Retrospective studies in patients with non-muscle invasive bladder cancer (NMIBC) have reported a significant reduction in Alzheimer's disease (AD) incidence (12-78%) among Bacillus Calmette-Guérin (BCG) recipients versus controls. To investigate the underlying mechanisms, we evaluated BCG in the PS19 mouse model of tauopathy. We found that BCG administration reduced hippocampal phospho-tau and microgliosis while preserving neuronal markers. In vivo volumetric T2-MRI demonstrated attenuation of brain atrophy accompanied by increased glutamate-weighted CEST-MRI signals. Functionally, BCG-treated mice showed improved performance in the novel object recognition test (NORT), as well as improved body-weight maintenance and survival. Transcriptomic profiling of the hippocampus revealed near complete normalization of the PS19 disease-associated gene expression signature towards that of healthy controls. Flow cytometric profiling of brain myeloid populations demonstrated a reduction in activated resident microglia, but total microglia cells remain elevated. Moreover, an increase of the co-stimulatory marker CD80 on the recruited peripheral myeloid cells ensues following BCG treatment. Consistent with this shift in myeloid state, primary brain myeloid cells from BCG-treated mice also exhibited enhanced phagocytosis of FITC-labeled tau fibrils and increased lactate production. Together, these findings indicate that BCG induces systemic and CNS myeloid cell reprogramming that limits neuroinflammation, enhances tau clearance, and rescues cognitive and neurodegenerative phenotypes in a tauopathy model. BCG is a safe, readily available therapy that merits consideration as a preventive agent against dementia. One sentence summary:BCG therapy prevents tauopathy in PS19 mouse model.

The development of granulomas with central necrosis harboring Mycobacterium tuberculosis (Mtb) is the hallmark of human tuberculosis (TB). New anti-TB therapies need to effectively penetrate the cellular and necrotic compartments of these lesions and reach sufficient concentrations to eliminate Mtb. BTZ-043 is a novel antibiotic showing good bactericidal activity in humans in a phase IIa trial. Here, we report on lesional BTZ-043 concentrations severalfold above the minimal-inhibitory-concentration and the substantial local efficacy of BTZ-043 in interleukin-13-overexpressing mice, which mimic human TB pathology of granuloma necrosis. High-resolution MALDI imaging further reveals that BTZ-043 diffuses and accumulates in the cellular compartment, and fully penetrates the necrotic center. This is the first study that visualizes an efficient penetration and accumulation of a clinical-stage TB drug in human-like centrally necrotizing granulomas and that also determines its lesional activity. Our results most likely predict a substantial bactericidal effect of BTZ-043 at these hard-to-reach sites in TB patients. Therapy of tuberculosis is challenging, mainly due to complex structures of necrotic granulomas that often impair drug delivery. In this work, the authors show that the drug BTZ-043 fully penetrates necrotic granulomas and has potent lesional antibacterial activity.

Background BTZ-043 is a promising novel drug candidate for antituberculosis treatment. This study aimed to apply a previously developed mouse-to-human translational modeling platform for antituberculosis drugs to predict phase 2A outcomes for BTZ-043 in humans and evaluate the impact of observed drug-drug interactions on the contribution of BTZ-043 to combotherapy in a mouse model.Methods The study utilized data from mouse experiments for BTZ-043 monotherapy and combotherapy with bedaquiline, pretomanid, and linezolid, and clinical information for BTZ-043 monotherapy. The translational models were applied to predict the colony-forming units as a measure of efficacy in humans treated with BTZ-043 monotherapy and evaluate the effect of BTZ-043 on the pharmacokinetics-pharmacodynamics of combotherapy bedaquiline, pretomanid, and linezolid.Results The mouse-pharmacokinetic and mouse-pharmacodynamic data for BTZ-043 monotherapy were best described by 2-compartmental and direct Emax models, respectively. The model-based prediction of efficacy in humans was comparable to the observed phase 2A efficacy. Single-compartmental models, developed separately, best described the mouse-pharmacokinetic data for bedaquiline, pretomanid, and linezolid in combotherapy. Coadministration with BTZ-043 was associated with at least a 2-fold reduction in bedaquiline, pretomanid, and linezolid exposures in mice, and model-based simulations suggested that the observed decreases in exposure to these drugs would have resulted in even lower efficacy than what was observed when bedaquiline, pretomanid, and linezolid are coadministered with BTZ-043.Conclusions The translational modeling platform adequately predicted the efficacy of BTZ-043 monotherapy. In the absence of drug-drug interactions, coadministration of BTZ-043 with bedaquiline, pretomanid, and linezolid in combotherapy is predicted to improve treatment efficacy. Clinical Trials Registration. NCT0404400.Conclusions The translational modeling platform adequately predicted the efficacy of BTZ-043 monotherapy. In the absence of drug-drug interactions, coadministration of BTZ-043 with bedaquiline, pretomanid, and linezolid in combotherapy is predicted to improve treatment efficacy. Clinical Trials Registration. NCT0404400. The previously developed translational modeling platform for antituberculosis drugs adequately predicted phase 2A efficacy outcomes for BTZ-043. In the absence of drug-drug interactions, BTZ-043 increases efficacy outcomes when coadministered with a combination regimen of bedaquiline, pretomanid, and linezolid.

