Discovery of Novel Oral Protein Synthesis Inhibitors of Mycobacterium tuberculosis That Target Leucyl-tRNA Synthetase

The recent development and spread of extensively drug-resistant and totally drug-resistant resistant (TDR) strains of Mycobacterium tuberculosis highlight the need for new antitubercular drugs. Protein synthesis inhibitors have played an important role in the treatment of tuberculosis (TB) starting with the inclusion of streptomycin in the first combination therapies. Although parenteral aminoglycosides are a key component of therapy for multidrug-resistant TB, the oxazolidinone linezolid is the only orally available protein synthesis inhibitor that is effective against TB. Here, we show that small-molecule inhibitors of aminoacyl-tRNA synthetases (AARSs), which are known to be excellent antibacterial protein synthesis targets, are orally bioavailable and effective against M. tuberculosis in TB mouse infection models. We applied the oxaborole tRNA-trapping (OBORT) mechanism, which was first developed to target fungal cytoplasmic leucyl-tRNA synthetase (LeuRS), to M. tuberculosis LeuRS. X-ray crystallography was used to guide the design of LeuRS inhibitors that have good biochemical potency and excellent whole-cell activity against M. tuberculosis. Importantly, their good oral bioavailability translates into in vivo efficacy in both the acute and chronic mouse models of TB with potency comparable to that of the frontline drug isoniazid.     

Development of new antituberculous drugs based on bacterial virulence factors interfering with host cytokine networks

The worldwide increase in the prevalence of tuberculosis (TB), especially multidrug-resistant TB and extensively drug-resistant TB, is an important global health concern, and new effective drugs are urgently needed. Information on the genome of Mycobacterium tuberculosis (MTB) and various mycobacterial virulence genes is leading to the identification of genes that code for new drug targets. Mycobacterium tuberculosis (MTB) is resistant to the antimicrobial mechanisms of host macrophages and can survive and replicate in macrophages for long periods, resulting in a persistent infection. Mycobacterial virulence factors suppress macrophage bactericidal functions partly via their downregulatory effects on the host antimicrobial cytokine networks, consisting of proinflammatory, immunopotentiating, and Th1-inducing cytokines. Thus, for the development of unique drugs that exhibit antimycobacterial action through novel mechanisms, it is reasonable to search for targets among bacterial genes encoding virulence factors which interfere with the host cytokine responses protective to mycobacterial pathogens. In this review, we discuss the profiles of cytokine networks related to host resistance to mycobacteria, including the mechanisms of downregulation of host antimycobacterial immunity due to immunosuppressive cytokines, which are occasionally induced in the advanced stages of TB. We also highlight the development of antituberculous drugs based on bacterial virulence factors interfering with the host antimycobacterial cytokine network.     

Molecular tools for rapid identification and novel effective therapy against MDRTB/XDRTB infections

Tuberculosis (TB) is mainly an intracellular infection of the lung alveolar macrophages, and any anti-TB agent must therefore be active at the macrophage. Among the available therapies, isoniazid and rifampicin are the most effective drugs against susceptible Mycobacterium tuberculosis, but they are ineffective against multidrug-resistant TB (MDRTB) strains. Rates of MDRTB in Portugal are the highest in Western Europe, demanding effective measures for their control. Our application of molecular techniques for the early identification of MDRTB assisted in the reduction of these rates. Further examination revealed that a large number of MDRTB cases were extensively-drug resistant (XDRTB), providing evidence for the urgent need of new and effective anti-MDRTB/XDRTB therapeutic strategies. This review describes in detail: the characteristics of the main M. tuberculosis strains circulating in Portugal; the creation of a Task Force for TB control, based on molecular tools that allow 1-day identification of an MDRTB patient; the usefulness of evaluating the ex vivo activity of anti-tubercular agents against the M. tuberculosis isolated from the patient’s sputum; and the mode of action by which phenothiazines have been shown to promote the killing of intracellular MDRTB/XDRTB by nonkilling macrophages.     

