Non-replicating Persistence Research Articles

Slow growth of Mycobacterium tuberculosis at acidic pH is regulated by phoPR and host-associated carbon sources.

During pathogenesis, Mycobacterium tuberculosis (Mtb) colonizes environments, such as the macrophage or necrotic granuloma, that are acidic and rich in cholesterol and fatty acids. The goal of this study was to examine how acidic pH and available carbon sources interact to regulate Mtb physiology. Here we report that Mtb growth at acidic pH requires host-associated carbon sources that function at the intersection of glycolysis and the TCA cycle, such as pyruvate, acetate, oxaloacetate and cholesterol. In contrast, in other tested carbon sources, Mtb fully arrests its growth at acidic pH and establishes a state of non-replicating persistence. Growth-arrested Mtb is resuscitated by the addition of pyruvate suggesting that growth arrest is due to a pH-dependent checkpoint on metabolism. Additionally, we demonstrate that the phoPR two-component regulatory system is required to slow Mtb growth at acidic pH and functions to maintain redox homeostasis. Transcriptional profiling and functional metabolic studies demonstrate that signals from acidic pH and carbon source are integrated to remodel pathways associated with anaplerotic central metabolism, lipid anabolism and the regeneration of oxidized cofactors. Because phoPR is required for Mtb virulence in animals, we suggest that pH-driven adaptation may be critical to Mtb pathogenesis.

Non-Replicating Mycobacterium tuberculosis Elicits a Reduced Infectivity Profile with Corresponding Modifications to the Cell Wall and Extracellular Matrix

A key feature of Mycobacterium tuberculosis is its ability to become dormant in the host. Little is known of the mechanisms by which these bacilli are able to persist in this state. Therefore, the focus of this study was to emulate environmental conditions encountered by M. tuberculosis in the granuloma, and determine the effect of such conditions on the physiology and infectivity of the organism. Non-replicating persistent (NRP) M. tuberculosis was established by the gradual depletion of nutrients in an oxygen-replete and controlled environment. In contrast to rapidly dividing bacilli, NRP bacteria exhibited a distinct phenotype by accumulating an extracellular matrix rich in free mycolate and lipoglycans, with increased arabinosylation. Microarray studies demonstrated a substantial down-regulation of genes involved in energy metabolism in NRP bacteria. Despite this reduction in metabolic activity, cells were still able to infect guinea pigs, but with a delay in the development of disease when compared to exponential phase bacilli. Using these approaches to investigate the interplay between the changing environment of the host and altered physiology of NRP bacteria, this study sheds new light on the conditions that are pertinent to M. tuberculosis dormancy and how this organism could be establishing latent disease.

A Small RNA Encoded in the Rv2660c Locus of Mycobacterium tuberculosis Is Induced during Starvation and Infection

Enhanced transcription of the Rv2660c locus in response to starvation of Mycobacterium tuberculosis H37Rv encouraged addition of the predicted Rv2660c protein to an improved vaccine formulation. Using strand-specific RNA sequencing, we show that the up-regulated transcript is in fact a small RNA encoded on the opposite strand to the annotated Rv2660c. The transcript originates within a prophage and is expressed only in strains that carry PhiRv2. The small RNA contains both host and phage sequences and provides a useful biomarker to monitor bacterial starvation during infection and/or non-replicating persistence. Using different approaches we do not find any evidence of Rv2660c at the level of mRNA or protein. Further efforts to understand the mechanism by which Rv2660c improves efficacy of the H56 vaccine are likely to provide insights into the pathology and immunology of tuberculosis.

