Mtb-Selective 5-Aminomethyl Oxazolidinone Prodrugs: Robust Potency and Potential Liabilities.

Linezolid is a drug with proven human antitubercular activity whose use is limited to highly drug-resistant patients because of its toxicity. This toxicity is related to its mechanism of action─linezolid inhibits protein synthesis in both bacteria and eukaryotic mitochondria. A highly selective and potent series of oxazolidinones, bearing a 5-aminomethyl moiety (in place of the typical 5-acetamidomethyl moiety of linezolid), was identified. Linezolid-resistant mutants were cross-resistant to these molecules but not vice versa. Resistance to the 5-aminomethyl molecules mapped to an N-acetyl transferase (Rv0133) and these mutants remained fully linezolid susceptible. Purified Rv0133 was shown to catalyze the transformation of the 5-aminomethyl oxazolidinones to their corresponding N-acetylated metabolites, and this transformation was also observed in live cells of Mycobacterium tuberculosis. Mammalian mitochondria, which lack an appropriate N-acetyltransferase to activate these prodrugs, were not susceptible to inhibition with the 5-aminomethyl analogues. Several compounds that were more potent than linezolid were taken into C3HeB/FeJ mice and were shown to be highly efficacious, and one of these (9) was additionally taken into marmosets and found to be highly active. Penetration of these 5-aminomethyl oxazolidinone prodrugs into caseum was excellent. Unfortunately, these compounds were rapidly converted into the corresponding 5-alcohols by mammalian metabolism which retained antimycobacterial activity but resulted in substantial mitotoxicity.

Drug distribution and efficacy of the DprE1 inhibitor BTZ-043 in the C3HeB/FeJ mouse tuberculosis model.

BTZ-043, a suicide inhibitor of the Mycobacterium tuberculosis cell wall synthesis decaprenylphosphoryl-beta-D-ribose 2' epimerase, is under clinical development as a potential new anti-tuberculosis agent. BTZ-043 is potent and bactericidal in vitro but has limited activity against non-growing bacilli in rabbit caseum. To better understand its behavior in vivo, BTZ-043 was evaluated for efficacy and spatial drug distribution as a single agent in the C3HeB/FeJ mouse model presenting with caseous necrotic pulmonary lesions upon Mycobacterium tuberculosis infection. BTZ-043 promoted significant reductions in lung and spleen bacterial burdens in the C3HeB/FeJ mouse model after 2 months of therapy. BTZ-043 penetrates cellular and necrotic lesions and was retained at levels above the serum-shifted minimal inhibitory concentration in caseum. The calculated rate of kill was found to be highest and dose-dependent during the second month of treatment. BTZ-043 treatment was associated with improved histology scores of pulmonary lesions, especially compared to control mice, which experienced advanced fulminant neutrophilic alveolitis in the absence of treatment. These positive treatment responses to BTZ-043 monotherapy in a mouse model of advanced pulmonary disease can be attributed to favorable distribution in tissues and lesions, retention in the caseum, and its high potency and bactericidal nature at drug concentrations achieved in necrotic lesions.

Lung microenvironments harbor Mycobacterium tuberculosis phenotypes with distinct treatment responses.

Tuberculosis lung lesions are complex and harbor heterogeneous microenvironments that influence antibiotic effectiveness. Major strides have been made recently in understanding drug pharmacokinetics in pulmonary lesions, but the bacterial phenotypes that arise under these conditions and their contribution to drug tolerance are poorly understood. A pharmacodynamic marker called the RS ratio® quantifies ongoing rRNA synthesis based on the abundance of newly synthesized precursor rRNA relative to mature structural rRNA. Application of the RS ratio in the C3HeB/FeJ mouse model demonstrated that Mycobacterium tuberculosis populations residing in different tissue microenvironments are phenotypically distinct and respond differently to drug treatment with rifampin, isoniazid, or bedaquiline. This work provides a foundational basis required to address how anatomic and pathologic microenvironmental niches may contribute to long treatment duration and drug tolerance during the treatment of human tuberculosis.

ADP-ribosylation-resistant rifabutin analogs show improved bactericidal activity against drug-tolerant M. abscessus in caseum surrogate.