Strategies for potentiation of ethionamide and folate antagonists against Mycobacterium tuberculosis

Antifolates inhibit de novo folate biosynthesis, whereas ethionamide targets the mycolate synthetic pathway in Mycobacterium tuberculosis. These antibiotics are effective against M. tuberculosis but their use has been hampered by concerns over toxicity and low therapeutic indexes. With the increasing spread of drug-resistant forms, interest in using old drugs for tuberculosis treatment has been renewed. Specific inhibitors targeting resistance mechanisms could sensitize M. tuberculosis to these available, clinically approved drugs. This review discusses recently developed strategies to boost the antituberculous activity of ethionamide and antifolates. These approaches might help broaden the currently limited chemotherapeutic options of not only drug-resistant but also drug-susceptible tuberculosis, which still remains one of the most common infectious diseases in the developing world.     

Mycobacterium caprae infection in humans

Mycobacterium caprae, a member of the Mycobacterium tuberculosis complex, causes tuberculosis (TB) in man and animals. Some features distinguish M. caprae from its epidemiological twin, Mycobacterium bovis: M. caprae is evolutionarily older, accounts for a smaller burden of zoonotic TB and is not globally distributed, but primarily restricted to European countries. M. caprae occurs only in a low proportion of human TB cases and this proportion may even decrease, if progress toward eradication of animal TB in Europe continues. So why bother, if M. caprae is not an enigma for diagnostic TB tests and if resistance against first-line drugs is a rarity with M. caprae? This ‘European’ pathogen of zoonotic TB asks interesting questions regarding the definition of a species. The latter, seemingly only an academic question, particularly requires and challenges the collaboration between human and veterinary medicine.     

New structural classes of antituberculosis agents

Tuberculosis (TB), one of the deadliest diseases is shattering the health and socioeconomic status of the society. The emergence of multidrug resistant (MDR) and extremely drug resistant (XDR) strains has provided unprecedented lethal character to TB. The development of MDR and XDR strains of TB results in more deaths, longer duration of therapy, and appearance of the disease in the immunocompromised patients. Because of the development of rapid resistance by Mycobacterium tuberculosis, researchers are confronted with serious challenges in combating TB. For instance, the need for potency and specificity in therapeutic agents approaching clinics, and the increasing demand of low toxicity due to long duration of treatment. Recently, it is proposed that such challenges could be addressed by a shift from contemporary or known classes of drugs to new scaffold‐containing or entirely new structural classes of drugs that possibly act on the previously unknown targets, resulting in possibly less instances of resistance development. The exploitation of advances made in the biology of TB in the last and present decades have created opportunities to discover a large number of new structural classes that specifically targets TB by molecular mechanism of action(s) unknown earlier. We have earlier reviewed new structural classes of anti‐TB agents up to year 2005. This review covers literature reports of the subsequent 10 years on the discovery of new structural classes of synthetic anti‐TB agents. Due to the availability of large number of research reports, we have divided new compounds in 38 structural classes, 368 structures, and 307 references.     

The development,evaluation and performance of molecular diagnostics for detection of Mycobacterium tuberculosis

The unique pathogenesis of tuberculosis (TB) poses several barriers to the development of accurate diagnostics: a) the establishment of life-long latency by Mycobacterium tuberculosis (M.tb) after primary infection confounds the development of classical antibody or antigen based assays; b) our poor understanding of the molecular pathways that influence progression from latent to active disease; c) the intracellular nature of M.tb infection in tissues means that M.tb and/or its components, are not readily detectable in peripheral specimens; and d) the variable presence of M.tb bacilli in specimens from patients with extrapulmonary TB or children. The literature on the current portfolio of molecular diagnostics tests for TB is reviewed here and the developmental pipeline is summarized. Also reviewed are data from recently published operational research on the GeneXpert MTB/RIF assay and discussed are the lessons that can be taken forward for the design of studies to evaluate the impact of TB diagnostics.     