MazF6 toxin of Mycobacterium tuberculosis demonstrates antitoxin specificity and is coupled to regulation of cell growth by a Soj-like protein

BackgroundMolecular programs employed by Mycobacterium tuberculosis (Mtb) for the establishment of non-replicating persistence (NRP) are poorly understood. In order to investigate mechanisms regulating entry into NRP, we asked how cell cycle regulation is linked to downstream adaptations that ultimately result in NRP. Based on previous reports and our recent studies, we reason that, in order to establish NRP, cells are halted in the cell cycle at the point of septum formation by coupled regulatory mechanisms.ResultsUsing bioinformatic consensus modeling, we identified an alternative cell cycle regulatory element, SojMtb encoded by rv1708. SojMtb coordinates a regulatory mechanism involving cell cycle control at the point of septum formation and elicits the induction of the MazF6 toxin. MazF6 functions as an mRNA interferase leading to bacteriostasis that can be prevented by interaction with its cognate antitoxin, MazE6. Further, MazEF6 acts independently of other Maz family toxin:antitoxin pairs. Notably, soj Mtb and mazEF6 transcripts where identified at 20, 40 and 100 days post-infection in increasing abundance indicating a role in adaption during chronic infection.ConclusionsHere we present the first evidence of a coupled regulatory system in which cell cycle regulation via SojMtb is linked to downstream adaptations that are facilitated through the activity of the MazEF6 TA pair.

A systems chemical biology study of malate synthase and isocitrate lyase inhibition in Mycobacterium tuberculosis during active and NRP growth

The ability of Mycobacterium tuberculosis (Mtb) to survive in low oxygen environments enables the bacterium to persist in a latent state within host tissues. In vitro studies of Mtb growth have identified changes in isocitrate lyase (ICL) and malate synthase (MS) that enable bacterial persistence under low oxygen and other environmentally limiting conditions. Systems chemical biology (SCB) enables us to evaluate the effects of small molecule inhibitors not only on the reaction catalyzed by malate synthase and isocitrate lyase, but the effect on the complete tricarboxylic acid cycle (TCA) by taking into account complex network relationships within that system.To study the kinetic consequences of inhibition on persistent bacilli, we implement a systems-chemical biology (SCB) platform and perform a chemistry-centric analysis of key metabolic pathways believed to impact Mtb latency. We explore consequences of disrupting the function of malate synthase (MS) and isocitrate lyase (ICL) during aerobic and hypoxic non-replicating persistence (NRP) growth by using the SCB method to identify small molecules that inhibit the function of MS and ICL, and simulating the metabolic consequence of the disruption.Results indicate variations in target and non-target reaction steps, clear differences in the normal and low oxygen models, as well as dosage dependent response. Simulation results from singular and combined enzyme inhibition strategies suggest ICL may be the more effective target for chemotherapeutic treatment against Mtb growing in a microenvironment where oxygen is slowly depleted, which may favor persistence.

Characterization of mycobacterial triacylglycerols and monomeromycolyl diacylglycerols from Mycobacterium smegmatis biofilm by electrospray ionization multiple-stage and high-resolution mass spectrometry

The storage of triacylglycerols (TAGs) is essential for non-replicating persistence relevant to survival and the re-growth of mycobacteria during their exit from non-replicating state stress conditions. However, the detailed structures of this lipid family in mycobacteria largely remain unexplored. In this contribution, we describe a multiple-stage linear ion-trap mass spectrometric approach with high resolution mass spectrometry toward direct structural analysis of the TAGs, including a novel lipid subclass previously defined as monomeromycolyl diacylglycerol (MMDAG) isolated from biofilm of Mycobacterium smegmatis, a rapidly growing, non-pathogenic mycobacterium that has been used as a tool for molecular analysis of mycobacteria. Our results demonstrate that the major isomer in each of the molecular species of TAGs and MMDAGs consists of the common structure in which Δ(9)18:1- and 16:0-fatty acyl substituents are exclusively located at sn-1 and sn-2, respectively. Several isomers were found for most of the molecular species, and thus hundreds of structures are present in this lipid family. More importantly, this study revealed the structures of MMDAG, a novel subclass of TAG that has not been previously reported by direct mass spectrometric approaches.

