structural attributes, namely a triple-ring scaffold with a central triazine moiety, substituted in positions 3 and 5 by two piperidine or pyrrolidine rings, and in position 1 by an amine group bearing a single aliphatic chain moiety. The most effective compound, ZINC00615682, inhibited intracellular replication of L. pneumophila with an IC50 of approximately 20 nM in Acanthamoeba castellanii and slightly less efficiently in Dictyostelium discoideum or macrophages. Pharmacological and genetic attempts to implicate calcineurin in the intracellular replication of L. pneumophila failed. Taken together, these results show that the amoebae-based screen and structure-activity relationship analysis is suitable for the identification of novel inhibitors of the intracellular replication of L. pneumophila. The most potent compound identified in this study targets (an) as yet unidentified host factor(s).
RESUMEN
Tuberculosis is considered to be one of the world's deadliest disease with 2 million deaths each year. The need for new antitubercular drugs is further exacerbated by the emergence of drug-resistance strains. Despite multiple recent efforts, the majority of the hits discovered by traditional target-based screening showed low efficiency in vivo. Therefore, there is heightened demand for whole-cell based approaches directly using host-pathogen systems. The phenotypic host-pathogen assay described here is based on the monitoring of GFP-expressing Mycobacterium marinum during infection of the amoeba Acanthamoeba castellanii. The assay showed straight-forward medium-throughput scalability, robustness and ease of manipulation, demonstrating its qualities as an efficient compound screening system. Validation with a series of known antitubercular compounds highlighted the advantages of the assay in comparison to previously published macrophage-Mycobacterium tuberculosis-based screening systems. Combination with secondary growth assays based on either GFP-expressing D. discoideum or M. marinum allowed us to further fine-tune compound characterization by distinguishing and quantifying growth inhibition, cytotoxic properties and antibiotic activities of the compounds. The simple and relatively low cost system described here is most suitable to detect anti-infective compounds, whether they present antibiotic activities or not, in which case they might exert anti-virulence or host defense boosting activities, both of which are largely overlooked by classical screening approaches.
Asunto(s)
Acanthamoeba castellanii/microbiología , Antibacterianos , Interacciones Huésped-Patógeno/efectos de los fármacos , Mycobacterium marinum/fisiología , Acanthamoeba castellanii/metabolismo , Antibacterianos/química , Antibacterianos/farmacología , Evaluación Preclínica de Medicamentos/métodos , FluorescenciaRESUMEN
Legionella pneumophila is a ubiquitous fresh-water bacterium which reproduces within its erstwhile predators, environmental amoeba, by subverting the normal pathway of phagocytosis and degradation. The molecular mechanisms which confer resistance to amoeba are apparently conserved and also allow replication within macrophages. Thus, L. pneumophila can act as an 'accidental' human pathogen and cause a severe pneumonia known as Legionnaires' disease. The intracellular localisation of L. pneumophila protects it from some antibiotics, and this fact must be taken into account to develop new anti-bacterial compounds. In addition, the intracellular lifestyle of L. pneumophila may render the bacteria susceptible to compounds diminishing bacterial virulence and decreasing intracellular survival and replication of this pathogen. The development of a single infection cycle intracellular replication assay using GFP-producing L. pneumophila and Acanthamoebacastellanii amoeba is reported here. This fluorescence-based assay allows for continuous monitoring of intracellular replication rates, revealing the effect of bacterial gene deletions or drug treatment. To examine how perturbations of the host cell affect L. pneumophila replication, several known host-targeting compounds were tested, including modulators of cytoskeletal dynamics, vesicle scission and Ras GTPase localisation. Our results reveal a hitherto unrealized potential antibiotic property of the ß-lactone-based Ras depalmitoylation inhibitor palmostatin M, but not the closely related inhibitor palmostatin B. Further characterisation indicated that this compound caused specific growth inhibition of Legionella and Mycobacterium species, suggesting that it may act on a common bacterial target.
Asunto(s)
Antibacterianos/farmacología , Espacio Intracelular/microbiología , Legionella pneumophila/efectos de los fármacos , Animales , Línea Celular , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Concentración 50 Inhibidora , Espacio Intracelular/efectos de los fármacos , Legionella pneumophila/crecimiento & desarrollo , Ratones , Pruebas de Sensibilidad Microbiana , Peso Molecular , Mycobacterium/efectos de los fármacos , Mycobacterium/crecimiento & desarrollo , Propiolactona/análogos & derivados , Propiolactona/farmacología , Especificidad de la Especie , Sulfonas/farmacologíaRESUMEN
Mycobacterium marinum is the causative agent of fish and amphibian tuberculosis in the wild. It is a genetically close cousin of Mycobacterium tuberculosis, and thereby the infection process remarkably shares many of the hallmarks of M. tuberculosis infection in human, at both the cellular and organism levels. Therefore, M. marinum is used as a model for the study of mycobacterial infection in various host organisms. Recently, the Dictyostelium-M. marinum system has been shown to be a valuable model that recapitulates the main features of the intracellular fate of M. marinum including phagosome maturation arrest, as well as its particular cell-to-cell dissemination mode. We present here a "starter kit" of detailed methods that allows to establish an infection of Dictyostelium with M. marinum and to monitor quantitatively the intracellular bacterial growth.