In Vitro Interactions between New Antitubercular Drug Candidates SQ109 and TMC207

The in vitro interactions of two new antitubercular drugs, SQ109 and TMC207, with each other and with rifampin (RIF) were evaluated. The combination of SQ109 with TMC207 (i) improved an already excellent TMC207 MIC for M. tuberculosis H37Rv by 4- to 8-fold, (ii) improved the rate of killing of bacteria over the rate of killing by each single drug, and (iii) enhanced the drug postantibiotic effect by 4 h. In no instance did we observe antagonistic activities with the combination of SQ109 and TMC207. Rifampin activates cytochrome P450 genes to reduce the area under the curve (AUC) for TMC207 in humans. The presence of RIF in three-drug combinations did not affect the synergistic activities of SQ109 and TMC207, and SQ109 also dramatically decreased the MIC of RIF. SQ109 was active by itself, and both its activity was improved by and it improved the in vitro activities of both RIF and TMC207.Pulmonary tuberculosis (TB) is currently treated with a regimen of four drugs discovered before 1970: rifampin (RIF), isoniazid (INH), pyrazinamide (PZA), and ethambutol (EMB) or streptomycin (STR). These four drugs are administered for 2 months (the intensive phase), followed by the administration of RIF and INH for 4 to 7 months (the continuation phase), for a total of 6 to 9 months of treatment (1, 19). Because of the difficulty in eradicating Mycobacterium tuberculosis from tissues with the currently available drugs and the long duration of treatment for TB, many patients fail to take all their drugs or terminate their therapy early, and noncompliance with therapy has fueled the development of M. tuberculosis drug resistance (3). Multidrug-resistant TB (MDR-TB), defined as infection with M. tuberculosis isolates resistant to both RIF and INH, requires up to 24 months of treatment with a cocktail of less effective and more toxic second-line drugs to achieve a cure.At least seven new antitubercular drugs are currently under evaluation in human clinical trials (18). The new drug candidates in development act on different metabolic or structural targets in M. tuberculosis, and all act on different targets than the current front-line TB drugs. SQ109 [N-geranyl-N′-(2-adamantyl) ethane-1,2-diamine] is a new diamine antitubercular drug candidate that interferes with cell wall synthesis in M. tuberculosis (9, 14). The exact mechanism of action (MOA) on the cell wall is not yet known. TMC207 (also known as R207910) is a diarylquinoline that specifically inhibits mycobacteria by inhibition of ATP synthesis (2). Individually, both SQ109 and TMC207 are bactericidal, effective against MDR-TB strains, and synergistic with (or can be synergistically enhanced by) other front-line anti-TB drugs (5, 8). SQ109 acts synergistically to improve the antitubercular action of INH and RIF (5), whereas TMC207 is synergistic with PZA (8). In humans, both drugs have a long half-life: SQ109 has a half-life of 60 h at 300 mg/day (unpublished data), and TMC207 has a half-life of 24 h at 400 mg/day (2). Therefore, both drugs have the potential for intermittent dosing. Of interest, the area under the curve (AUC) for TMC207 in humans was decreased by the activation of certain cytochrome P450 enzymes by RIF (10). Since SQ109 can lower by 4-fold the MIC of RIF, we were interested to see whether SQ109 in combination with TMC207 might mitigate the adverse drug-drug interaction that occurs with TMC207 in humans in the presence of RIF.In this study we investigated the in vitro efficacy of combinations of TMC207 and SQ109 at killing M. tuberculosis, with and without the presence of RIF.     

Mechanism of thioamide drug action against tuberculosis and leprosy

Thioamide drugs, ethionamide (ETH) and prothionamide (PTH), are clinically effective in the treatment of Mycobacterium tuberculosis, M. leprae, and M. avium complex infections. Although generally considered second-line drugs for tuberculosis, their use has increased considerably as the number of multidrug resistant and extensively drug resistant tuberculosis cases continues to rise. Despite the widespread use of thioamide drugs to treat tuberculosis and leprosy, their precise mechanisms of action remain unknown. Using a cell-based activation method, we now have definitive evidence that both thioamides form covalent adducts with nicotinamide adenine dinucleotide (NAD) and that these adducts are tight-binding inhibitors of M. tuberculosis and M. leprae InhA. The crystal structures of the inhibited M. leprae and M. tuberculosis InhA complexes provide the molecular details of target–drug interactions. The purified ETH-NAD and PTH-NAD adducts both showed nanomolar K_is against M. tuberculosis and M. leprae InhA. Knowledge of the precise structures and mechanisms of action of these drugs provides insights into designing new drugs that can overcome drug resistance.     

