RESUMEN
In the current study, we describe a novel DNA sensor system for specific and quantitative detection of mycobacteria, which is the causative agent of tuberculosis. Detection is achieved by using the enzymatic activity of the mycobacterial encoded enzyme topoisomerase IA (TOP1A) as a biomarker. The presented work is the first to describe how the catalytic activities of a member of the type IA family of topoisomerases can be exploited for specific detection of bacteria. The principle for detection relies on a solid support anchored DNA substrate with dual functions namely: (1) the ability to isolate mycobacterial TOP1A from crude samples and (2) the ability to be converted into a closed DNA circle upon reaction with the isolated enzyme. The DNA circle can act as a template for rolling circle amplification generating a tandem repeat product that can be visualized at the single molecule level by fluorescent labelling. This reaction scheme ensures specific, sensitive, and quantitative detection of the mycobacteria TOP1A biomarker as demonstrated by the use of purified mycobacterial TOP1A and extracts from an array of non-mycobacteria and mycobacteria species. When combined with mycobacteriophage induced lysis as a novel way of effective yet gentle extraction of the cellular content from the model Mycobacterium smegmatis, the DNA sensor system allowed detection of mycobacteria in small volumes of cell suspensions. Moreover, it was possible to detect M. smegmatis added to human saliva. Depending on the composition of the sample, we were able to detect 0.6 or 0.9 million colony forming units (CFU) per mL of mycobacteria, which is within the range of clinically relevant infection numbers. We, therefore, believe that the presented assay, which relies on techniques that can be adapted to limited resource settings, may be the first step towards the development of a new point-of-care diagnostic test for tuberculosis.
Asunto(s)
Proteínas Bacterianas/análisis , Técnicas Biosensibles/métodos , ADN-Topoisomerasas de Tipo I/análisis , Ácidos Nucleicos Inmovilizados/metabolismo , Mycobacterium/aislamiento & purificación , Patología Molecular/métodos , Proteínas Bacterianas/metabolismo , Biomarcadores/análisis , Biomarcadores/metabolismo , ADN-Topoisomerasas de Tipo I/aislamiento & purificación , ADN-Topoisomerasas de Tipo I/metabolismo , Colorantes Fluorescentes/química , Humanos , Ácidos Nucleicos Inmovilizados/química , Mycobacterium/enzimología , Sensibilidad y EspecificidadRESUMEN
The search for compounds with biological activity for many diseases is turning increasingly to drug repurposing. In this study, we have focused on the European Union-approved antimalarial pyronaridine which was found to have in vitro activity against Mycobacterium tuberculosis (MIC 5⯵g/mL). In macromolecular synthesis assays, pyronaridine resulted in a severe decrease in incorporation of 14C-uracil and 14C-leucine similar to the effect of rifampicin, a known inhibitor of M. tuberculosis RNA polymerase. Surprisingly, the co-administration of pyronaridine (2.5⯵g/ml) and rifampicin resulted in in vitro synergy with an MIC 0.0019-0.0009⯵g/mL. This was mirrored in a THP-1 macrophage infection model, with a 16-fold MIC reduction for rifampicin when the two compounds were co-administered versus rifampicin alone. Docking pyronaridine in M. tuberculosis RNA polymerase suggested the potential for it to bind outside of the RNA polymerase rifampicin binding pocket. Pyronaridine was also found to have activity against a M. tuberculosis clinical isolate resistant to rifampicin, and when combined with rifampicin (10% MIC) was able to inhibit M. tuberculosis RNA polymerase in vitro. All these findings, and in particular the synergistic behavior with the antitubercular rifampicin, inhibition of RNA polymerase in combination in vitro and its current use as a treatment for malaria, may suggest that pyronaridine could also be used as an adjunct for treatment against M. tuberculosis infection. Future studies will test potential for in vivo synergy, clinical utility and attempt to develop pyronaridine analogs with improved potency against M. tuberculosis RNA polymerase when combined with rifampicin.