Necrotic lesions and cavities filled with caseum are a hallmark of mycobacterial pulmonary disease. Bronchocavitary Mycobacterium abscessus disease is associated with poor treatment outcomes. In caseum surrogate, M. abscessus entered an extended stationary phase showing tolerance to killing by most current antibiotics, suggesting that caseum persisters contribute to the poor performance of available treatments. Novel ADP-ribosylation-resistant rifabutin analogs exhibited bactericidal activity against these M. abscessus persisters at concentrations achievable by rifamycins in caseum.

Investigation into the Mechanism of Action of the Tuberculosis Drug Candidate SQ109 and Its Metabolites and Analogues in Mycobacteria.

We tested a series of SQ109 analogues against Mycobacterium tuberculosis and M. smegmatis, in addition to determining their uncoupling activity. We then investigated potential protein targets, involved in quinone and cell wall biosynthesis, using "rescue" experiments. There was little effect of menaquinone on growth inhibition by SQ109, but there were large increases in the IC50 of SQ109 and its analogues (up to 20×) on addition of undecaprenyl phosphate (Up), a homologue of the mycobacterial decaprenyl (C50) diphosphate. Inhibition of an undecaprenyl diphosphate phosphatase, an ortholog of the mycobacterial phosphatase, correlated with cell growth inhibition, and we found that M. smegmatis cell growth inhibition could be well predicted by using uncoupler and Up-rescue results. We also investigated whether SQ109 was metabolized inside Mycobacterium tuberculosis, finding only a single metabolite, previously shown to be inactive. The results are of general interest since they help explain the mechanism of SQ109 in mycobacteria.

A Novel Tool to Identify Bactericidal Compounds against Vulnerable Targets in Drug-Tolerant M. tuberculosis found in Caseum.

Caseous necrosis is a hallmark of tuberculosis (TB) pathology and creates a niche for drug-tolerant persisters within the host. Cavitary TB and high bacterial burden in caseum require longer treatment duration. An in vitro model that recapitulates the major features of Mycobacterium tuberculosis (Mtb) in caseum would accelerate the identification of compounds with treatment-shortening potential. We have developed a caseum surrogate model consisting of lysed and denatured foamy macrophages. Upon inoculation of Mtb from replicating cultures, the pathogen adapts to the lipid-rich matrix and gradually adopts a nonreplicating state. We determined that the lipid composition of ex vivo caseum and the surrogate matrix are similar. We also observed that Mtb in caseum surrogate accumulates intracellular lipophilic inclusions (ILI), a distinctive characteristic of quiescent and drug-tolerant Mtb. Expression profiling of a representative gene subset revealed common signatures between the models. Comparison of Mtb drug susceptibility in caseum and caseum surrogate revealed that both populations are similarly tolerant to a panel of TB drugs. By screening drug candidates in the surrogate model, we determined that the bedaquiline analogs TBAJ876 and TBAJ587, currently in clinical development, exhibit superior bactericidal against caseum-resident Mtb, both alone and as substitutions for bedaquiline in the bedaquiline-pretomanid-linezolid regimen approved for the treatment of multidrug-resistant TB. In summary, we have developed a physiologically relevant nonreplicating persistence model that reflects the distinct metabolic and drug-tolerant state of Mtb in caseum. IMPORTANCE M. tuberculosis (Mtb) within the caseous core of necrotic granulomas and cavities is extremely drug tolerant and presents a significant hurdle to treatment success and relapse prevention. Many in vitro models of nonreplicating persistence have been developed to characterize the physiologic and metabolic adaptations of Mtb and identify compounds active against this treatment-recalcitrant population. However, there is little consensus on their relevance to in vivo infection. Using lipid-laden macrophage lysates, we have designed and validated a surrogate matrix that closely mimics caseum and in which Mtb develops a phenotype similar to that of nonreplicating bacilli in vivo. The assay is well suited to screen for bactericidal compounds against caseum-resident Mtb in a medium-throughput format, allowing for reduced reliance on resource intensive animal models that present large necrotic lesions and cavities. Importantly, this approach will aid the identification of vulnerable targets in caseum Mtb and can accelerate the development of novel TB drugs with treatment-shortening potential.