Abstract Introduction BTZ-043 is a promising novel drug candidate for anti-tuberculosis treatment. This study aimed to apply a previously developed mouse-to-human translational modeling platform for anti-tuberculosis drugs to predict phase IIA outcomes for BTZ-043 in humans and evaluate the impact of observed drug-drug interactions on the contribution of BTZ-043 to combotherapy in a mouse model. Methods The study utilized data from mouse experiments for BTZ-043 monotherapy and combotherapy with bedaquiline, pretomanid, and linezolid, and clinical information for BTZ-043 monotherapy. The translational models were applied to predict the colony-forming units as a measure of efficacy in humans treated with BTZ-043 monotherapy and evaluate the effect of BTZ-043 on the pharmacokinetics-pharmacodynamics of combotherapy bedaquiline, pretomanid, and linezolid. Results The mouse-pharmacokinetic and mouse-pharmacodynamic data for BTZ-043 monotherapy were best described by two-compartmental and direct Emax models, respectively. The model-based prediction of efficacy in humans was comparable to the observed phase IIA efficacy. Single-compartmental models, developed separately, best described the mouse-pharmacokinetic data for bedaquiline, pretomanid, and linezolid in combotherapy. Co-administration with BTZ-043 was associated with at least a 2-fold reduction in bedaquiline, pretomanid, and linezolid exposures in mice, and model-based simulations suggested that the observed decreases in exposure to these drugs would have resulted in even lower efficacy than what was observed when BPaL is co-administered with BTZ-043. Conclusion The translational modeling platform adequately predicted the efficacy of BTZ-043 monotherapy. In the absence of drug-drug interactions, co-administration of BTZ-043 with bedaquiline, pretomanid, and linezolid in combotherapy is predicted to improve treatment efficacy.

JSF-3285 is a promising preclinical candidate for tuberculosis that potently targets the Mycobacterium tuberculosis β-ketoacyl-ACP synthase KasA. In mouse models of acute, sub-acute, chronic, and relapse infection, JSF-3285 offers substantial activity in combination with bedaquiline and pretomanid, which could be applicable for both drug-sensitive and drug-resistant infections.

Isoniazid is an important first-line medicine to treat tuberculosis (TB). Isoniazid resistance increases the risk of poor treatment outcomes and development of multidrug resistance, and is driven primarily by mutations involving katG, encoding the prodrug-activating enzyme, rather than its validated target, InhA. The chemical tractability of InhA has fostered efforts to discover direct inhibitors of InhA (DIIs). In this study, we bridge the gap in understanding the potential contribution of DIIs to novel combination regimens and demonstrate a clear distinction of DIIs, like GSK693 and the newly described GSK138, from isoniazid, based on activity against clinical isolates and contribution to novel drug regimens. The results suggest that DIIs, specifically GSK138 and GSK693, could be promising partners in novel drug regimens, including those used against isoniazid-resistant TB, potentially enhancing their efficacy and/or preventing the selection of resistant mutants and supporting the continued exploration of InhA as a promising target for TB drug development.