Use of whole genome sequencing in surveillance of drug resistant tuberculosis

Introduction: The threat of resistance to anti-tuberculosis drugs is of global concern. Current efforts to monitor resistance rely on phenotypic testing where cultured bacteria are exposed to critical concentrations of the drugs. Capacity for such testing is low in TB endemic countries. Drug resistance is caused by mutations in the Mycobacterium tuberculosis genome and whole genome sequencing to detect these mutations offers an alternative means of assessing resistance.

Areas covered: The challenges of assessing TB drug resistance are discussed. Progress in elucidating the M. tuberculosis resistome and evidence of the accuracy of next generation sequencing for detecting resistance is reviewed.

Expert Commentary: There are considerable advantages to using next generation sequencing for TB drug resistance surveillance. Accuracy is high for detecting resistance to the major first-line drugs but is currently lower for the second-line drugs due to our incomplete knowledge regarding resistance causing mutations. With the advances in sequencing technology and the opportunity to replace phenotypic drug susceptibility testing with safer and more cost effective methods it would appear that the question is when to implement. Current bottlenecks are sample extraction to allow whole genome sequencing directly from sputum and the lack of bioinformatics expertise in some TB endemic countries.     

Mycobacterium tuberculosis Folate Metabolism and the Mechanistic Basis for para-Aminosalicylic Acid Susceptibility and Resistance

para-Aminosalicylic acid (PAS) entered clinical use in 1946 as the second exclusive drug for the treatment of tuberculosis (TB). While PAS was initially a first-line TB drug, the introduction of more potent antitubercular agents relegated PAS to the second-line tier of agents used for the treatment of drug-resistant Mycobacterium tuberculosis infections. Despite the long history of PAS usage, an understanding of the molecular and biochemical mechanisms governing the susceptibility and resistance of M. tuberculosis to this drug has lagged behind that of most other TB drugs. Herein, we discuss previous studies that demonstrate PAS-mediated disruption of iron acquisition, as well as recent genetic, biochemical, and metabolomic studies that have revealed that PAS is a prodrug that ultimately corrupts one-carbon metabolism through inhibition of the formation of reduced folate species. We also discuss findings from laboratory and clinical isolates that link alterations in folate metabolism to PAS resistance. These advancements in our understanding of the basis of the susceptibility and resistance of M. tuberculosis to PAS will enable the development of novel strategies to revitalize this and other antimicrobial agents for use in the global effort to eradicate TB.     

The Antituberculosis Antibiotic Capreomycin Inhibits Protein Synthesis by Disrupting Interaction between Ribosomal Proteins L12 and L10

Capreomycin is a second-line drug for multiple-drug-resistant tuberculosis (TB). However, with increased use in clinics, the therapeutic efficiency of capreomycin is decreasing. To better understand TB resistance to capreomycin, we have done research to identify the molecular target of capreomycin. Mycobacterium tuberculosis ribosomal proteins L12 and L10 interact with each other and constitute the stalk of the 50S ribosomal subunit, which recruits initiation and elongation factors during translation. Hence, the L12-L10 interaction is considered to be essential for ribosomal function and protein synthesis. Here we provide evidence showing that capreomycin inhibits the L12-L10 interaction by using an established L12-L10 interaction assay. Overexpression of L12 and/or L10 in M. smegmatis, a species close to M. tuberculosis, increases the MIC of capreomycin. Moreover, both elongation factor G-dependent GTPase activity and ribosome-mediated protein synthesis are inhibited by capreomycin. When protein synthesis was blocked with thiostrepton, however, the bactericidal activity of capreomycin was restrained. All of these results suggest that capreomycin seems to inhibit TB by interrupting the L12-L10 interaction. This finding might provide novel clues for anti-TB drug discovery.     