Macrophage internalization creates a multidrug-tolerant fungal persister reservoir and facilitates the emergence of drug resistance.

Candida glabrata is a major fungal pathogen notable for causing recalcitrant infections, rapid emergence of drug-resistant strains, and its ability to survive and proliferate within macrophages. Resembling bacterial persisters, a subset of genetically drug-susceptible C. glabrata cells can survive lethal exposure to the fungicidal echinocandin drugs. Herein, we show that macrophage internalization induces cidal drug tolerance in C. glabrata, expanding the persister reservoir from which echinocandin-resistant mutants emerge. We show that this drug tolerance is associated with non-proliferation and is triggered by macrophage-induced oxidative stress, and that deletion of genes involved in reactive oxygen species detoxification significantly increases the emergence of echinocandin-resistant mutants. Finally, we show that the fungicidal drug amphotericin B can kill intracellular C. glabrata echinocandin persisters, reducing emergence of resistance. Our study supports the hypothesis that intra-macrophage C. glabrata is a reservoir of recalcitrant/drug-resistant infections, and that drug alternating strategies can be developed to eliminate this reservoir.

Adaptation to the intracellular environment of primary human macrophages influences drug susceptibility of Mycobacterium tuberculosis.

As a facultative intracellular pathogen, M. tuberculosis (Mtb) is highly adapted to evading antibacterial mechanisms in phagocytic cells. Both the macrophage and pathogen experience transcriptional and metabolic changes from the onset of phagocytosis. To account for this interaction in the assessment of intracellular drug susceptibility, we allowed a 3-day preadaptation phase post-macrophage infection prior to drug treatment. We found that intracellular Mtb in human monocyte-derived macrophages (MDM) presents dramatic alterations in susceptibility to isoniazid, sutezolid, rifampicin and rifapentine when compared to axenic culture. Infected MDM gradually accumulate lipid bodies, adopting a characteristic appearance reminiscent of foamy macrophages in granulomas. Furthermore, TB granulomas in vivo develop hypoxic cores with decreasing oxygen tension gradients across their radii. Accordingly, we evaluated the effects of hypoxia on preadapted intracellular Mtb in our MDM model. We observed that hypoxia induced greater lipid body formation and no additional shifts in drug tolerance, suggesting that the adaptation of intracellular Mtb to baseline host cell conditions under normoxia dominates changes to intracellular drug susceptibility. Using unbound plasma concentrations in patients as surrogates for free drug concentrations in lung interstitial fluid, we estimate that intramacrophage Mtb in granulomas are exposed to bacteriostatic concentrations of most study drugs.

CinA mediates multidrug tolerance in Mycobacterium tuberculosis.

The ability of Mycobacterium tuberculosis (Mtb) to resist and tolerate antibiotics complicates the development of improved tuberculosis (TB) chemotherapies. Here we define the Mtb protein CinA as a major determinant of drug tolerance and as a potential target to shorten TB chemotherapy. By reducing the fraction of drug-tolerant persisters, genetic inactivation of cinA accelerated killing of Mtb by four antibiotics in clinical use: isoniazid, ethionamide, delamanid and pretomanid. Mtb ΔcinA was killed rapidly in conditions known to impede the efficacy of isoniazid, such as during nutrient starvation, during persistence in a caseum mimetic, in activated macrophages and during chronic mouse infection. Deletion of CinA also increased in vivo killing of Mtb by BPaL, a combination of pretomanid, bedaquiline and linezolid that is used to treat highly drug-resistant TB. Genetic and drug metabolism studies suggest that CinA mediates drug tolerance via cleavage of NAD-drug adducts.

A Rabbit Model to Study Antibiotic Penetration at the Site of Infection for Nontuberculous Mycobacterial Lung Disease: Macrolide Case Study.

