Contribution of direct InhA inhibitors to novel drug regimens in a mouse model of tuberculosis.

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.

Dose optimization of TBI-223 for enhanced therapeutic benefit compared to linezolid in antituberculosis regimen.

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.

Prevalence of unexpected red blood cell antibodies in pregnant women and follow-up of pregnancy outcome in pregnant women treated with intra-uterine transfusion.

BACKGROUND: For the management of hemolytic disease of the fetus and newborn (HDFN), it is important to detect unexpected red cell antibody in pregnant women. We assessed the prevalence of unexpected red cell antibodies in consecutive pregnant women attending antenatal clinic (ANC). More importantly, cases with unexpected antibody causing severe anemia were followed-up for intervention (Intra-uterine transfusion {IUT}) and outcome of pregnancy (still-birth/live-healthy). AIMS AND OBJECTIVES: The study was conducted with an objective to find the prevalence of unexpected RBC antibodies in pregnant women, their specificity and to do the follow-up for IUT and outcome of pregnancy (still-birth, live-birth) in antibody positive women. MATERIALS AND METHODS: This was a prospective study from January 2021 to May 2022 at two tertiary care centres. All antenatal samples received by the laboratory were screened for unexpected red cell antibody. Whenever antibody screen was positive, antibody identification was performed. Patients, positive for unexpected antibody and anemia were followed up for any transfusion-based intervention and outcome of pregnancy. RESULTS: A total of 539 consecutive samples were worked up and among these, 10 samples (1.85%) were found to be antibody positive. The antibodies identified were Anti-D (n=6), anti-Leb (n=1), anti-M (n=1), anti-C (n=1) and anti-E (n=1).The prevalence of unexpected antibodies in Rh positive and Rh negative pregnant women was 0.83% and 10.9% respectively. Follow-up was done for all 10 cases with unexpected antibody and anemia was monitored by MCA PSV (middle cerebral artery peak systolic velocity).Two women developed severe anemia thus requiring single intrauterine transfusion (at 26 weeks and 28 weeks respectively) each, for correction of anemia. In both these cases, healthy male child was delivered. At 3-month follow-up both children were alive and healthy. CONCLUSION: The study found prevalence of unexpected RBC antibodies in pregnant women as 1.85%. The study also underlined importance of transfusion-based interventions contributing to successful outcome in couple of cases with severe anemia.

Bactericidal and sterilizing activity of novel regimens combining bedaquiline or TBAJ-587 with GSK2556286 and TBA-7371 in a mouse model of tuberculosis.

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.

Renormalized charge and dielectric effects in colloidal interactions: a numerical solution of the nonlinear Poisson-Boltzmann equation for unknown boundary conditions.

 The Derjaguin-Landau-Verwey-Overbeek (DLVO) theory, introduced more than 70 years ago, is a hallmark of colloidal particle modeling. For highly charged particles in the dilute regime, it is often supplemented by Alexander's prescription (Alexander et al. in J Chem Phys 80:5776, 1984) for using a renormalized charge. Here, we solve the problem of the interaction between two charged colloids at finite ionic strength, including dielectric mismatch effects, using an efficient numerical scheme to solve the nonlinear Poisson-Boltzmann (NPB) equation with unknown boundary conditions. Our results perfectly match the analytical predictions for the renormalized charge by Trizac and coworkers (Aubouy et al. in J Phys A 36:5835, 2003). Moreover, they allow us to reinterpret previous molecular dynamics (MD) simulation results by Kreer et al. (Phys Rev E 74:021401, 2006), rendering them now in agreement with the expected behavior. We furthermore find that the influence of polarization becomes important only when the Debye layers overlap significantly.

Discovery of natural-product-derived sequanamycins as potent oral anti-tuberculosis agents.

The emergence of drug-resistant tuberculosis has created an urgent need for new anti-tubercular agents. Here, we report the discovery of a series of macrolides called sequanamycins with outstanding in vitro and in vivo activity against Mycobacterium tuberculosis (Mtb). Sequanamycins are bacterial ribosome inhibitors that interact with the ribosome in a similar manner to classic macrolides like erythromycin and clarithromycin, but with binding characteristics that allow them to overcome the inherent macrolide resistance of Mtb. Structures of the ribosome with bound inhibitors were used to optimize sequanamycin to produce the advanced lead compound SEQ-9. SEQ-9 was efficacious in mouse models of acute and chronic TB as a single agent, and it demonstrated bactericidal activity in a murine TB infection model in combination with other TB drugs. These results support further investigation of this series as TB clinical candidates, with the potential for use in new regimens against drug-susceptible and drug-resistant TB.