Asunto(s)
Antibióticos Antituberculosos/farmacología , Antimaláricos/farmacología , Antituberculosos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , ARN Polimerasas Dirigidas por ADN/antagonistas & inhibidores , Mycobacterium tuberculosis/efectos de los fármacos , Naftiridinas/farmacología , Rifampin/farmacología , Antimaláricos/química , Antituberculosos/química , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Sitios de Unión , ARN Polimerasas Dirigidas por ADN/química , ARN Polimerasas Dirigidas por ADN/metabolismo , Reposicionamiento de Medicamentos , Farmacorresistencia Bacteriana , Sinergismo Farmacológico , Quimioterapia Combinada , Humanos , Pruebas de Sensibilidad Microbiana , Simulación del Acoplamiento Molecular , Mycobacterium tuberculosis/enzimología , Mycobacterium tuberculosis/crecimiento & desarrollo , Naftiridinas/química , Unión Proteica , Conformación Proteica , Relación Estructura-Actividad , Células THP-1RESUMEN
There is a shortage of compounds that are directed towards new targets apart from those targeted by the FDA approved drugs used against Mycobacterium tuberculosis. Topoisomerase I (Mttopo I) is an essential mycobacterial enzyme and a promising target in this regard. However, it suffers from a shortage of known inhibitors. We have previously used computational approaches such as homology modeling and docking to propose 38 FDA approved drugs for testing and identified several active molecules. To follow on from this, we now describe the in vitro testing of a library of 639 compounds. These data were used to create machine learning models for Mttopo I which were further validated. The combined Mttopo I Bayesian model had a 5 fold cross validation receiver operator characteristic of 0.74 and sensitivity, specificity and concordance values above 0.76 and was used to select commercially available compounds for testing in vitro. The recently described crystal structure of Mttopo I was also compared with the previously described homology model and then used to dock the Mttopo I actives norclomipramine and imipramine. In summary, we describe our efforts to identify small molecule inhibitors of Mttopo I using a combination of machine learning modeling and docking studies in conjunction with screening of the selected molecules for enzyme inhibition. We demonstrate the experimental inhibition of Mttopo I by small molecule inhibitors and show that the enzyme can be readily targeted for lead molecule development.
Asunto(s)
Antituberculosos/farmacología , ADN-Topoisomerasas de Tipo I/metabolismo , Diseño de Fármacos , Aprendizaje Automático , Simulación del Acoplamiento Molecular , Mycobacterium tuberculosis/efectos de los fármacos , Inhibidores de Topoisomerasa I/farmacología , Tuberculosis/tratamiento farmacológico , Antituberculosos/química , Antituberculosos/metabolismo , Teorema de Bayes , Diseño Asistido por Computadora , ADN-Topoisomerasas de Tipo I/química , Relación Dosis-Respuesta a Droga , Humanos , Terapia Molecular Dirigida , Mycobacterium smegmatis/efectos de los fármacos , Mycobacterium smegmatis/enzimología , Mycobacterium smegmatis/crecimiento & desarrollo , Mycobacterium tuberculosis/enzimología , Mycobacterium tuberculosis/crecimiento & desarrollo , Conformación Proteica , Relación Estructura-Actividad , Inhibidores de Topoisomerasa I/química , Inhibidores de Topoisomerasa I/metabolismo , Tuberculosis/microbiologíaRESUMEN
Mycobacterium tuberculosis (Mtb) is a formidable pathogen which has the ability to survive the hostile environment of the host by evading the host defense system. The re-configuration of its transcriptional and metabolic process allows the pathogen to confront the adverse environment within the host macrophages. The factors that assist the transcription and modulate the DNA topology would have to play a key role in the regulation of global gene expression of the organism. How transcription of these essential housekeeping genes alters in response to growth conditions and environmental stress has not been addressed together in a set of experimental conditions in Mtb. Now, we have mapped the transcription start sites (TSS) and promoters of several genes that play a central role in the regulation of DNA topology and transcription in Mtb. Using in vivo reporter assays, we validated the activity of the identified promoter elements in different growth conditions. The variation in transcript abundance of these essential genes was also analyzed in growth phase-dependent manner. These data provide the first glimpse into the specific adaptive changes in the expression of genes involved in transcription and DNA topology modulation in Mtb.