Isoniazid is an important first-line medicine to treat tuberculosis (TB). Isoniazid resistance increases the risk of poor treatment outcomes and development of multidrug resistance, and is driven primarily by mutations involving katG , encoding the pro-drug activating enzyme, rather than its validated target, InhA. The chemical tractability of InhA has fostered efforts to discover direct inhibitors of InhA (DIIs). During the past five years, successful target engagement and in vivo efficacy have been demonstrated by diverse DIIs. In this study, we bridge the gap in understanding the potential contribution of DIIs to novel combination regimens and demonstrate a clear distinction of DIIs, like GSK693 and the newly described GSK138, from isoniazid, based on activity against clinical isolates and contribution to novel drug regimens. The results presented increase the understanding of DII mechanism of action and provide further impetus to continue exploiting InhA as a promising target for TB drug development.

The combination of bedaquiline, pretomanid, and linezolid (BPaL) has become a preferred regimen for treating multidrug- and extensively drug-resistant tuberculosis (TB). However, treatment-limiting toxicities of linezolid and reports of emerging bedaquiline and pretomanid resistance necessitate efforts to develop new short-course oral regimens. We recently found that the addition of GSK2556286 increases the bactericidal and sterilizing activity of BPa-containing regimens in a well-established BALB/c mouse model of tuberculosis. Here, we used this model to evaluate the potential of new regimens combining bedaquiline or the more potent diarylquinoline TBAJ-587 with GSK2556286 and the DprE1 inhibitor TBA-7371, all of which are currently in early-phase clinical trials. We found the combination of bedaquiline, GSK2556286, and TBA-7371 to be more active than the first-line regimen and nearly as effective as BPaL in terms of bactericidal and sterilizing activity. In addition, we found that GSK2556286 and TBA-7371 were as effective as pretomanid and the novel oxazolidinone TBI-223 when either drug pair was combined with TBAJ-587 and that the addition of GSK2556286 increased the bactericidal activity of the TBAJ-587, pretomanid, and TBI-223 combination. We conclude that GSK2556286 and TBA-7371 have the potential to replace pretomanid, an oxazolidinone, or both components, in combination with bedaquiline or TBAJ-587.

Abstract TBI-223, a novel oxazolidinone for tuberculosis, is designed to provide improved efficacy and safety compared to linezolid in combination with bedaquiline and pretomanid (BPaL). We aim to optimize the dosing of TBI-223 within the BPaL regimen for enhanced therapeutic outcomes. TBI-223 is investigated in preclinical monotherapy, multidrug therapy, and lesion penetration experiments to describe its efficacy and safety versus linezolid. A translational platform incorporating linezolid and BPaL data from preclinical experiments and 4 clinical trials (NCT00396084, NCT02333799, NCT03086486, NCT00816426) is developed, enabling validation of the framework. TBI-223 preclinical and Phase 1 data (NCT03758612) are applied to the translational framework to predict clinical outcomes and optimize TBI-223 dosing in combination with bedaquiline and pretomanid. Results indicate that daily doses of 1200–2400 mg TBI-223 may achieve efficacy comparable to the BPaL regimen, with >90% of patients predicted to reach culture conversion by two months.

The clinical efficacy of combination drug regimens containing the first-generation diarylquinoline (DARQ) bedaquiline in the treatment of multidrug-resistant tuberculosis has validated ATP synthesis as a vulnerable pathway in Mycobacterium tuberculosis. New DARQs in clinical development may be even more effective than bedaquiline, including against emerging bedaquiline-resistant strains. Telacebec (T) is a novel cytochrome bc(1):aa(3) oxidase inhibitor that also inhibits ATP synthesis. Based on its demonstrated efficacy as a monotherapy in mice and in a phase 2a clinical trial, we tested the contribution of T to novel combination therapies against two strains of M. tuberculosis (H37Rv and HN878) in an established BALB/c mouse model of tuberculosis in an effort to find more effective regimens. Overall, T was more effective in regimens against the HN878 strain than against the H37Rv strain, a finding supported by the greater vulnerability of the former strain to T and to genetic depletion of QcrB. Against both strains, combinations of a DARQ, clofazimine, and T were highly bactericidal. However, only against HN878 did T contribute synergistically, whereas an antagonistic effect was observed against H37Rv. These results demonstrate the therapeutic potential of T and highlight how differences in the susceptibility of M. tuberculosis strains could lead to different conclusions about a drug's potential contribution to novel drug regimens.