GSK2556286 Is a Novel Antitubercular Drug Candidate Effective In Vivo with the Potential To Shorten Tuberculosis Treatment.

As a result of a high-throughput compound screening campaign using Mycobacterium tuberculosis-infected macrophages, a new drug candidate for the treatment of tuberculosis has been identified. GSK2556286 inhibits growth within human macrophages (50% inhibitory concentration [IC50] = 0.07 µM), is active against extracellular bacteria in cholesterol-containing culture medium, 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-resistant (MDR) or extensively drug-resistant (XDR) and drug-sensitive (DS) M. tuberculosis with the potential to shorten the duration of treatment in novel combination drug regimens. (This study has been registered at ClinicalTrials.gov under identifier NCT04472897).

Pharmacodynamic Correlates of Linezolid Activity and Toxicity in Murine Models of Tuberculosis.

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 for Ultrashort Treatment of Buruli Ulcer in a Mouse Model.

Telacebec (Q203) is a new antitubercular drug with extremely potent activity against Mycobacterium ulcerans Here, we explored the treatment-shortening potential of Q203 alone or in combination with rifampin (RIF) in a mouse footpad infection model. The first study compared Q203 at 5 and 10 mg/kg doses alone and with rifampin. Q203 alone rendered most mouse footpads culture negative in 2 weeks. Combining Q203 with rifampin resulted in a relapse-free cure 24 weeks after completing 2 weeks of treatment, compared to a 25% relapse rate in mice receiving RIF with clarithromycin, the current standard of care, for 4 weeks. The second study explored the dose-ranging activity of Q203 alone and with RIF, including the extended activity of Q203 after treatment discontinuation. The bactericidal activity of Q203 persisted for ≥ 4 weeks beyond the last dose. All mice receiving just 1 week of Q203 at 2 to 10 mg/kg were culture negative 4 weeks after stopping treatment. Mice receiving 2 weeks of Q203 at 0.5, 2, and 10 mg/kg were culture negative 4 weeks after treatment. RIF did not increase the efficacy of Q203. A pharmacokinetics substudy revealed that Q203 doses of 2 to 10 mg/kg in mice produce plasma concentrations similar to those produced by 100 to 300 mg doses in humans, with no adverse effect of RIF on Q203 concentrations. These results indicate the extraordinary potential of Q203 to reduce the duration of treatment necessary for a cure to ≤ 1 week (or 5 doses of 2 to 10 mg/kg) in our mouse footpad infection model and warrant further evaluation of Q203 in clinical trials.

Mechanistic Modeling of Mycobacterium tuberculosis Infection in Murine Models for Drug and Vaccine Efficacy Studies.

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.

Shortening Buruli Ulcer Treatment with Combination Therapy Targeting the Respiratory Chain and Exploiting Mycobacterium ulcerans Gene Decay.

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.

Activity of a Long-Acting Injectable Bedaquiline Formulation in a Paucibacillary Mouse Model of Latent Tuberculosis Infection.

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. Following immunization with Mycobacterium bovis rBCG30, BALB/c mice were challenged by aerosol infection with M. tuberculosis H37Rv. Treatment began 13 weeks after challenge infection with one of the following regimens: an untreated negative-control regimen; positive-control regimens of daily rifampin (10 mg/kg of body weight), once-weekly rifapentine (15 mg/kg) and isoniazid (50 mg/kg), or daily bedaquiline (25 mg/kg); test regimens of one, two, or three monthly doses of LAI bedaquiline at 160 mg/dose (BLAI-160); and test regimens of daily bedaquiline at 2.67 mg/kg (B2.67), 5.33 mg/kg (B5.33), or 8 mg/kg (B8) to deliver the same total amount of bedaquiline as one, two, or three doses of BLAI-160, respectively. All drugs were administered orally, except for BLAI-160 (intramuscular injection). The primary outcome was the decline in M. tuberculosis lung CFU counts during 12 weeks of treatment. The negative- and positive-control regimens performed as expected. One, two, and three doses of BLAI-160 resulted in decreases of 2.9, 3.2, and 3.5 log10 CFU/lung, respectively, by week 12. Daily oral dosing with B2.67, B5.33, and B8 decreased lung CFU counts by 1.6, 2.8, and 4.1 log10, respectively. One dose of BLAI-160 exhibited activity for at least 12 weeks. The sustained activity of BLAI-160 indicates that it shows promise as a short-course LTBI treatment requiring few patient encounters to ensure treatment completion.