Expression of non-replicating persistence associated genes of Mycobacterium bovis in lymph nodes from skin test-reactor cattle

Upon oxygen shift-down, Mycobacterium tuberculosis complex bacteria can induce a genetic program characterized by halted duplication, which is called Non-replicating persistence (NRP). During this phase, at least 48 genes, collectively named Dormancy survival regulator (DosR) regulon, are important for the long-term survival of bacilli under a non-respiring state, a condition that bacilli encounter inside granulomatous lesions. It remains unclear whether expression of NRP genes occurs within the tissue of Mycobacterium bovis naturally infected cattle. In order to start dissecting this question, total RNA from bovine lymph node tissues of sacrificed tuberculin reacting animals was isolated and transcription of genes required for in vivo duplication (esxB and fbpB) and in vitro NRP (hspX, pfkB, and mb2660c) were analyzed by RT-PCR approaches. Detection of transcripts was positive in bovine tissue samples for genes hspX, pfkB, and mb2660c in 84, 32, and 21%, respectively. NRP genes were upregulated even in animals with a negative IFN-γ in vitro test, and the expression of NRP genes occurred more often than expression of the esxB gene.

Synthesis and antimycobacterial activities of non-purine analogs of 6-aryl-9-benzylpurines: Imidazopyridines, pyrrolopyridines, benzimidazoles, and indoles

6,9-Disubstituted purines and 7-deazapurines are known to be powerful inhibitors of Mycobacterium tuberculosis ( Mtb) in vitro. Analogs modified in the six-membered ring (imidazopyridines, pyrrolopyridines, benzimidazoles, and indoles) were synthesized and evaluated as Mtb inhibitors. The targets were prepared by functionalization on the bicyclic heterocycle or from simple pyridines. The results reported herein, indicate that the purine N-1, but not N-3, is important for binding to the unknown target. The 3-deazapurines appears to be slightly more active compared to the parent purines and slightly less active than their 7-deazapurine isomers. Removal of both the purine N-3 and N-7 did not result in further enhanced antimycobacterial activity but the toxicity towards mammalian cells was increased. Both 3-deaza and 3,7-dideazapurines exhibited a modest activity against of the Mtb isolate in the state of non-replicating persistence.

Phosphodiesterase 4 Inhibition Reduces Innate Immunity and Improves Isoniazid Clearance of Mycobacterium tuberculosis in the Lungs of Infected Mice

Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) is one of the leading infectious disease causes of morbidity and mortality worldwide. Though current antibiotic regimens can cure the disease, treatment requires at least six months of drug therapy. One reason for the long duration of therapy is that the currently available TB drugs were selected for their ability to kill replicating organisms and are less effective against subpopulations of non-replicating persistent bacilli. Evidence from in vitro models of Mtb growth and mouse infection studies suggests that host immunity may provide some of the environmental cues that drive Mtb towards non-replicating persistence. We hypothesized that selective modulation of the host immune response to modify the environmental pressure on the bacilli may result in better bacterial clearance during TB treatment. For this proof of principal study, we compared bacillary clearance from the lungs of Mtb-infected mice treated with the anti-TB drug isoniazid (INH) in the presence and absence of an immunomodulatory phosphodiesterase 4 inhibitor (PDE4i), CC-3052. The effects of CC-3052 on host global gene expression, induction of cytokines, and T cell activation in the lungs of infected mice were evaluated. We show that CC-3052 modulates the innate immune response without causing generalized immune suppression. Immune modulation combined with INH treatment improved bacillary clearance and resulted in smaller granulomas and less lung pathology, compared to treatment with INH alone. This novel strategy of combining anti-TB drugs with an immune modulating molecule, if applied appropriately to patients, may shorten the duration of TB treatment and improve clinical outcome.