As a result of a high-throughput compound screening campaign of Mycobacterium tuberculosis infected macrophages, a new preclinical drug candidate for the treatment of tuberculosis has been identified. GSK2556286 inhibits growth within human macrophages (IC50 = 0.07 µM), is active against extracellular bacteria in cholesterol-containing culture media and exhibits no cross-resistance with known antitubercular drugs. In addition, it has shown efficacy in different mouse models of tuberculosis (TB) and has an adequate safety profile in two preclinical species. These features indicate a compound with a novel mode of action, although still not fully defined, that is effective against both multidrug or extensively-resistant (M/XDR) and drug-sensitive (DS) M. tuberculosis with the potential to shorten the duration of treatment in novel combination drug regimens. One Sentence Summary GSK2556286 is a novel preclinical drug candidate for the treatment of tuberculosis with a new mode of action potentially able to contribute to the shortening of TB chemotherapy.

BACKGROUND:Linezolid (LZD) is bactericidal against Mycobacterium tuberculosis, but it has treatment-limiting toxicities. A better understanding of exposure-response relationships governing LZD efficacy and toxicity will inform dosing strategies. Because in vitro monotherapy studies yielded conflicting results, we explored LZD pharmacokinetic/pharmacodynamic (PK/PD) relationships in vivo against actively and nonactively multiplying bacteria, including in combination with pretomanid.METHODS:Linezolid multidose pharmacokinetics were modeled in mice. Dose-fractionation studies were performed in acute (net bacterial growth) and chronic (no net growth) infection models. In acute models, LZD was administered alone or with bacteriostatic or bactericidal pretomanid doses. Correlations between PK/PD parameters and lung colony-forming units (CFUs) and complete blood counts were assessed.RESULTS:Overall, time above minimum inhibitory concentration (T>MIC) correlated best with CFU decline. However, in growth-constrained models (ie, chronic infection, coadministration with pretomanid 50 mg/kg per day), area under the concentration-time curve over MIC (AUC/MIC) had similar explanatory power. Red blood cell counts correlated strongly with LZD minimum concentration (Cmin).CONCLUSIONS:Although T>MIC was the most consistent correlate of efficacy, AUC/MIC was equally predictive when bacterial multiplication was constrained by host immunity or pretomanid. In effective combination regimens, administering the same total LZD dose less frequently may be equally effective and cause less Cmin-dependent toxicity.

Telacebec (Q203) is a new anti-tuberculosis drug in clinical development with extremely potent activity against Mycobacterium ulcerans , the causative agent of Buruli ulcer (BU). The potency of Q203 has prompted investigation of its potential role in ultra-short, even single-dose, treatment regimens for BU in mouse models. However, the relationships of Q203 dose and duration and host immune status to treatment outcomes remain unclear, as does the risk of emergence of drug resistance with Q203 monotherapy. In the present study, immunocompetent BALB/c and immunocompromised SCID-beige mice were infected in both hind footpads and treated eight weeks later. In both mouse strains, controls received rifampin-clarithromycin; others received Q203 at 0.5 or 2 mg/kg/d for 5 or 10 days. Additionally, BALB/c mice received a single dose of 2.5 or 10 mg/kg or 3.3 mg/kg/d for 3 days. Treatment response was based on changes in footpad swelling and CFU counts at the end of treatment as well as 4 and 13 weeks after stopping treatment. Efficacy depended on total dose more than duration. Total doses of 5-20 mg/kg rendered nearly all BALB/c mice culture-negative 13 weeks post-treatment without selection of Q203-resistant bacteria. Although less potent in SCID-beige mice, Q203 still rendered the majority of footpads culture-negative at total doses of 10-20 mg/kg. However, Q203 resistance was identified in relapse isolates from some SCID-beige mice. Overall, these results support the potential of Q203 monotherapy for single-dose or other ultra-short therapy for BU, although highly immunocompromised hosts may require higher doses or durations and/or combination therapy.