Asunto(s)
Regulación Bacteriana de la Expresión Génica , Mycobacterium tuberculosis/genética , Tuberculosis/microbiología , Proteínas Bacterianas/genética , Perfilación de la Expresión Génica , Humanos , Mycobacterium tuberculosis/crecimiento & desarrollo , Regiones Promotoras Genéticas , Sitio de Iniciación de la Transcripción , Transcripción GenéticaRESUMEN
We present a quantum dot based DNA nanosensor specifically targeting the cleavage step in the reaction cycle of the essential DNA-modifying enzyme, mycobacterial topoisomerase I. The design takes advantages of the unique photophysical properties of quantum dots to generate visible fluorescence recovery upon specific cleavage by mycobacterial topoisomerase I. This report, for the first time, demonstrates the possibility to quantify the cleavage activity of the mycobacterial enzyme without the pre-processing sample purification or post-processing signal amplification. The cleavage induced signal response has also proven reliable in biological matrices, such as whole cell extracts prepared from Escherichia coli and human Caco-2 cells. It is expected that the assay may contribute to the clinical diagnostics of bacterial diseases, as well as the evaluation of treatment outcomes.
Asunto(s)
Proteínas Bacterianas/análisis , Técnicas Biosensibles/métodos , ADN-Topoisomerasas de Tipo I/análisis , ADN/química , Mycobacterium/enzimología , Puntos Cuánticos/química , Proteínas Bacterianas/química , Células CACO-2 , ADN-Topoisomerasas de Tipo I/química , HumanosRESUMEN
Discovering new drugs to treat tuberculosis more efficiently and to overcome multidrug resistance is a world health priority. To find novel antitubercular agents several approaches have been used in various institutions worldwide, including target-based approaches against several validated mycobacterial enzymes and phenotypic screens. We screened more than 17,000 compounds from Vichem's Nested Chemical Library™ using an integrated strategy involving whole cell-based assays with Corynebacterium glutamicum and Mycobacterium tuberculosis, and target-based assays with protein kinases PknA, PknB and PknG as well as other targets such as PimA and bacterial topoisomerases simultaneously. With the help of the target-based approach we have found very potent hits inhibiting the selected target enzymes, but good minimal inhibitory concentrations (MIC) against M. tuberculosis were not achieved. Focussing on the whole cell-based approach several potent hits were found which displayed minimal inhibitory concentrations (MIC) against M. tuberculosis below 10 µM and were non-mutagenic, non-cytotoxic and the targets of some of the hits were also identified. The most active hits represented various scaffolds. Medicinal chemistry-based lead optimization was performed applying various strategies and, as a consequence, a series of novel potent compounds were synthesized. These efforts resulted in some effective potential antitubercular lead compounds which were confirmed in phenotypic assays.
Asunto(s)
Antituberculosos/aislamiento & purificación , Diseño de Fármacos , Bibliotecas de Moléculas Pequeñas , Tuberculosis Resistente a Múltiples Medicamentos/tratamiento farmacológico , Girasa de ADN/efectos de los fármacos , ADN-Topoisomerasas/efectos de los fármacos , Pruebas de Enzimas , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/aislamiento & purificación , Humanos , Manosiltransferasas/antagonistas & inhibidores , Pruebas de Sensibilidad Microbiana , Terapia Molecular Dirigida/métodos , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/aislamiento & purificación , Inhibidores de Topoisomerasa/química , Inhibidores de Topoisomerasa/aislamiento & purificaciónRESUMEN
We describe inhibition of Mycobacterium tuberculosis topoisomerase I (MttopoI), an essential mycobacterial enzyme, by two related compounds, imipramine and norclomipramine, of which imipramine is clinically used as an antidepressant. These molecules showed growth inhibition of both Mycobacterium smegmatis and M. tuberculosis cells. The mechanism of action of these two molecules was investigated by analyzing the individual steps of the topoisomerase I (topoI) reaction cycle. The compounds stimulated cleavage, thereby perturbing the cleavage-religation equilibrium. Consequently, these molecules inhibited the growth of the cells overexpressing topoI at a low MIC. Docking of the molecules on the MttopoI model suggested that they bind near the metal binding site of the enzyme. The DNA relaxation activity of the metal binding mutants harboring mutations in the DxDxE motif was differentially affected by the molecules, suggesting that the metal coordinating residues contribute to the interaction of the enzyme with the drug. Taken together, the results highlight the potential of these small molecules, which poison the M. tuberculosis and M. smegmatis topoisomerase I, as leads for the development of improved molecules to combat mycobacterial infections. Moreover, targeting metal coordination in topoisomerases might be a general strategy to develop new lead molecules.