Role of the Mycobacterium tuberculosis P55 Efflux Pump in Intrinsic Drug Resistance,Oxidative Stress Responses,and Growth

Bacterial efflux pumps have traditionally been studied as low-level drug resistance determinants. Recent insights have suggested that efflux systems are often involved with fundamental cellular physiological processes, suggesting that drug extrusion may be a secondary function. In Mycobacterium tuberculosis, little is known about the physiological or drug resistance roles of efflux pumps. Using Mycobacterium bovis BCG as a model system, we showed that deletion of the Rv1410c gene encoding the P55 efflux pump made the strain more susceptible to a range of toxic compounds, including rifampin (rifampicin) and clofazimine, which are first- and second-line antituberculosis drugs. The efflux pump inhibitors carbonyl cyanide m-chlorophenylhydrazone (CCCP) and valinomycin inhibited the P55-determined drug resistance, suggesting the active export of the compounds by use of the transmembrane proton and electrochemical gradients as sources of energy. In addition, the P55 efflux pump mutant was more susceptible to redox compounds and displayed increased intracellular redox potential, suggesting an essential role of the efflux pump in detoxification processes coupled to oxidative balance within the cell. Finally, cells that lacked the p55 gene displayed smaller colony sizes and had a growth defect in liquid culture. This, together with an increased susceptibility to the cell wall-targeting compounds bacitracin and vancomycin, suggested that P55 is needed for proper cell wall assembly and normal growth in vitro. Thus, P55 plays a fundamental role in oxidative stress responses and in vitro cell growth, in addition to contributing to intrinsic antibiotic resistance. Inhibitors of the P55 efflux pump could help to improve current treatments for tuberculosis.Tuberculosis (TB) continues to be one of the main causes of morbidity and mortality worldwide. The most recent WHO report estimates that there are 9.2 million new cases of TB and 1.7 million deaths per year (41), significant numbers of which occur among human immunodeficiency virus-positive patients. Along with human immunodeficiency virus coinfection, multidrug-resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB) pose major threats that challenge TB control, especially in those regions of the world with the highest burden of TB.Efflux pumps are membrane proteins that export substrates from bacterial and eukaryotic cells. They confer resistance to anticancer drugs in tumor cells and to antibiotics in bacteria, often providing low levels of intrinsic multidrug resistance (25). Their activities allow better tolerance of drugs and thus may potentiate the acquisition of chromosomal mutations that provide higher levels of resistance (29, 30). In recent years, it has become evident that efflux pumps have important functions in many other cellular processes, such as physiological homeostasis, resistance to stress conditions, lipid transport, and virulence (24).While drug resistance in Mycobacterium tuberculosis clinical isolates is often due to the acquisition of mutations in genes encoding drug targets or enzymes activating prodrugs, such mutations are not found in many low-level-drug-resistant isolates, suggesting the contribution of efflux pumps (11). In fact, many M. tuberculosis efflux pumps contribute to drug resistance under laboratory conditions, and there are reports describing increases in the levels of expression of efflux pumps in various drug-resistant M. tuberculosis isolates (11, 15, 17, 32, 33, 38). In addition, the inactivation of certain efflux pumps attenuates M. tuberculosis, indicating that, like other bacterial pathogens, efflux pumps also contribute to virulence (10).We have previously characterized the contribution of the M. tuberculosis P55 efflux pump to drug resistance in Mycobacterium smegmatis (39). The gene encoding the P55 efflux pump, Rv1410c, forms an operon with Rv1411c (5), which encodes the lipoprotein LprG. Both genes are predicted to support the growth of M. tuberculosis in vivo (6, 37). A recent report has demonstrated that this operon is required for survival in the presence of ethidium bromide and for maintenance of a normal cell surface composition in M. smegmatis (13).In the study described here, we characterized P55 in the TB vaccine strain, M. bovis BCG. Our results demonstrate that P55 plays a role in at least three important processes: it extrudes and thus provides resistance to several drugs (including rifampin [rifampicin], one of the most important frontline TB drugs), it is part of the oxidative stress response, and it is needed to maintain normal growth characteristics both on solid medium and in liquid medium.     