Shorter-course treatment for Mycobacterium ulcerans disease with high-dose rifamycins and clofazimine in a mouse model of Buruli ulcer.

TRIAL REGISTRATION: ClinicalTrials.gov NCT03474198, NCT01659437.

Impact of Clofazimine Dosing on Treatment Shortening of the First-Line Regimen in a Mouse Model of Tuberculosis.

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.

A multi-antigenic MVA vaccine increases efficacy of combination chemotherapy against Mycobacterium tuberculosis.

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.

Bactericidal and Sterilizing Activity of a Novel Regimen with Bedaquiline, Pretomanid, Moxifloxacin, and Pyrazinamide in a Murine Model of Tuberculosis.

New regimens based on 2 or more novel agents are sought to shorten or to simplify treatment of tuberculosis (TB), including drug-resistant forms. Prior studies showed that the novel combinations of bedaquiline (BDQ) plus pretomanid (PMD) plus pyrazinamide (PZA) and PMD plus moxifloxacin (MXF) plus PZA shortened the treatment duration necessary to prevent relapse by 2 to 3 months and 1 to 2 months, respectively, compared with the current first-line regimen, in a murine TB model. These 3-drug combinations are now being studied in clinical trials. Here, the 4-drug combination of BDQ+PMD+MXF+PZA was compared to its 3-drug component regimens and different treatment durations of PZA and MXF were explored, to identify the optimal regimens and treatment times and to estimate the likelihood of success against drug-resistant strains. BDQ+PMD+MXF+PZA rendered all mice relapse-free after 2 months of treatment. PZA administration could be discontinued after the first month of treatment without worsening outcomes, whereas the absence of MXF, PZA, or BDQ administration from the beginning necessitated approximately 0.5, 1, or 2 months, respectively, of additional treatment to attain the same outcome.

Mutations in pepQ Confer Low-Level Resistance to Bedaquiline and Clofazimine in Mycobacterium tuberculosis.

The novel ATP synthase inhibitor bedaquiline recently received accelerated approval for treatment of multidrug-resistant tuberculosis and is currently being studied as a component of novel treatment-shortening regimens for drug-susceptible and multidrug-resistant tuberculosis. In a limited number of bedaquiline-treated patients reported to date, ≥4-fold upward shifts in bedaquiline MIC during treatment have been attributed to non-target-based mutations in Rv0678 that putatively increase bedaquiline efflux through the MmpS5-MmpL5 pump. These mutations also confer low-level clofazimine resistance, presumably by a similar mechanism. Here, we describe a new non-target-based determinant of low-level bedaquiline and clofazimine cross-resistance in Mycobacterium tuberculosis: loss-of-function mutations in pepQ (Rv2535c), which corresponds to a putative Xaa-Pro aminopeptidase. pepQ mutants were selected in mice by treatment with clinically relevant doses of bedaquiline, with or without clofazimine, and were shown to have bedaquiline and clofazimine MICs 4 times higher than those for the parental H37Rv strain. Coincubation with efflux inhibitors verapamil and reserpine lowered bedaquiline MICs against both mutant and parent strains to a level below the MIC against H37Rv in the absence of efflux pump inhibitors. However, quantitative PCR (qPCR) revealed no significant differences in expression of Rv0678, mmpS5, or mmpL5 between mutant and parent strains. Complementation of a pepQ mutant with the wild-type gene restored susceptibility, indicating that loss of PepQ function is sufficient for reduced susceptibility both in vitro and in mice. Although the mechanism by which mutations in pepQ confer bedaquiline and clofazimine cross-resistance remains unclear, these results may have clinical implications and warrant further evaluation of clinical isolates with reduced susceptibility to either drug for mutations in this gene.