Tuberculosis (TB) drug, regimen, and vaccine development rely heavily on preclinical animal experiments, and quantification of bacterial and immune response dynamics is essential for understanding drug and vaccine efficacy. A mechanism-based model was built to describe Mycobacterium tuberculosis H37Rv infection over time in BALB/c and athymic nude mice, which consisted of bacterial replication, bacterial death, and adaptive immune effects. The adaptive immune effect was best described by a sigmoidal function on both bacterial load and incubation time. Applications to demonstrate the utility of this baseline model showed (i) the important influence of the adaptive immune response on pyrazinamide (PZA) drug efficacy, (ii) a persistent adaptive immune effect in mice relapsing after chemotherapy cessation, and (iii) the protective effect of vaccines after M. tuberculosis challenge. These findings demonstrate the utility of our model for describing M. tuberculosis infection and corresponding adaptive immune dynamics for evaluating the efficacy of TB drugs, regimens, and vaccines.

New drugs are required to counter the tuberculosis (TB) pandemic. Here, we describe the synthesis and characterization of 1,3-benzothiazin-4-ones (BTZs), a new class of antimycobacterial agents that kill Mycobacterium tuberculosis in vitro, ex vivo, and in mouse models of TB. Using genetics and biochemistry, we identified the enzyme decaprenylphosphorylβ-D-ribose 2′-epimerase as a major BTZ target. Inhibition of this enzymatic activity abolishes the formation of decaprenylphosphoryl arabinose, a key precursor that is required for the synthesis of the cell-wall arabinans, thus provoking cell lysis and bacterial death. The most advanced compound, BTZ043, is a candidate for inclusion in combination therapies for both drug-sensitive and extensively drug-resistant TB. The loss of human lives to tuberculosis (TB) continues essentially unabated as a result of poverty, synergy with the HIV/AIDS pandemic, and the emergence of multidrugand extensively drug-resistant strains of Mycobacterium tuberculosis (1-3). Despite some recent successes, such as the discovery of the diarylquinoline drug TMC207 (4) and the promise of the bicyclic nitroimidazole compounds (5-8), and because of the high attrition rate in drug development (9), much greater effort is required to find better drugs in order to meet the Europe PMC Funders Group Author Manuscript Science. Author manuscript; available in PMC 2011 July 01. Published in final edited form as: Science. 2009 May 8; 324(5928): 801–804. doi:10.1126/science.1171583. Copyright 2009 by the American Association for the Advancement of Science; all rights reserved. E uope PM C Fuders A uhor M ancripts E uope PM C Fuders A uhor M ancripts desired goals of killing persistent tubercle bacilli and reducing TB treatment duration from 6 to less than 3 months (10, 11). A series of sulfur-containing heterocycles was synthesized and tested for antibacterial and antifungal activity (12, 13). Among their derivatives, compounds belonging to the nitrobenzothiazinone (BTZ) class showed particular promise in terms of their potency and specificity for mycobacteria. One of them, 2-[2-methyl-1,4-dioxa-8-azaspiro[4.5]dec-8yl]-8-nitro-6-(trifluoromethyl)-4H-1,3-benzothiazin-4-one (BTZ038), was selected for further studies. This compound (series number 10526038; C17H16F3N3O5S, with a molecular weight of 431.4; logP =2.84) (Fig. 1A) was synthesized in seven steps with a yield of 36%. Structure activity relationship work showed that the sulfur atom and the nitro group at positions 1 and 8, respectively, were critical for activity. BTZ038 has a single chiral center, and both enantiomers, BTZ043 (S) and BTZ044 (R), were found to be equipotent in vitro. Because early metabolic studies with bacteria or mice indicated that the nitro group could be reduced to an amino group, and because many TB drugs are prodrugs that require activation by M. tuberculosis (14), the S and R enantiomers of the amino derivatives and the likely hydroxylamine intermediate were synthesized and tested for antimycobacterial activity in vitro (table S1). The amino (BTZ045, S and R) and hydroxylamine (BTZ046) derivatives were substantially less active (500to 5000-fold). The minimal inhibitory concentrations (MICs) of a variety of BTZs against different mycobacteria were very low, ranging from ~0.1 to 80 ng/ml for fast growers and from 1 to 30 ng/ml for members of the M. tuberculosis complex (13). The MIC of BTZ043 against M. tuberculosis H37Rv and Mycobacterium smegmatis were 1 ng/ml (2.3 nM) and 4 ng/ml (9.2 nM), respectively (Table 1), which compares favorably with those of the existing TB drugs isoniazid (INH) (0.02 to 0.2 μg/ml) and ethambutol (EMB) (1 to 5 μg/ml) (14). From structure activity relationship studies, >30 different BTZ derivatives showed MICs of <50 ng/ml against tubercle bacilli (examples are shown in table S2). Crucially, BTZ043 displayed similar activity against all clinical isolates of M. tuberculosis that were tested, including multidrug-resistant and extensively drug-resistant strains, indicating that it targets a previously unknown biological function (table S3). BTZ043 is bactericidal, reducing viability in vitro by more than 1000-fold in under 72 hours (Fig. 1B), which is comparable to the killing effect seen with INH. In two different model systems (auxotrophy and starvation) involving metabolically inert M. tuberculosis, BTZ043 was less effective, which implies that it blocks a