A case of prosthetic joint infection caused by Mycobacterium tuberculosis complicated secondary bacterial infection after knee joint replacement surgery

Mycobacterium tuberculosis (M. tuberculosis) is a rare cause of prosthetic joint infection (PJI). Previous studies have reported that many cases of PJI caused by M. tuberculosis have no medical history of active tuberculosis (TB) or other localization, which contributes to diagnostic difficulties. Furthermore, owing to the limited number of studies on treatment, appropriate treatment strategies, such as the duration of anti-tuberculosis (anti-TB) drugs and surgical indications, remain unclear.We report a case of PJI caused by M. tuberculosis and secondary pyogenic arthritis caused by Staphylococcus aureus and Streptococcus dysgalactiae in a 67-year-old man after knee joint replacement surgery in Japan, which was a moderately endemic country until 2020 and a low endemic country since 2021. Although he had no past medical history or close contact with TB, he was diagnosed with PJI caused by M. tuberculosis, following the culture of a synovectomy specimen. He underwent two-stage surgery and was treated with anti-TB drugs for a total of 12 months and recovered without recurrence.Based on our case and previous studies, there are three points of clinical significance for PJI caused by M. tuberculosis. First, about one year of anti-TB drugs with two staged joint revision resulted in a good course of treatment. Second, surgical treatment might be considered in cases complicated by secondary bacterial infection. Third, because the diagnosis of PJI caused by M. tuberculosis is difficult, TB should be considered in the differential diagnosis of routine bacterial culture-negative PJI, especially in endemic areas.     

Prevalence and Genetic Characterization of Second-Line Drug-Resistant and Extensively Drug-Resistant Mycobacterium tuberculosis in Rural China

This study aimed to investigate the prevalence of resistance to second-line antituberculosis (anti-TB) drugs and its association with resistance-related mutations in Mycobacterium tuberculosis isolated in China. In the present study, we collected 380 isolates from a population-based study in China and tested the drug susceptibility to first- and selected second-line drugs. These results were compared with polymorphisms in the DNA sequences of genes associated with drug resistance and MIC values of the studied second-line drugs. Of 43 multidrug-resistant M. tuberculosis isolates, 13 showed resistance to fluoroquinolones or injectable second-line drugs (preextensively drug-resistant TB [pre-XDR-TB]), and 4 were resistant to both and thus defined as extensively drug-resistant TB (XDR-TB). Age and previous TB therapy, including use of second-line drugs, were two independent factors associated with increased resistance to both first- and second-line drugs. Molecular analysis identified the most frequent mutations in the resistance-associated genes: D94G in gyrA (29.1%) and A1401G in rrs (30.8%). Meanwhile, all 4 XDR-TB isolates had a mutation in gyrA, and 3 of them carried the A1401G mutation in rrs. Mutations in gyrA and rrs were associated with high-level resistance to fluoroquinolones and the second-line injectable drugs. In addition to the identification of resistance-associated mutations and development of a rapid molecular test to diagnose the second-line drug resistance, it should be a priority to strictly regulate the administration of second-line drugs to maintain their efficacy to treat multidrug-resistant TB.     

Analysis of drug resistance and mutation profiles in Mycobacterium tuberculosis isolates in a surveillance site in Beijing,China

ObjectiveThis study analyzed drug resistance and mutations profiles in Mycobacterium tuberculosis isolates in a surveillance site in Huairou District, Beijing, China.MethodsThe proportion method was used to assess drug resistance profiles for four first-line and seven second-line anti-tuberculosis (TB) drugs. Molecular line probe assays were used for the rapid detection of resistance to rifampicin (RIF) and isoniazid (INH).ResultsAmong 235 strains of M. tuberculosis, 79 (33.6%) isolates were resistant to one or more drugs. The isolates included 18 monoresistant (7.7%), 19 polyresistant (8.1%), 28 RIF-resistant (11.9%), 24 multidrug-resistant (MDR) (10.2%), 7 pre-extensively drug-resistant (XDR, 3.0%), and 2 XDR strains (0.9%). A higher rate of MDR-TB was detected among previously treated patients than among patients with newly diagnosed TB (34.5% vs. 6.8%). The majority (62.5%) of RIF-resistant isolates exhibited a mutation at S531L in the DNA-dependent RNA polymerase gene. Meanwhile, 62.9% of INH-resistant isolates carried a mutation at S315T1 in the katG gene.ConclusionOur results confirmed the high rate of drug-resistant TB, especially MDR-TB, in Huairou District, Beijing, China. Therefore, detailed drug testing is crucial in the evaluation of MDR-TB treatment.     