Nontuberculous mycobacterial pulmonary disease (NTM-PD) is a potentially fatal infectious disease requiring long treatment duration with multiple antibiotics and against which there is no reliable cure. Among the factors that have hampered the development of adequate drug regimens is the lack of an animal model that reproduces the NTM lung pathology required for studying antibiotic penetration and efficacy. Given the documented similarities between tuberculosis and NTM immunopathology in patients, we first determined that the rabbit model of active tuberculosis reproduces key features of human NTM-PD and provides an acceptable surrogate model to study lesion penetration. We focused on clarithromycin, a macrolide and pillar of NTM-PD treatment, and explored the underlying causes of the disconnect between its favorable potency and pharmacokinetics and inconsistent clinical outcome. To quantify pharmacokinetic-pharmacodynamic target attainment at the site of disease, we developed a translational model describing clarithromycin distribution from plasma to lung lesions, including the spatial quantitation of clarithromycin and azithromycin in mycobacterial lesions of two patients on long-term macrolide therapy. Through clinical simulations, we visualized the coverage of clarithromycin in plasma and four disease compartments, revealing heterogeneous bacteriostatic and bactericidal target attainment depending on the compartment and the corresponding potency against nontuberculous mycobacteria in clinically relevant assays. Overall, clarithromycin's favorable tissue penetration and lack of bactericidal activity indicated that its clinical activity is limited by pharmacodynamic, rather than pharmacokinetic, factors. Our results pave the way toward the simulation of lesion pharmacokinetic-pharmacodynamic coverage by multidrug combinations to enable the prioritization of promising regimens for clinical trials.

On-Slide Heat Sterilization Enables Mass Spectrometry Imaging of Tissue Infected with High-Threat Pathogens Outside of Biocontainment: A Study Directed at Mycobacterium tuberculosis.

Mass spectrometry imaging investigations of tissues infected with agents that require high-security biocontainment, such as Mycobacterium tuberculosis, have been limited due to incompatible sterilization techniques. Here we describe an on-slide heat sterilization method that enables mass spectrometry imaging investigations of pharmaceuticals, lipids, and metabolites in infected tissue samples outside of biocontainment. An evaluation of different temperatures and incubation times determined that 100 °C for 1 h was essential to sterilize 5 times the bacterial burden observed in tuberculosis (TB) cavity sections. Laser-capture microdissection combined with liquid chromatography with tandem mass spectrometry quantitation, in addition to mass spectrometry imaging, showed that no degradation was observed following the on-slide heat sterilization protocol for a variety of drug classes covering a range of physicochemical properties. Utilizing the tissue mimetic model, we demonstrated that the detection of lipid and metabolite ions was not impacted by heat sterilization and that, for several metabolites, the on-slide heat sterilization method improved the sensitivity when compared to control samples. An application of the on-slide heat sterilization to M. tuberculosis infected tissue enabled the first detection and spatial distribution of lipids indicative of a lysosomal storage disease phenotype within TB granuloma macrophages, in addition to the differential distribution of metabolites central to the fatty acid oxidation pathway. These initial investigations detected a pronounced heterogeneity within the cellular regions and necrotic cores of individual TB granulomas and across different evolving granulomas. This study provides the framework for mass spectrometry imaging investigations of high-threat pathogens outside of biocontainment.

Lesion Penetration and Activity Limit the Utility of Second-Line Injectable Agents in Pulmonary Tuberculosis.

Amikacin and kanamycin are second-line injectables used in the treatment of multidrug-resistant tuberculosis (MDR-TB) based on the clinical utility of streptomycin, another aminoglycoside and first-line anti-TB drug. While streptomycin was tested as a single agent in the first controlled TB clinical trial, introduction of amikacin and kanamycin into MDR-TB regimens was not preceded by randomized controlled trials. A recent large retrospective meta-analysis revealed that compared with regimens without any injectable drug, amikacin provided modest benefits, and kanamycin was associated with worse outcomes. Although their long-term use can cause irreversible ototoxicity, they remain part of MDR-TB regimens because they have a role in preventing emergence of resistance to other drugs. To quantify the contribution of amikacin and kanamycin to second-line regimens, we applied two-dimensional matrix-assisted laser desorption ionization (MALDI) mass spectrometry imaging in large lung lesions, quantified drug exposure in lung and in lesions of rabbits with active TB, and measured the concentrations required to kill or inhibit growth of the resident bacterial populations. Using these metrics, we applied site-of-action pharmacokinetic and pharmacodynamic (PK-PD) concepts and simulated drug coverage in patients' lung lesions. The results provide a pharmacological explanation for the limited clinical utility of both agents and reveal better PK-PD lesion coverage for amikacin than kanamycin, consistent with retrospective data of contribution to treatment success. Together with recent mechanistic studies dissecting antibacterial activity from aminoglycoside ototoxicity, the limited but rapid penetration of streptomycin, amikacin, and kanamycin to the sites of TB disease supports the development of analogs with improved efficacy and tolerability.