Higher human CD4 T cell response to novel Mycobacterium tuberculosis latency associated antigens Rv2660 and Rv2659 in latent infection compared with tuberculosis disease

One third of the world's population is infected with Mycobacterium tuberculosis ( M.tb). A vaccine that would prevent progression to TB disease will have a dramatic impact on the global TB burden. We propose that antigens of M.tb that are preferentially expressed during latent infection will be excellent candidates for post-exposure vaccination. We therefore assessed human T cell recognition of two such antigens, Rv2660 and Rv2659. Expression of these was shown to be associated with non-replicating persistence in vitro. After six days incubation of PBMC from persons with latent tuberculosis infection (LTBI) and tuberculosis (TB) disease, Rv2660 and Rv2659 induced IFN-γ production in a greater proportion of persons with LTBI, compared with TB diseased patients. Persons with LTBI also had increased numbers of viable T cells, and greater specific CD4 + T cell proliferation and cytokine expression capacity. Persons with LTBI preferentially recognize Rv2659 and Rv2660, compared with patients with TB disease. These results suggest promise of these antigens for incorporation into post-exposure TB vaccines.

Synthesis of non-purine analogs of 6-aryl-9-benzylpurines, and their antimycobacterial activities. Compounds modified in the imidazole ring

Purine analogs modified in the five-membered ring have been synthesized and examined for antibacterial activity against Mycobacterium tuberculosis H 37Rv in vitro employing the microplate alamar blue assay (MABA). The 9-deaza analogs were only found to be weak inhibitors, but the 8-aza-, 7-deaza- and 8-aza-7-deazapurine analogs studied displayed excellent antimycobacterial activities, some even substantially better than the parent purine. In the 7-deazapurine series, MIC values between 0.08 and 0.35 μM, values comparable or better than the reference drugs used in the study (MIC rifampicin 0.09 μM, MIC isoniazid 0.28 μM and MIC PA-824 0.44 μM). The five most active compounds were also examined against a panel of drug-resistant Mtb strain, and they all retained their activity. The compounds examined were significantly less active against M. tuberculosis in a state of non-replicating persistence (NRP). MIC in the low-oxygen-recovery assay (LORA) ⩾60 μM. The 7-deazapurines were somewhat more toxic towards mammalian cells, but still the selectivity indexes were excellent. The non-purine analogs exhibit a selective antimycobacterial activity. They were essentially inactive against Staphylococcus aureus and Escherichia coli.

Lipid Droplet-associated Proteins Are Involved in the Biosynthesis and Hydrolysis of Triacylglycerol in Mycobacterium bovis Bacillus Calmette-Guérin

Mycobacteria store triacylglycerols (TGs) in the form of intracellular lipid droplets (LDs) during hypoxia-induced nonreplicating persistence. These bacteria are phenotypically drug-resistant and therefore are believed to be the cause for prolonged tuberculosis treatment. LDs are also associated with bacilli in tuberculosis patient sputum and hypervirulent strains. Although proteins bound to LDs are well characterized in eukaryotes, the identities and functions of such proteins have not been described in mycobacteria. Here, we have identified five proteins: Tgs1 (BCG3153c), Tgs2 (BCG3794c), BCG1169c, BCG1489c, and BCG1721, which are exclusively associated with LDs purified from hypoxic nonreplicating Mycobacterium bovis bacillus Calmette-Guérin (BCG). Disruption of genes tgs1, tgs2, BCG1169c, and BCG1489c in M. bovis BCG revealed that they are indeed involved in TG metabolism. We also characterized BCG1721, an essential bi-functional enzyme capable of promoting buildup and hydrolysis of TGs, depending on the metabolic state. Nonreplicating mycobacteria overexpressing a BCG1721 construct with an inactive lipase domain displayed a phenotype of attenuated TG breakdown and regrowth upon resuscitation. In addition, by heterologous expression in baker's yeast, these mycobacterial proteins also co-localized with LDs and complemented a lipase-deficient yeast strain, indicating that neutral lipid deposition and homeostasis in eukaryotic and prokaryotic microorganisms are functionally related. The demonstrated functional role of BCG1721 to support growth upon resuscitation makes this novel LD-associated factor a potential new target for therapeutic intervention.