Asunto(s)
Proteínas Bacterianas/antagonistas & inhibidores , ADN-Topoisomerasas de Tipo I/metabolismo , Mycobacterium tuberculosis/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Inhibidores de Topoisomerasa I/farmacología , Secuencias de Aminoácidos , Proteínas Bacterianas/metabolismo , Sitios de Unión/genética , ADN/genética , ADN Bacteriano/genética , Imipramina/farmacología , Mutación/genética , Mycobacterium smegmatis/efectos de los fármacos , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/metabolismo , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Tuberculosis/tratamiento farmacológico , Tuberculosis/microbiologíaRESUMEN
Topoisomerases are an important class of enzymes for regulating the DNA transaction processes. Mycobacterium tuberculosis (Mtb) is one of the most formidable pathogens also posing serious challenges for therapeutic interventions. The organism contains only one type IA topoisomerase (Rv3646c), offering an opportunity to test its potential as a candidate drug target. To validate the essentiality of M. tuberculosis topoisomerase I (TopoI(Mt) ) for bacterial growth and survival, we have generated a conditionally regulated strain of topoI in Mtb. The conditional knockdown mutant exhibited delayed growth on agar plate. In liquid culture, the growth was drastically impaired when TopoI expression was suppressed. Additionally, novobiocin and isoniazid showed enhanced inhibitory potential against the conditional mutant. Analysis of the nucleoid revealed its altered architecture upon TopoI depletion. These studies establish the essentiality of TopoI for the M. tuberculosis growth and open up new avenues for targeting the enzyme.
Asunto(s)
Antibacterianos/farmacología , Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Mycobacterium tuberculosis/genética , Tuberculosis/microbiología , Proteínas Bacterianas/metabolismo , ADN-Topoisomerasas de Tipo I/genética , ADN-Topoisomerasas de Tipo I/metabolismo , Regulación Enzimológica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Genes Esenciales , Humanos , Isoniazida/farmacología , Pruebas de Sensibilidad Microbiana , Viabilidad Microbiana , Mutación , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/crecimiento & desarrollo , Novobiocina/farmacología , Oxazinas/farmacología , Tetraciclinas/metabolismo , Xantenos/farmacologíaRESUMEN
m-AMSA, an established inhibitor of eukaryotic type II topoisomerases, exerts its cidal effect by binding to the enzyme-DNA complex thus inhibiting the DNA religation step. The molecule and its analogues have been successfully used as chemotherapeutic agents against different forms of cancer. After virtual screening using a homology model of the Mycobacterium tuberculosis topoisomerase I, we identified m-AMSA as a high scoring hit. We demonstrate that m-AMSA can inhibit the DNA relaxation activity of topoisomerase I from M. tuberculosis and Mycobacterium smegmatis. In a whole cell assay, m-AMSA inhibited the growth of both the mycobacteria.