Pharmacokinetics and Target Attainment of SQ109 in Plasma and Human-Like Tuberculosis Lesions in Rabbits.

SQ109 is a novel well-tolerated drug candidate in clinical development for the treatment of drug-resistant tuberculosis (TB). It is the only inhibitor of the MmpL3 mycolic acid transporter in clinical development. No SQ109-resistant mutant has been directly isolated thus far in vitro, in mice, or in patients, which is tentatively attributed to its multiple targets. It is considered a potential replacement for poorly tolerated components of multidrug-resistant TB regimens. To prioritize SQ109-containing combinations with the best potential for cure and treatment shortening, one must understand its contribution against different bacterial populations in pulmonary lesions. Here, we have characterized the pharmacokinetics of SQ109 in the rabbit model of active TB and its penetration at the sites of disease-lung tissue, cellular and necrotic lesions, and caseum. A two-compartment model with first-order absorption and elimination described the plasma pharmacokinetics. At the human-equivalent dose, parameter estimates fell within the ranges published for preclinical species. Tissue concentrations were modeled using an "effect" compartment, showing high accumulation in lung and cellular lesion areas with penetration coefficients in excess of 1,000 and lower passive diffusion in caseum after 7 daily doses. These results, together with the hydrophobic nature and high nonspecific caseum binding of SQ109, suggest that multiweek dosing would be required to reach steady state in caseum and poorly vascularized compartments, similar to bedaquiline. Linking lesion pharmacokinetics to SQ109 potency in assays against replicating, nonreplicating, and intracellular M. tuberculosis showed SQ109 concentrations markedly above pharmacokinetic-pharmacodynamic targets in lung and cellular lesions throughout the dosing interval.

Blocking Bacterial Naphthohydroquinone Oxidation and ADP-Ribosylation Improves Activity of Rifamycins against Mycobacterium abscessus.

Rifampicin is an effective drug for treating tuberculosis (TB) but is not used to treat Mycobacterium abscessus infections due to poor in vitro activity. While rifabutin, another rifamycin, has better anti-M. abscessus activity, its activity is far from the nanomolar potencies of rifamycins against Mycobacterium tuberculosis. Here, we asked (i) why is rifabutin more active against M. abscessus than rifampicin, and (ii) why is rifabutin's anti-M. abscessus activity poorer than its anti-TB activity? Comparative analysis of naphthoquinone- versus naphthohydroquinone-containing rifamycins suggested that the improved activity of rifabutin over rifampicin is linked to its less readily oxidizable naphthoquinone core. Although rifabutin is resistant to bacterial oxidation, metabolite and genetic analyses showed that this rifamycin is metabolized by the ADP-ribosyltransferase ArrMab like rifampicin, preventing it from achieving the nanomolar activity that it displays against M. tuberculosis. Based on the identified dual mechanism of intrinsic rifamycin resistance, we hypothesized that rifamycins more potent than rifabutin should contain the molecule's naphthoquinone core plus a modification that blocks ADP-ribosylation at its C-23. To test these predictions, we performed a blinded screen of a diverse collection of 189 rifamycins and identified two molecules more potent than rifabutin. As predicted, these compounds contained both a more oxidatively resistant naphthoquinone core and C-25 modifications that blocked ADP-ribosylation. Together, this work revealed dual bacterial metabolism as the mechanism of intrinsic resistance of M. abscessus to rifamycins and provides proof of concept for the repositioning of rifamycins for M. abscessus disease by developing derivatives that resist both bacterial oxidation and ADP-ribosylation.

