Therapeutic options for tuberculosis (TB) are limited and notoriously ineffective despite the wide variety of potent antibiotics available for treating other bacterial infections. We investigated an approach that enables an expansion of TB therapeutic strategies by using synergistic combinations of drugs. To achieve this, we devised a high-throughput synergy screen (HTSS) of chemical libraries having known pharmaceutical properties, including thousands that are clinically approved. Spectinomycin was used to test the concept that clinically available antibiotics with limited efficacy against Mycobacterium tuberculosis might be used for TB treatment when coadministered with a synergistic partner compound used as a sensitizer. Screens using Mycobacterium smegmatis revealed many compounds in our libraries that acted synergistically with spectinomycin. Among them, several families of antimicrobial compounds, including macrolides and azoles, were also synergistic against M. tuberculosis in vitro and in a macrophage model of M. tuberculosis infection. Strikingly, each sensitizer identified for synergy with spectinomycin uniquely enhanced the activities of other clinically used antibiotics, revealing a remarkable number of unexplored synergistic drug combinations. HTSS also revealed a novel activity for bromperidol, a butyrophenone used as an antipsychotic drug, which was discovered to be bactericidal and greatly enhanced the activities of several antibiotics and drug combinations against M. tuberculosis. Our results suggest that many compounds in the currently available pharmacopoeia could be readily mobilized for TB treatment, including disease caused by multi- and extensively drug-resistant strains for which there are no effective therapies.

Tuberculosis, caused by Mycobacterium tuberculosis infection, is a major cause of morbidity and mortality in the world today. M. tuberculosis hijacks the phagosome-lysosome trafficking pathway to escape clearance from infected macrophages. There is increasing evidence that manipulation of autophagy, a regulated catabolic trafficking pathway, can enhance killing of M. tuberculosis. Therefore, pharmacological agents that induce autophagy could be important in combating tuberculosis. We report that the antiprotozoal drug nitazoxanide and its active metabolite tizoxanide strongly stimulate autophagy and inhibit signaling by mTORC1, a major negative regulator of autophagy. Analysis of 16 nitazoxanide analogues reveals similar strict structural requirements for activity in autophagosome induction, EGFP-LC3 processing and mTORC1 inhibition. Nitazoxanide can inhibit M. tuberculosis proliferation in vitro. Here we show that it inhibits M. tuberculosis proliferation more potently in infected human THP-1 cells and peripheral monocytes. We identify the human quinone oxidoreductase NQO1 as a nitazoxanide target and propose, based on experiments with cells expressing NQO1 or not, that NQO1 inhibition is partly responsible for mTORC1 inhibition and enhanced autophagy. The dual action of nitazoxanide on both the bacterium and the host cell response to infection may lead to improved tuberculosis treatment.