Safety,Tolerability, and Pharmacokinetics of PA-824 in Healthy Subjects

PA-824 is a novel antibacterial agent that has shown in vitro activity against both drug-sensitive and drug-resistant Mycobacterium tuberculosis. The compound''s MIC is between 0.015 and 0.25 μg/ml for drug-sensitive strains and between 0.03 and 0.53 μg/ml for drug-resistant strains. In addition, it is active against nonreplicating anaerobic Mycobacterium tuberculosis. The safety, tolerability, and pharmacokinetics of PA-824 were evaluated in two escalating-dose clinical studies, one a single-dose study and the other a multiple-dose study (up to 7 days of daily dosing). In 58 healthy subjects dosed with PA-824 in these studies, the drug candidate was well tolerated, with no significant or serious adverse events. In both studies, following oral administration PA-824 reached maximal plasma levels in 4 to 5 h independently of the dose. Maximal blood levels averaged approximately 3 μg/ml (1,500-mg dose) in the single-dose study and 3.8 μg/ml (600-mg dose) in the multiple-dose study. Steady state was achieved after 5 to 6 days of daily dosing, with an accumulation ratio of approximately 2. The elimination half-life averaged 16 to 20 h. Overall, PA-824 was well tolerated following oral doses once daily for up to 7 days, and pharmacokinetic parameters were consistent with a once-a-day regimen. The results of these studies, combined with the demonstrated activity of PA-824 against drug-sensitive and multidrug-resistant Mycobacterium tuberculosis, support the investigation of this novel compound for the treatment of tuberculosis.According to the World Health Organization, there were 9.27 million new tuberculosis (TB) cases worldwide in 2007, which claimed the lives of approximately 1.77 million people, including 456,000 patients coinfected with human immunodeficiency virus (11). In addition, global increases in cases of multidrug-resistant TB and, more recently, extensively drug-resistant TB pose serious treatment challenges (12). New anti-TB drugs are needed that can shorten the duration of treatment, improve the treatment of resistant disease, facilitate the treatment of TB patients coinfected with human immunodeficiency virus, and shorten the treatment of latent TB infection.The 4-nitroimidazo-oxazoles (a subclass of nitroimidazoles) have potent sterilizing activity against Mycobacterium tuberculosis, as first demonstrated in 1993 (1). The further investigation of nitroimidazoles in an anaerobic model of M. tuberculosis dormancy demonstrated that metronidazole is active against slow-growing M. tuberculosis, suggesting the potential for the treatment of latent TB infection and for shortening the treatment of active TB disease (10). The further development of the nitroimidazole class by Pathogenesis, Inc., led to the discovery of another subclass, 4-nitroimidazo-oxazines, with promising activity against M. tuberculosis. PA-824, with the full chemical name (S)-2-nitro-6-[4-(trifluoromethoxy)benzyloxy]-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine), was identified as the lead 4-nitroimidazo-oxazine. Stover et al. (8) reported that the MIC of PA-824 under aerobic conditions against a variety of drug-sensitive clinical isolates was similar to the MIC of isoniazid (MIC of PA-824, 0.015 to 0.25 μg/ml; MIC of isoniazid, 0.03 to 0.06 μg/ml). PA-824 also was found to be active against all single-drug- and multidrug-resistant clinical isolates of M. tuberculosis tested, with MICs of 0.03 to 0.53 μg/ml. Additional studies using microaerophilic and anaerobic culture models indicated that PA-824 also is active against both replicating and nonreplicating or infrequently replicating M. tuberculosis isolates (3, 8).Like metronidazole, PA-824 requires metabolic activation by M. tuberculosis through an F420-dependent nitroreduction (4, 5, 8). Although not thoroughly elucidated at this time, PA-824''s novel mechanism of action involves the inhibition of the synthesis of both protein and lipids but not nucleic acid. Studies by Stover et al. (8) demonstrated that PA-824 inhibits the oxidation of hydroxymycolate to ketomycolate, an essential lipid for M. tuberculosis cell wall function. Recent work by Singh et al. (7) indicates that the reduction of PA-824 to its des-nitroimidazole metabolite by a deazaflavin (F420)-dependent nitroreductase is associated with the generation of reactive nitrogen species, including nitric oxide, which may represent important effectors of PA-824 killing of M. tuberculosis under anaerobic conditions. In an experimental mouse model of infection, Tyagi et al. (9) demonstrated that, at a dose of 100 mg/kg of body weight, PA-824 has substantial bactericidal activity during both the initial and continuation phases of TB treatment. Using a short-course mouse infection model that employs 9 days of the drug treatment of gamma-interferon knockout mice infected with M. tuberculosis 14 days before treatment initiation, Lenaerts et al. (3) found that at 100 mg/kg PA-824 was as active as isoniazid at 25 mg/kg, rifampin (rifampicin) at 10 mg/kg, and moxifloxacin at 100 mg/kg. Additional studies of a mouse model of TB examined the activity of PA-824 administered in combination with current TB drugs. When substituted for isoniazid in standard therapy, PA-824 resulted in significantly fewer CFU after 2 months of therapy and a higher rate of conversion to culture negativity than that of the standard drug combination. Relapse rates after 6 months of treatment were not different in the experimental and control treatment arms in this study, but the study design was such that an improved relapse rate relative to the control could not have been demonstrated (6). Pharmacokinetic analyses reported by Nuermberger et al. (6) demonstrated in mice that the standard rifampin-isoniazid-pyrazinamide regimen does not affect core PA-824 pharmacokinetic parameters, such as C_max (maximum concentration observed), AUC_0—24 (total area under concentration-time curve from 0 to 24 h), or t_1/2 (half-life). Further nonclinical studies are under way to characterize PA-824''s activity and interactions in novel drug combinations.     