Structure, In Vivo Detection, and Antibacterial Activity of Metabolites of SQ109, an Anti-Infective Drug Candidate.

SQ109 is a drug candidate for the treatment of tuberculosis (TB). It is thought to target primarily the protein MmpL3 in Mycobacterium tuberculosis, but it also inhibits the growth of some other bacteria. SQ109 is metabolized by the liver, and it has been proposed that some of its metabolites might be responsible for its activity against TB. Here, we synthesized six potential P450 metabolites of SQ109 and used these as well as 10 other likely metabolites as standards in a mass spectrometry study of M. tuberculosis-infected rabbits treated with SQ109, in addition to testing all 16 putative metabolites for antibacterial activity. We found that there were just two major metabolites in lung tissue: a hydroxy-adamantyl analog of SQ109 and N'-adamantylethylenediamine. Neither of these, or the other potential metabolites tested, inhibited the growth of M. tuberculosis or of M. smegmatis, Bacillus subtilis, or E. coli, making it unlikely that an SQ109 metabolite contributes to its antibacterial activity. In the rabbit TB model, it is thus the gradual accumulation of nonmetabolized SQ109 in tissues to therapeutic levels that leads to good efficacy. Our results also provide new insights into how SQ109 binds to its target MmpL3, based on our mass spectroscopy results which indicate that the charge in SQ109 is primarily localized on the geranyl nitrogen, explaining the very short distance to a key Asp found in the X-ray structure of SQ109 bound to MmpL3.

Caseum: a Niche for Mycobacterium tuberculosis Drug-Tolerant Persisters.

Caseum, the central necrotic material of tuberculous lesions, is a reservoir of drug-recalcitrant persisting mycobacteria. Caseum is found in closed nodules and in open cavities connecting with an airway. Several commonly accepted characteristics of caseum were established during the preantibiotic era, when autopsies of deceased tuberculosis (TB) patients were common but methodologies were limited. These pioneering studies generated concepts such as acidic pH, low oxygen tension, and paucity of nutrients being the drivers of nonreplication and persistence in caseum. Here we review widely accepted beliefs about the caseum-specific stress factors thought to trigger the shift of Mycobacterium tuberculosis to drug tolerance. Our current state of knowledge reveals that M. tuberculosis is faced with a lipid-rich diet rather than nutrient deprivation in caseum. Variable caseum pH is seen across lesions, possibly transiently acidic in young lesions but overall near neutral in most mature lesions. Oxygen tension is low in the avascular caseum of closed nodules and high at the cavity surface, and a gradient of decreasing oxygen tension likely forms toward the cavity wall. Since caseum is largely made of infected and necrotized macrophages filled with lipid droplets, the microenvironmental conditions encountered by M. tuberculosis in foamy macrophages and in caseum bear many similarities. While there remain a few knowledge gaps, these findings constitute a solid starting point to develop high-throughput drug discovery assays that combine the right balance of oxygen tension, pH, lipid abundance, and lipid species to model the profound drug tolerance of M. tuberculosis in caseum.

Tissue Distribution of Doxycycline in Animal Models of Tuberculosis.

Doxycycline, an FDA-approved tetracycline, is used in tuberculosis in vivo models for the temporal control of mycobacterial gene expression. In these models, animals are infected with recombinant Mycobacterium tuberculosis carrying genes of interest under transcriptional control of the doxycycline-responsive TetR-tetO unit. To minimize fluctuations of plasma levels, doxycycline is usually administered in the diet. However, tissue penetration studies to identify the minimum doxycycline content in food achieving complete repression of TetR-controlled genes in tuberculosis (TB)-infected organs and lesions have not been conducted. Here, we first determined the tetracycline concentrations required to achieve silencing of M. tuberculosis target genes in vitro Next, we measured doxycycline concentrations in plasma, major organs, and lung lesions in TB-infected mice and rabbits and compared these values to silencing concentrations measured in vitro We found that 2,000 ppm doxycycline supplemented in mouse and rabbit feed is sufficient to reach target concentrations in TB lesions. In rabbit chow, the calcium content had to be reduced 5-fold to minimize chelation of doxycycline and deliver adequate oral bioavailability. Clearance kinetics from major organs and lung lesions revealed that doxycycline levels fall below concentrations that repress tet promoters within 7 to 14 days after doxycycline is removed from the diet. In summary, we have shown that 2,000 ppm doxycycline supplemented in standard mouse diet and in low-calcium rabbit diet delivers concentrations adequate to achieve full repression of tet promoters in infected tissues of mice and rabbits.