Mycobacterium tuberculosis Transcriptional Adaptation, Growth Arrest and Dormancy Phenotype Development Is Triggered by Vitamin C

BackgroundTubercle bacilli are thought to persist in a dormant state during latent tuberculosis (TB) infection. Although little is known about the host factors that induce and maintain Mycobacterium tuberculosis (M. tb) within latent lesions, O2 depletion, nutrient limitation and acidification are some of the stresses implicated in bacterial dormancy development/growth arrest. Adaptation to hypoxia and exposure to NO/CO is implemented through the DevRS/DosT two-component system which induces the dormancy regulon.Methodology/Principal FindingsHere we show that vitamin C (ascorbic acid/AA) can serve as an additional signal to induce the DevR regulon. Physiological levels of AA scavenge O2 and rapidly induce the DevR regulon at an estimated O2 saturation of <30%. The kinetics and magnitude of the response suggests an initial involvement of DosT and a sustained DevS-mediated response during bacterial adaptation to increasing hypoxia. In addition to inducing DevR regulon mechanisms, vitamin C induces the expression of selected genes previously shown to be responsive to low pH and oxidative stress, triggers bacterial growth arrest and promotes dormancy phenotype development in M. tb grown in axenic culture and intracellularly in THP-1 cells.Conclusions/SignificanceVitamin C mimics multiple intracellular stresses and has wide-ranging regulatory effects on gene expression and physiology of M. tb which leads to growth arrest and a ‘dormant’ drug-tolerant phenotype, but in a manner independent of the DevRS/DosT sytem. The ‘AA-dormancy infection model’ offers a potential alternative to other models of non-replicating persistence of M. tb and may be useful for investigating host-‘dormant’ M. tb interactions. Our findings offer a new perspective on the role of nutritional factors in TB and suggest a possible role for vitamin C in TB.

海洋生物からの医薬シーズの探索

In search of new pharmaceutically valuable substances from marine organisms, we have been engaged in chemical studies 1] structural elucidation; 2] asymmetric total synthesis; 3] elucidation of pharmacophore by analysis of structure–activity relationship; 4] action mechanism and search for target protein on the bioactive substances, which were isolated from marine invertebrates and marine microorganisms at Japanese coasts and Indonesian coral reefs on the guidance of new practical bioassay. a) Anti–angiogenic substance: selective growth–inhibitor against human umbilical vein endothelial cells (HUVECs) (e.g. cortistatin A: 9(10–19)–abeo–androstane–type steroidal alkaloid having isoquinoline unit) b) Hypoxia–selective growth inhibitor: growth inhibitor against tumor cells under hypoxic environment selectively (e.g. furospinosulin–1: furanosesterterpene) c) Anti–mycobacterial substances, which are effective to both active and non–replicating persistent (NRP) states of tuberculosis (e.g. halicyclamine A: macrocyclic alkaloid)

Triacylglycerol Utilization Is Required for Regrowth of In Vitro Hypoxic Nonreplicating Mycobacterium bovis Bacillus Calmette-Guerin

Mycobacteria store triacylglycerols (TGs) under various stress conditions, such as hypoxia, exposure to nitric oxide, and acidic environments. These stress conditions are known to induce nonreplicating persistence in mycobacteria. The importance of TG accumulation and utilization during regrowth is not clearly understood. Here we specifically determined the levels of accumulated TG and TG lipase activity in Mycobacterium bovis bacillus Calmette-Guerin (BCG) in various different physiological states (logarithmic growth, aerated stationary phase, hypoxia-induced dormancy, and regrowth from dormancy). We found extensive accumulation and degradation of TGs in the bacilli during entry into and exit from hypoxia-induced dormancy, respectively. These processes are accompanied by dynamic appearance and disappearance of intracellular TG lipid particles. The reduction in TG levels coincides with an increase in cellular TG lipase activity in the regrowing bacilli. Tetrahydrolipstatin, an inhibitor of TG lipases, reduces total lipase activity, prevents breakdown of TGs, and blocks the growth of mycobacteria upon resuscitation with air. Our results demonstrate that utilization of TGs is essential for the regrowth of mycobacteria during their exit from the hypoxic nonreplicating state.