step in active metabolism, similar to INH (14). Observation with time-lapse fluorescence microscopy of individual M. smegmatis cells [expressing green fluorescent protein (GFP)] growing in a microfluidic device (15) revealed that upon exposure to BTZ043, the growth rate decreased rapidly followed by a swelling of the poles and lysis of the cells after a few hours (movie S1). M. tuberculosis showed similar but delayed behavior (movie S2 and fig. S1). Comparative transcriptome analysis of M. tuberculosis offered no evidence of mutagenic or nitrosative gene expression signatures after treatment with BTZ043, although expression of 60 genes was induced (table S4), and this was corroborated with proteomics. The transcriptional signature most resembled that generated by the cell wall inhibitors INH, isoxyl, and ethionamide, with the greatest overlap seen with the response to EMB treatment (16, 17). This is consistent with cell lysis and indicated that BTZ targets cell wall biogenesis. We then tested the uptake, intracellular killing, and potential cytotoxicity of BTZ compounds in an ex vivo model using a high-content screening approach (18, 19) in order to monitor macrophages infected with M. tuberculosis expressing GFP. Macrophages treated Makarov et al. Page 2 Science. Author manuscript; available in PMC 2011 July 01. E uope PM C Fuders A uhor M ancripts E uope PM C Fuders A uhor M ancripts with BTZ043 were protected (fig. S2) as compared with those treated with the amino derivative BTZ045 or the negative controls [dimethyl sulfoxide (DMSO)]. The deduced MIC of BTZ043 was <10 ng/ml, indicating that this compound is more potent than INH (100 ng/ml) and rifampin (>1 μg/ml) against intracellular bacteria (Fig. 1C). In contrast, the amino metabolite BTZ045 had an MIC of >1μg/ml, which is consistent with the in vitro findings (table S1). As a direct correlate of the antibacterial effect, there was extensive macrophage survival when all compounds were used at doses well above the MIC. BTZ043 was more cytotoxic than INH (Fig. 1C) at the highest concentration tested (10 μg/ml) but nonetheless has a favorable selectivity index of >100. Additional in vitro toxicology tests revealed no particularly unfavorable effects (table S4). The in vivo efficacy of BTZ043 was assessed 4 weeks after a low-dose aerosol infection of BALB/c mice in the chronic model of TB. Four weeks of treatment with BTZ043 reduced the bacterial burden in the lungs and spleens by 1 and 2 logs, respectively, at the concentrations used (Fig. 1D). Additional results suggest that BTZ efficacy is timerather than dose-dependent. Acute (5 g/kg) and chronic (25 and 250 mg/kg) toxicology studies in uninfected mice showed that, even at the highest dose tested, there were no adverse anatomical, behavioral, or physiological effects after one month (table S5). To find the target for BTZ, we employed two independent genetic approaches. First, we identified cosmids bearing DNA from M. smegmatis that confer increased resistance on M. smegmatis, and we pinpointed the region responsible by subcloning. Second, we isolated and characterized mutants of M. smegmatis, M. bovis Bacille Calmette-Guérin (BCG), and M. tuberculosis displaying high-level BTZ resistance. The first approach revealed that the MSMEG_6382 gene of M. smegmatis or its M. tuberculosis ortholog rv3790 mediated increased resistance (Fig. 2A), whereas the second showed that drug-resistant mutants harbor missense mutations in the same gene (Table 1). Biochemical studies showed that rv3790 and the neighboring gene rv3791 code for proteins that act in concert to catalyze the epimerization of decaprenylphosphoryl ribose (DPR) to decaprenylphosphoryl arabinose (DPA) (20), a precursor for arabinan synthesis without which a complete mycobacterial cell wall cannot be produced. These essential membrane-associated enzymes (20-23) have been suggested to act as decaprenylphosphoryl-β-D-ribose oxidase and decaprenylphosphoryl-D-2keto erythro pentose reductase, respectively, and we propose naming them DprE1 and DprE2. In all of the drug-resistant mutants we examined, the same codon of rv3790 (dprE1) was affected, in which Cys387 was replaced by Ser or Gly codons, respectively (Table 1). Mutants, harboring alleles such as those in MN47 or MN84, were rare, arising at a frequency of <10−8, and were dominant over the wild-type gene upon introduction into a BTZ-susceptible mycobacterium. Comparative genomics revealed that the BTZ resistance– determining region of rv3790 was highly conserved in orthologous genes from various actinobacteria, except that in a few cases Cys387 was replaced by Ser or Ala (Fig. 2B). The corresponding bacteria, M. avium and M. aurum, were found to be naturally resistant to BTZ (table S3), thus supporting the identification of DprE1/Rv3790 as the target. Further corroboration was obtained biochemically (Fig. 3) by using membrane preparations from M. smegmatis to catalyze the epimerization reaction from radiolabeled DPR precursor, which was produced in situ from 5-phosphoribose diphosphate (20), in the presence or absence of BTZ. Addition of BTZ038, or its enantiomers BTZ043 and BTZ044, abolished the production of DPA from DPR. This reaction was scarcely affected by either the S or R forms of BTZ045 (Fig. 3A) or by BTZ046 (fig. S3). Using recombinant proteins, we found that the reaction requires both DprE1 and E2 (Rv3790 and Rv3791) (Fig. 3B), with neither enzyme alone capable of catalyzing DPA formation. Furthermore, when BN2, the highly