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Signal transduction in Mycobacterium tuberculosis is mediated primarily by the Ser/Thr protein kinases and the two-component systems. The Ser/Thr kinase PknH has been shown to regulate growth of M. tuberculosis in a mouse model and in response to NO stress in vitro. Comparison of a pknH deletion mutant (⌬pknH) with its parental M. tuberculosis H37Rv strain using iTRAQ enabled us to quantify >700 mycobacterial proteins. Among these, members of the hypoxia-and NO-inducible dormancy (DosR) regulon were disregulated in the ⌬pknH mutant. Mycobacterium tuberculosis, the causative agent of tuberculosis, is a human intracellular pathogen that is phagocytosed by alveolar macrophages and subsequently "walled off" by the host immune response within granulomas (1). M. tuberculosis is able to persist within the hostile microenvironment of the granuloma, which is thought to include hypoxic, acidic, and nutrient-poor conditions and immune effectors such as nitric oxide (NO) 5 (2). The survival and persistence of M. tuberculosis in this environment requires the ability to sense external signals and mount an effective adaptive response. M. tuberculosis possesses multiple families of signal transduction systems, including the Ser/Thr protein kinases (STPKs) and the two-component regulatory systems (TCSs) (3).In a previous study, we found that the STPK PknH functions as an in vivo growth regulator (4). Hypervirulence was consistently detected in BALB/c mice infected with a pknH deletion mutant in M. tuberculosis after 3-4 weeks of infection (4), corresponding to the onset of adaptive immunity. Therefore, we hypothesized that M. tuberculosis uses the PknH kinase-mediated pathways to respond to host-induced signals to regulate its in vivo growth. Nitric oxide produced by the inducible nitricoxide synthase of the host macrophages plays a key role in controlling bacillary growth during the chronic phase of infection following activation of the host immune response (5). In vitro experiments revealed that the ⌬pknH mutant is more resistant to NO compared with WT (4), indicating that PknH may act as a sensor of NO to regulate M. tuberculosis growth in vivo.Predictions from bioinformatics analysis and studies using in vitro kinase assays have identified three endogenous substrates of PknH kinase: EmbR (6), a transcriptional regulator of the embCAB genes involved in lipoarabinomannan and arabinogalactan synthesis; DacB1, a cell division-related protein; and Rv0681, a putative transcriptional regulator (7). However, the substrates and downstream effectors of PknH signaling in response to NO stimulus have yet to be discovered.The DosR system, also known as DevR, is one of 11 pairs of TCSs present in M. tuberculosis (3). It is well established that DosR responds to hypoxia, NO, and CO via signaling through two cognate sensor kinases, DosS (DevS) and DosT (8,9) to activate transcription of a defined set of ϳ50 genes termed the "dormancy" or DosR regulon (10). Genes belonging to the DosR regulon, including dosR, are up-regulated in the Wayne m...

Here we describe the development and validation of an intracellular high-throughput screening assay for finding new antituberculosis compounds active in human macrophages. The assay consists of a luciferase-based primary identification assay, followed by a green fluorescent protein-based secondary profiling assay. Standard tuberculosis drugs and 158 previously recognized active antimycobacterial compounds were used to evaluate assay robustness. Data show that the assay developed is a short and valuable tool for the discovery of new antimycobacterial compounds.T uberculosis (TB) caused by Mycobacterium tuberculosis affects 9.0 million people annually, with 1.5 million deaths in 2013 (1). Standard TB treatment involves a regimen of four antibiotics taken daily for 6 to 9 months. However, the long treatment duration, toxicity, and interaction with antiretrovirals lead to poor patient compliance and treatment failure. Novel TB drug regimens are therefore urgently needed to treat both standard and drug-resistant forms of TB. Two new drugs, bedaquiline (2) and delamanid (3), were recently approved for the treatment of multidrug-resistant (MDR) TB, and other compounds are in the clinical development pipeline (4). Yet, the search for new TB drug candidates with different modes of action seeks to increase the chances of finding new drugs.Screening of chemical libraries is the first crucial step in the antimicrobial discovery process. Potential antimycobacterial agents are identified by testing chemicals for the ability to inhibit M. tuberculosis growth under in vitro growth conditions in culture medium. However, in vitro screening results are often misleading, as the culture broth does not reflect the environment M. tuberculosis encounters in vivo during the natural course of the disease, neglecting important factors such as compound activation, membrane permeability, removal by efflux pump, and toxicity to mammalian cells (4). Furthermore, adaptive metabolic changes that M. tuberculosis undergoes within the host may affect compound activity (5). Ex vivo screening, in the macrophage, may represent physiological conditions that mimic disease and take into consideration the favorable contribution of host cells in the process of eradicating M. tuberculosis.M. tuberculosis's intracellular lifestyle presents an attractive area for new drug discovery programs. A successful example is the intracellular high-content screening campaign that led to the discovery of Q203 (6). Image-based high-content screening technologies are being adopted more frequently to evaluate the activities of compounds against M. tuberculosis by using various cell types (7-9) or the granuloma infection model (10).High-content screening against M. tuberculosis is a robust and informative assay; however, it is still lacking in terms of speed and simplicity since the endpoint assay requires multiple steps for staining, image acquisition, and cumbersome data analysis. In addition, most of the intracellular compound screening done so far was performed inside e...