Structural Insight into the Heme-based Redox Sensing by DosS from Mycobacterium tuberculosis

Mycobacterium tuberculosis is thought to undergo transformation into its non-replicating persistence state under the influence of hypoxia or nitric oxide (NO). This transformation is thought to be mediated via two sensor histidine kinases, DosS and DosT, each of which contains two GAF domains that are responsible for detecting oxygen tension. In this study we determined the crystal structures of the first GAF domain (GAF-A) of DosS, which shows an interaction with a heme. A b-type heme was embedded in a hydrophobic cavity of the GAF-A domain and was roughly perpendicular to the beta-sheet of the GAF domain. The heme iron was liganded by His-149 at the proximal heme axial position. The iron, in the oxidized form, was six-coordinated with a water molecule at the distal position. Upon reduction, the iron, in ferrous form, was five-coordinated, and when the GAF domain was exposed to atmospheric O(2), the ferrous form was oxidized to generate the Met form rather than a ferrous O(2)-bound form. Because the heme is isolated inside the GAF domain, its accessibility is restricted. However, a defined hydrogen bond network found at the heme site could accelerate the electron transferability and would explain why DosS was unable to bind O(2). Flavin nucleotides were shown to reduce the heme iron of DosS while NADH was unable to do so. These results suggest that DosS is a redox sensor and detects hypoxic conditions by its reduction.

Menaquinone synthesis is critical for maintaining mycobacterial viability during exponential growth and recovery from non‐replicating persistence

Understanding the basis of bacterial persistence in latent infections is critical for eradication of tuberculosis. Analysis of Mycobacterium tuberculosis mRNA expression in an in vitro model of non-replicating persistence indicated that the bacilli require electron transport chain components and ATP synthesis for survival. Additionally, low microM concentrations of aminoalkoxydiphenylmethane derivatives inhibited both the aerobic growth and survival of non-replicating, persistent M. tuberculosis. Metabolic labelling studies and quantification of cellular menaquinone levels suggested that menaquinone synthesis, and consequently electron transport, is the target of the aminoalkoxydiphenylmethane derivatives. This hypothesis is strongly supported by the observations that treatment with these compounds inhibits oxygen consumption and that supplementation of growth medium with exogenous menaquinone rescued both growth and oxygen consumption of treated bacilli. In vitro assays indicate that the aminoalkoxydiphenylmethane derivatives specifically inhibit MenA, an enzyme involved in the synthesis of menaquinone. Thus, the results provide insight into the physiology of mycobacterial persistence and a basis for the development of novel drugs that enhance eradication of persistent bacilli and latent tuberculosis.

Synthesis and evaluation of rifabutin analogs against Mycobacteriumavium and H 37Rv, MDR and NRP Mycobacterium tuberculosis

Clinical utility of rifabutin 1 (RBT), a potent antibiotic used in multidrug regimens for tuberculosis (TB) as well as for infections caused by Mycobacterium avium complex (MAC), has been hampered due to dose-limiting toxicity. RBT analogs 2–11 were synthesized and evaluated against M. avium 1581 and Mycobacterium tuberculosis susceptible and resistant strains in vitro. A selection of candidates were also assayed against non-replicating persistent (NRP) M. tuberculosis. Subsequent in vivo studies with the best preclinical candidate drugs 5 and 8, in a model of progressive pulmonary tuberculosis of Balb/C mice infected either with H 37Rv drug–sensible strain or with multidrug resistant (MDR) clinical isolates, resistant to all primary antibiotics including rifampicin, were performed. The results disclosed here suggest that 5 and 8 have potential for clinical application.