Buruli ulcer is treatable with antibiotics. An 8-week course of rifampin (RIF) and either streptomycin (STR) or clarithromycin (CLR) cures over 90% of patients. ABSTRACT Buruli ulcer is treatable with antibiotics. An 8-week course of rifampin (RIF) and either streptomycin (STR) or clarithromycin (CLR) cures over 90% of patients. However, STR requires injections and may be toxic, and CLR shares an adverse drug-drug interaction with RIF and may be poorly tolerated. Studies in a mouse footpad infection model showed that increasing the dose of RIF or using the long-acting rifamycin rifapentine (RPT), in combination with clofazimine (CFZ), a relatively well-tolerated antibiotic, can shorten treatment to 4 weeks. CFZ is reduced by a component of the electron transport chain (ETC) to produce reactive oxygen species toxic to bacteria. Synergistic activity of CFZ with other ETC-targeting drugs, the ATP synthase inhibitor bedaquiline (BDQ) and the bc1:aa3 oxidase inhibitor Q203 (now named telacebec), was recently described against Mycobacterium tuberculosis. Recognizing that M. tuberculosis mutants lacking the alternative bd oxidase are hypersusceptible to Q203 and that Mycobacterium ulcerans is a natural bd oxidase-deficient mutant, we tested the in vitro susceptibility of M. ulcerans to Q203 and evaluated the treatment-shortening potential of novel 3- and 4-drug regimens combining RPT, CFZ, Q203, and/or BDQ in a mouse footpad model. The MIC of Q203 was extremely low (0.000075 to 0.00015 μg/ml). Footpad swelling decreased more rapidly in mice treated with Q203-containing regimens than in mice treated with RIF and STR (RIF+STR) and RPT and CFZ (RPT+CFZ). Nearly all footpads were culture negative after only 2 weeks of treatment with regimens containing RPT, CFZ, and Q203. No relapse was detected after only 2 weeks of treatment in mice treated with any of the Q203-containing regimens. In contrast, 15% of mice receiving RIF+STR for 4 weeks relapsed. We conclude that it may be possible to cure patients with Buruli ulcer in 14 days or less using Q203-containing regimens rather than currently recommended 56-day regimens.