Asunto(s)
Amsacrina/farmacología , Antituberculosos/farmacología , ADN-Topoisomerasas de Tipo I/metabolismo , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/enzimología , Inhibidores de Topoisomerasa I/farmacología , Inhibidores de Topoisomerasa II/farmacología , Amsacrina/química , Antituberculosos/química , ADN Bacteriano/metabolismo , Humanos , Simulación del Acoplamiento Molecular , Mycobacterium smegmatis/efectos de los fármacos , Mycobacterium smegmatis/enzimología , Mycobacterium smegmatis/crecimiento & desarrollo , Mycobacterium tuberculosis/crecimiento & desarrollo , Inhibidores de Topoisomerasa I/química , Inhibidores de Topoisomerasa II/química , Tuberculosis/tratamiento farmacológico , Tuberculosis/microbiologíaRESUMEN
Bacterial DNA topoisomerase I (topoI) catalyzes relaxation of negatively supercoiled DNA. The enzyme alters DNA topology through protein-operated DNA gate, switching between open and closed conformations during its reaction. We describe the mechanism of inhibition of Mycobacterium smegmatis and Mycobacterium tuberculosis topoI by monoclonal antibodies (mAbs) that bind with high affinity and inhibit at 10-50 nM concentration. Unlike other inhibitors of topoisomerases, the mAbs inhibited several steps of relaxation reaction, namely DNA binding, cleavage, strand passage, and enzyme-DNA dissociation. The enhanced religation of the cleaved DNA in presence of the mAb indicated closing of the enzyme DNA gate. The formation of enzyme-DNA heterocatenane in the presence of the mAbs as a result of closing the gate could be inferred by the salt resistance of the complex, visualized by atomic force microscopy and confirmed by fluorescence measurements. Locking the enzyme-DNA complex as a closed clamp restricted the movements of the DNA gate, affecting all of the major steps of the relaxation reaction. Enzyme trapped on DNA in closed clamp conformation formed roadblock for the elongating DNA polymerase. The unusual multistep inhibition of mycobacterial topoisomerases may facilitate lead molecule development, and the mAbs would also serve as valuable tools to probe the enzyme mechanism.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Proteínas Bacterianas/metabolismo , ADN-Topoisomerasas de Tipo I/metabolismo , Inhibidores de Topoisomerasa/farmacología , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Biocatálisis/efectos de los fármacos , ADN/química , ADN/genética , ADN/metabolismo , División del ADN/efectos de los fármacos , ADN-Topoisomerasas de Tipo I/genética , ADN-Topoisomerasas de Tipo I/inmunología , Modelos Biológicos , Modelos Moleculares , Mutación , Mycobacterium smegmatis/enzimología , Mycobacterium tuberculosis/enzimología , Conformación de Ácido Nucleico , Oligonucleótidos/química , Oligonucleótidos/genética , Oligonucleótidos/metabolismo , Unión Proteica/efectos de los fármacos , Estructura Terciaria de Proteína , Espectrometría de Fluorescencia , Inhibidores de Topoisomerasa/química , Inhibidores de Topoisomerasa/inmunologíaRESUMEN
Type I DNA topoisomerases from bacteria catalyse relaxation of negatively supercoiled DNA in a Mg(2+) dependent manner. Although topoisomerases of distinct classes have been subjected for anti-cancer and anti-infective drug development, bacterial type I enzymes are way behind in this regard. Our studies with Mycobacterium smegmatis topoisomerase I (MstopoI) revealed several of its distinct properties compared to the well studied Escherichia coli topoisomerase I (EctopoI) suggesting the possibility of targeting the mycobacterial enzyme for inhibitor development. Here, we describe Mycobacterium tuberculosis topoisomerase I (MttopoI) and compare its properties with MstopoI and EctopoI. The enzyme cleaves DNA at preferred sites in a pattern similar to its ortholog from M. smegmatis. Oligonucleotides containing the specific recognition sequence inhibited the activity of the enzyme in a manner similar to that of MstopoI. Substitution of the acidic residues, D111 and E115 which are involved in Mg(2+) co-ordination, to alanines affected the DNA relaxation activity. Unlike the wild type enzyme, D111A was dependent on Mg(2+) for DNA cleavage and both the mutants were compromised in religation. The monoclonal antibody (mAb), 2F3G4, developed against MstopoI inhibited the relaxation activity of MttopoI. These studies affirm the characteristics of MttopoI to be similar to MstopoI and set a stage to target it for the development of specific small molecule inhibitors.