A Distal Tyrosine Residue Is Required for Ligand Discrimination in DevS from Mycobacterium tuberculosis

DevS is a heme-based sensor kinase required for sensing environmental conditions leading to nonreplicating persistence in Mycobacterium tuberculosis. Kinase activity is observed when the heme is a ferrous five-coordinate high-spin or six-coordinate low-spin CO or NO complex but is strongly inhibited in the oxy complex. Discrimination between these exogenous ligands has been proposed to depend on a specific hydrogen bond network with bound oxygen. Here we report resonance Raman data and autophosphorylation assays of wild-type and Y171F DevS in various heme complexes. The Y171F mutation eliminates ligand discrimination for CO, NO, and O2, resulting in equally inactive complexes. In contrast, the ferrous-deoxy Y171F variant exhibits autokinase activity equivalent to that of the wild type. Raman spectra of the oxy complex of Y171F indicate that the environment of the oxy group is significantly altered from that in the wild type. They also suggest that a solvent molecule in the distal pocket substitutes for the Tyr hydroxyl group to act as a poorer hydrogen bond donor to the oxy group. The wild-type CO and NO complexes exist as a major population in which the CO or NO groups are free of hydrogen bonds, while the Y171F mutation results in a mild increase in the distal pocket polarity. The Y171F mutation has no impact on the proximal environment of the heme, and the activity observed with the five-coordinate ferrous-deoxy wild type is conserved in the Y171F variant. Thus, while the absence of an exogenous ligand in the ferrous-deoxy proteins leads to a moderate kinase activity, interactions between Tyr171 and distal diatomic ligands turn the kinase activity on and off. The Y171F mutation disrupts the on-off switch and renders all states with a distal ligand inactive. This mechanistic model is consistent with Tyr171 being required for distal ligand discrimination, but nonessential for autophosphorylation activity.

In vitro and in vivo antimycobacterial activities of ketone and amide derivatives of quinoxaline 1,4-di-N-oxide

To evaluate a novel series of quinoxaline 1,4-di-N-oxides for in vitro activity against Mycobacterium tuberculosis and for efficacy in a mouse model of tuberculosis (TB). Ketone and amide derivatives of quinoxaline 1,4-di-N-oxide were evaluated in in vitro and in vivo tests including: (i) activity against M. tuberculosis resistant to currently used antitubercular drugs including multidrug-resistant strains (MDR-TB resistant to isoniazid and rifampicin); (ii) activity against non-replicating persistent (NRP) bacteria; (iii) MBC; (iv) maximum tolerated dose, oral bioavailability and in vivo efficacy in mice; and (v) potential for cross-resistance with another bioreduced drug, PA-824. Ten compounds were tested on single drug-resistant M. tuberculosis. In general, all compounds were active with ratios of MICs against resistant and non-resistant strains of <or=4.00. One compound, 5, was orally active in a murine model of TB, bactericidal, active against NRP bacteria and active on MDR-TB and poly drug-resistant clinical isolates (resistant to 3-5 antitubercular drugs). Quinoxaline 1,4-di-N-oxides represent a new class of orally active antitubercular drugs. They are likely bioreduced to an active metabolite, but the pathway of bacterial activation was different from PA-824, a bioreducible nitroimidazole in clinical trials. Compound 5 was bactericidal and active on NRP organisms indicating that activation occurred in both growing and non-replicating bacteria leading to cell death. The presence of NRP bacteria is believed to be a major factor responsible for the prolonged nature of antitubercular therapy. If the bactericidal activity and activity on non-replicating bacteria in vitro translate to in vivo conditions, quinoxaline 1,4-di-N-oxides may offer a path to shortened therapy.