Contribution of Oxazolidinones to the Efficacy of Novel Regimens Containing Bedaquiline and Pretomanid in a Mouse Model of Tuberculosis.

New regimens based on two or more novel agents are sought to shorten or simplify treatment of tuberculosis (TB). Pretomanid (PMD) is a nitroimidazole in phase 3 trials that has significant bactericidal activity alone and in combination with bedaquiline (BDQ) and/or pyrazinamide (PZA). We previously showed that the novel combination of BDQ+PMD plus the oxazolidinone sutezolid (SZD) had sterilizing activity superior to that of the first-line regimen in a murine model of TB. The present experiments compared the activity of different oxazolidinones in combination with BDQ+PMD with or without PZA in the same model. The 3-drug regimen of BDQ+PMD plus linezolid (LZD) had sterilizing activity approaching that of BDQ+PMD+SZD and superior to that of the first-line regimen. The addition of PZA further enhanced activity. Reducing the duration of LZD to 1 month did not significantly affect the activity of the regimen. Halving the LZD dose or replacing LZD with RWJ-416457 modestly reduced activity over the first month but not after 2 months. AZD5847 and tedizolid also increased the bactericidal activity of BDQ+PMD, but they were less effective than the other oxazolidinones. These results provide optimism for safe, short-course oral regimens for drug-resistant TB that may also be superior to the current first-line regimen for drug-susceptible TB.

Efficacy of Rifampin Plus Clofazimine in a Murine Model of Mycobacterium ulcerans Disease.

Treatment of Buruli ulcer, or Mycobacterium ulcerans disease, has shifted from surgical excision and skin grafting to antibiotic therapy usually with 8 weeks of daily rifampin (RIF) and streptomycin (STR). Although the results have been highly favorable, administration of STR requires intramuscular injection and carries the risk of side effects, such as hearing loss. Therefore, an all-oral, potentially less toxic, treatment regimen has been sought and encouraged by the World Health Organization. A combination of RIF plus clarithromycin (CLR) has been successful in patients first administered RIF+STR for 2 or 4 weeks. Based on evidence of efficacy of clofazimine (CFZ) in humans and mice with tuberculosis, we hypothesized that the combination of RIF+CFZ would be effective against M. ulcerans in the mouse footpad model of M. ulcerans disease because CFZ has similar MIC against M. tuberculosis and M. ulcerans. For comparison, mice were also treated with the gold standard of RIF+STR, the proposed RIF+CLR alternative regimen, or CFZ alone. Treatment was initiated after development of footpad swelling, when the bacterial burden was 4.64±0.14log10 CFU. At week 2 of treatment, the CFU counts had increased in untreated mice, remained essentially unchanged in mice treated with CFZ alone, decreased modestly with either RIF+CLR or RIF+CFZ, and decreased substantially with RIF+STR. At week 4, on the basis of footpad CFU counts, the combination regimens were ranked as follows: RIF+STR>RIF+CLR>RIF+CFZ. At weeks 6 and 8, none of the mice treated with these regimens had detectable CFU. Footpad swelling declined comparably with all of the combination regimens, as did the levels of detectable mycolactone A/B. In mice treated for only 6 weeks and followed up for 24 weeks, there were no relapses in RIF+STR treated mice, one (5%) relapse in RIF+CFZ-treated mice, but >50% in RIF+CLR treated mice. On the basis of these results, RIF+CFZ has potential as a continuation phase regimen for treatment of M. ulcerans disease.

Antibiotics. Targeting DnaN for tuberculosis therapy using novel griselimycins.

The discovery of Streptomyces-produced streptomycin founded the age of tuberculosis therapy. Despite the subsequent development of a curative regimen for this disease, tuberculosis remains a worldwide problem, and the emergence of multidrug-resistant Mycobacterium tuberculosis has prioritized the need for new drugs. Here we show that new optimized derivatives from Streptomyces-derived griselimycin are highly active against M. tuberculosis, both in vitro and in vivo, by inhibiting the DNA polymerase sliding clamp DnaN. We discovered that resistance to griselimycins, occurring at very low frequency, is associated with amplification of a chromosomal segment containing dnaN, as well as the ori site. Our results demonstrate that griselimycins have high translational potential for tuberculosis treatment, validate DnaN as an antimicrobial target, and capture the process of antibiotic pressure-induced gene amplification.