The potent antituberculosis activity and long half-life of bedaquiline make it an attractive candidate for use in long-acting/extended-release formulations for the treatment of latent tuberculosis infection (LTBI). Our objective was to evaluate a long-acting injectable (LAI) bedaquiline formulation in a validated paucibacillary mouse model of LTBI.

Despite the existence of the prophylactic Bacille Calmette-Guérin (BCG) vaccine, infection by Mycobacterium tuberculosis (Mtb) remains a major public health issue causing up to 1.8 million annual deaths worldwide. Increasing prevalence of Mtb strains resistant to antibiotics represents an urgent threat for global health that has prompted a search for alternative treatment regimens not subject to development of resistance. Immunotherapy constitutes a promising approach to improving current antibiotic treatments through engagement of the host's immune system. We designed a multi-antigenic and multiphasic vaccine, based on the Modified Vaccinia Ankara (MVA) virus, denoted MVATG18598, which expresses ten antigens classically described as representative of each of different phases of Mtb infection. In vitro analysis coupled with multiple-passage evaluation demonstrated that this vaccine is genetically stable, i.e. fit for manufacturing. Using different mouse strains, we show that MVATG18598 vaccination results in both Th1-associated T-cell responses and cytolytic activity, targeting all 10 vaccine-expressed Mtb antigens. In chronic post-exposure mouse models, MVATG18598 vaccination in combination with an antibiotic regimen decreases the bacterial burden in the lungs of infected mice, compared with chemotherapy alone, and is associated with long-lasting antigen-specific Th1-type T cell and antibody responses. In one model, co-treatment with MVATG18598 prevented relapse of the disease after treatment completion, an important clinical goal. Overall, results demonstrate the capacity of the therapeutic MVATG18598 vaccine to improve efficacy of chemotherapy against TB. These data support further development of this novel immunotherapeutic in the treatment of Mtb infections.

The antileprosy drug clofazimine was recently repurposed as part of a newly endorsed short-course regimen for multidrug-resistant tuberculosis. It also enables significant treatment shortening when added to the first-line regimen for drug-susceptible tuberculosis in a mouse model. However, clofazimine causes dose- and duration-dependent skin discoloration in patients, and the optimal clofazimine dosing strategy in the context of the first-line regimen is unknown. We utilized a well-established mouse model to systematically address the impacts of duration, dose, and companion drugs on the treatment-shortening activity of clofazimine in the first-line regimen. In all studies, the primary outcome was relapse-free cure (culture-negative lungs) 6 months after stopping treatment, and the secondary outcome was bactericidal activity, i.e., the decline in the lung bacterial burden during treatment. Our findings indicate that clofazimine activity is most potent when coadministered with first-line drugs continuously throughout treatment and that equivalent treatment-shortening results are obtained with half the dose commonly used in mice. However, our studies also suggest that clofazimine at low exposures may have negative impacts on treatment outcomes, an effect that was evident only after the first 3 months of treatment. These data provide a sound evidence base to inform clofazimine dosing strategies to optimize the antituberculosis effect while minimizing skin discoloration. The results also underscore the importance of conducting long-term studies to allow the full evaluation of drugs administered in combination over long durations.