The Cyclic Peptide Ecumicin Targeting ClpC1 Is Active against Mycobacterium tuberculosis In Vivo

Drug-resistant tuberculosis (TB) has lent urgency to finding new drug leads with novel modes of action. A high-throughput screening campaign of >65,000 actinomycete extracts for inhibition of Mycobacterium tuberculosis viability identified ecumicin, a macrocyclic tridecapeptide that exerts potent, selective bactericidal activity against M. tuberculosis in vitro, including nonreplicating cells. Ecumicin retains activity against isolated multiple-drug-resistant (MDR) and extensively drug-resistant (XDR) strains of M. tuberculosis. The subcutaneous administration to mice of ecumicin in a micellar formulation at 20 mg/kg body weight resulted in plasma and lung exposures exceeding the MIC. Complete inhibition of M. tuberculosis growth in the lungs of mice was achieved following 12 doses at 20 or 32 mg/kg. Genome mining of lab-generated, spontaneous ecumicin-resistant M. tuberculosis strains identified the ClpC1 ATPase complex as the putative target, and this was confirmed by a drug affinity response test. ClpC1 functions in protein breakdown with the ClpP1P2 protease complex. Ecumicin markedly enhanced the ATPase activity of wild-type (WT) ClpC1 but prevented activation of proteolysis by ClpC1. Less stimulation was observed with ClpC1 from ecumicin-resistant mutants. Thus, ClpC1 is a valid drug target against M. tuberculosis, and ecumicin may serve as a lead compound for anti-TB drug development.     

Why and how thioridazine in combination with antibiotics to which the infective strain is resistant will cure totally drug-resistant tuberculosis

Over a period of 14 years, the authors have studied thioridazine, an old neuroleptic, that has been shown to have in vitro activity against intracellular Mycobacterium tuberculosis, regardless of its antibiotic resistance status, thioridazine cures infected mice of antibiotic-susceptible and multidrug-resistant tuberculosis (TB) infections and, when used in combination with antibiotics used for therapy of TB, renders the organism significantly more susceptible. This article will describe the authors’ further work and the mechanisms by which thioridazine alone and in combination with antibiotics cures an extensively drug-resistant infection and why it is expected to cure totally drug-resistant TB infections as well. The concepts presented are entirely new and because they focus on the activation of killing by nonkilling macrophages where M. tuberculosis normally resides during infection, and coupled to the inhibition of efflux pumps which contribute to the antibiotic-resistant status, effective therapy of any antibiotic-resistant TB infection is possible.