RESUMEN
New antibiotics are needed to combat rising levels of resistance, with new Mycobacterium tuberculosis (Mtb) drugs having the highest priority. However, conventional whole-cell and biochemical antibiotic screens have failed. Here we develop a strategy termed PROSPECT (primary screening of strains to prioritize expanded chemistry and targets), in which we screen compounds against pools of strains depleted of essential bacterial targets. We engineered strains that target 474 essential Mtb genes and screened pools of 100-150 strains against activity-enriched and unbiased compound libraries, probing more than 8.5 million chemical-genetic interactions. Primary screens identified over tenfold more hits than screening wild-type Mtb alone, with chemical-genetic interactions providing immediate, direct target insights. We identified over 40 compounds that target DNA gyrase, the cell wall, tryptophan, folate biosynthesis and RNA polymerase, as well as inhibitors that target EfpA. Chemical optimization yielded EfpA inhibitors with potent wild-type activity, thus demonstrating the ability of PROSPECT to yield inhibitors against targets that would have eluded conventional drug discovery.
Asunto(s)
Antituberculosos/clasificación , Antituberculosos/aislamiento & purificación , Descubrimiento de Drogas/métodos , Eliminación de Gen , Pruebas de Sensibilidad Microbiana/métodos , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/genética , Bibliotecas de Moléculas Pequeñas/farmacología , Antituberculosos/farmacología , Girasa de ADN/metabolismo , Farmacorresistencia Microbiana , Ácido Fólico/biosíntesis , Terapia Molecular Dirigida , Mycobacterium tuberculosis/citología , Mycobacterium tuberculosis/enzimología , Ácidos Micólicos/metabolismo , Reproducibilidad de los Resultados , Bibliotecas de Moléculas Pequeñas/clasificación , Bibliotecas de Moléculas Pequeñas/aislamiento & purificación , Especificidad por Sustrato , Inhibidores de Topoisomerasa II/aislamiento & purificación , Inhibidores de Topoisomerasa II/farmacología , Triptófano/biosíntesis , Tuberculosis/tratamiento farmacológico , Tuberculosis/microbiologíaRESUMEN
A key to the pathogenic success of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is the capacity to survive within host macrophages. Although several factors required for this survival have been identified, a comprehensive knowledge of such factors and how they work together to manipulate the host environment to benefit bacterial survival are not well understood. To systematically identify Mtb factors required for intracellular growth, we screened an arrayed, non-redundant Mtb transposon mutant library by high-content imaging to characterize the mutant-macrophage interaction. Based on a combination of imaging features, we identified mutants impaired for intracellular survival. We then characterized the phenotype of infection with each mutant by profiling the induced macrophage cytokine response. Taking a systems-level approach to understanding the biology of identified mutants, we performed a multiparametric analysis combining pathogen and host phenotypes to predict functional relationships between mutants based on clustering. Strikingly, mutants defective in two well-known virulence factors, the ESX-1 protein secretion system and the virulence lipid phthiocerol dimycocerosate (PDIM), clustered together. Building upon the shared phenotype of loss of the macrophage type I interferon (IFN) response to infection, we found that PDIM production and export are required for coordinated secretion of ESX-1-substrates, for phagosomal permeabilization, and for downstream induction of the type I IFN response. Multiparametric clustering also identified two novel genes that are required for PDIM production and induction of the type I IFN response. Thus, multiparametric analysis combining host and pathogen infection phenotypes can be used to identify novel functional relationships between genes that play a role in infection.
Asunto(s)
Antígenos Bacterianos/genética , Proteínas Bacterianas/genética , Mycobacterium tuberculosis/patogenicidad , Fagosomas/microbiología , Tuberculosis/microbiología , Animales , Antígenos Bacterianos/metabolismo , Proteínas Bacterianas/metabolismo , Línea Celular , Citocinas/inmunología , Citocinas/metabolismo , Biblioteca de Genes , Interacciones Huésped-Patógeno , Macrófagos/inmunología , Macrófagos/microbiología , Ratones , Mutación , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/crecimiento & desarrollo , Mycobacterium tuberculosis/inmunología , Fagosomas/inmunología , Fenotipo , Tuberculosis/inmunología , VirulenciaRESUMEN
Previous work established a coumarin scaffold as a starting point for inhibition of Mycobacterium tuberculosis (Mtb) FadD32 enzymatic activity. After further profiling of the coumarin inhibitor 4 revealed chemical instability, we discovered that a quinoline ring circumvented this instability and had the advantage of offering additional substitution vectors to further optimize. Ensuing SAR studies gave rise to quinoline-2-carboxamides with potent anti-tubercular activity. Further optimization of ADME/PK properties culminated in 21b that exhibited compelling in vivo efficacy in a mouse model of Mtb infection.
Asunto(s)
Antituberculosos/química , Proteínas Bacterianas/antagonistas & inhibidores , Cumarinas/química , Animales , Antituberculosos/metabolismo , Antituberculosos/farmacología , Antituberculosos/uso terapéutico , Proteínas Bacterianas/metabolismo , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Ratones , Pruebas de Sensibilidad Microbiana , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/metabolismo , Quinolinas/química , Relación Estructura-Actividad , Tuberculosis/tratamiento farmacológico , Tuberculosis/microbiologíaRESUMEN
BACKGROUND: Amplification of single-stranded DNA circles has wide utility for a variety of applications. The two-primer ramified rolling circle amplification (RAM) reaction provides exponential DNA amplification under isothermal conditions, creating a regular laddered series of double-stranded DNA products. However, the molecular mechanism of the RAM reaction remains unexplained. RESULTS: A RAM reaction model predicts exponential accumulation of a double-stranded DNA product size series, and product-size ratios, that are consistent with observed RAM reaction products. The mechanism involves generation of a series of increasing size intermediate templates; those templates produce RAM products and recursively generate smaller intermediate templates. The model allows prediction of the number of rounds of circular template replication. Real-time RAM reaction data are consistent with the model. Analysis of RAM reaction products shows exponential growth limitation consistent with the model's predictions. CONCLUSIONS: The model provides a rationale for the observed products of the RAM reaction, and the molecular yield among those products. Experimental results are consistent with the model.
Asunto(s)
ADN/metabolismo , Técnicas de Amplificación de Ácido Nucleico/métodos , Cartilla de ADN/química , ADN Circular/genética , ADN Circular/metabolismo , ADN de Cadena Simple/metabolismoRESUMEN
Successful treatment of Mycobacterium tuberculosis infection typically requires a complex regimen administered over at least 6 months. Interestingly, many of the antibiotics used to treat M. tuberculosis are prodrugs that require intracellular activation. Here, we describe three small molecules, active against both replicating and non-replicating M. tuberculosis, that require activation by Baeyer-Villiger monooxygenases (BVMOs). Two molecules require BVMO EthA (Rv3854c) for activation and the third molecule requires the BVMO MymA (Rv3083). While EthA is known to activate the antitubercular drug ethionamide, this is the first description of MymA as an activating enzyme of a prodrug. Furthermore, we found that MymA also plays a role in activating ethionamide, with loss of MymA function resulting in ethionamide-resistant M. tuberculosis. These findings suggest overlap in function and specificity of the BVMOs in M. tuberculosis.
Asunto(s)
Antituberculosos/farmacología , Etionamida/farmacología , Oxigenasas de Función Mixta/metabolismo , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/crecimiento & desarrollo , Oxidorreductasas/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Antituberculosos/química , Relación Dosis-Respuesta a Droga , Farmacorresistencia Bacteriana/efectos de los fármacos , Etionamida/química , Oxigenasas de Función Mixta/genética , Estructura Molecular , Mycobacterium tuberculosis/enzimología , Mycobacterium tuberculosis/metabolismo , Oxidorreductasas/genética , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-ActividadRESUMEN
During Mycobacterium tuberculosis infection, a population of bacteria is thought to exist in a nonreplicating state, refractory to antibiotics, which may contribute to the need for prolonged antibiotic therapy. The identification of inhibitors of the nonreplicating state provides tools that can be used to probe this hypothesis and the physiology of this state. The development of such inhibitors also has the potential to shorten the duration of antibiotic therapy required. Here we describe the development of a novel nonreplicating assay amenable to high-throughput chemical screening coupled with secondary assays that use carbon starvation as the in vitro model. Together these assays identify compounds with activity against replicating and nonreplicating M. tuberculosis as well as compounds that inhibit the transition from nonreplicating to replicating stages of growth. Using these assays we successfully screened over 300,000 compounds and identified 786 inhibitors of nonreplicating M. tuberculosis In order to understand the relationship among different nonreplicating models, we tested 52 of these molecules in a hypoxia model, and four different chemical scaffolds in a stochastic persister model, and a streptomycin-dependent model. We found that compounds display varying levels of activity in different models for the nonreplicating state, suggesting important differences in bacterial physiology between models. Therefore, chemical tools identified in this assay may be useful for determining the relevance of different nonreplicating in vitro models to in vivo M. tuberculosis infection. Given our current limited understanding, molecules that are active across multiple models may represent more promising candidates for further development.