Fluoroquinolone Efficacy against Tuberculosis Is Driven by Penetration into Lesions and Activity against Resident Bacterial Populations.

Fluoroquinolones represent the pillar of multidrug-resistant tuberculosis (MDR-TB) treatment, with moxifloxacin, levofloxacin, or gatifloxacin being prescribed to MDR-TB patients. Recently, several clinical trials of "universal" drug regimens, aiming to treat drug-susceptible and drug-resistant TB, have included a fluoroquinolone. In the absence of clinical data comparing their side-by-side efficacies in controlled MDR-TB trials, a pharmacological rationale is needed to guide the selection of the most efficacious fluoroquinolone. The present studies were designed to test the hypothesis that fluoroquinolone concentrations (pharmacokinetics) and activity (pharmacodynamics) at the site of infection are better predictors of efficacy than the plasma concentrations and potency measured in standard growth inhibition assays and are better suited to determinations of whether one of the fluoroquinolones outperforms the others in rabbits with active TB. We first measured the penetration of these fluoroquinolones in lung lesion compartments, and their potency against bacterial populations that reside in each compartment, to compute lesion-centric pharmacokinetic-pharmacodynamic (PK/PD) parameters. PK modeling methods were used to quantify drug penetration from plasma to tissues at human-equivalent doses. On the basis of these metrics, moxifloxacin emerged with a clear advantage, whereas plasma-based PK/PD favored levofloxacin (the ranges of the plasma AUC/MIC ratio [i.e., the area under the concentration-time curve over 24 h in the steady state divided by the MIC] are 46 to 86 for moxifloxacin and 74 to 258 for levofloxacin). A comparative efficacy trial in the rabbit model of active TB demonstrated the superiority of moxifloxacin in reducing bacterial burden at the lesion level and in sterilizing cellular and necrotic lesions. Collectively, these results show that PK/PD data obtained at the site of infection represent an adequate predictor of drug efficacy against TB and constitute the baseline required to explore synergies, antagonism, and drug-drug interactions in fluoroquinolone-containing regimens.

Synergistic Lethality of a Binary Inhibitor of Mycobacterium tuberculosis KasA.

We report GSK3011724A (DG167) as a binary inhibitor of ß-ketoacyl-ACP synthase (KasA) in Mycobacterium tuberculosis Genetic and biochemical studies established KasA as the primary target. The X-ray crystal structure of the KasA-DG167 complex refined to 2.0-Å resolution revealed two interacting DG167 molecules occupying nonidentical sites in the substrate-binding channel of KasA. The binding affinities of KasA to DG167 and its analog, 5g, which binds only once in the substrate-binding channel, were determined, along with the KasA-5g X-ray crystal structure. DG167 strongly augmented the in vitro activity of isoniazid (INH), leading to synergistic lethality, and also synergized in an acute mouse model of M. tuberculosis infection. Synergistic lethality correlated with a unique transcriptional signature, including upregulation of oxidoreductases and downregulation of molecular chaperones. The lead structure-activity relationships (SAR), pharmacokinetic profile, and detailed interactions with the KasA protein that we describe may be applied to evolve a next-generation therapeutic strategy for tuberculosis (TB).IMPORTANCE Cell wall biosynthesis inhibitors have proven highly effective for treating tuberculosis (TB). We discovered and validated members of the indazole sulfonamide class of small molecules as inhibitors of Mycobacterium tuberculosis KasA-a key component for biosynthesis of the mycolic acid layer of the bacterium's cell wall and the same pathway as that inhibited by the first-line antitubercular drug isoniazid (INH). One lead compound, DG167, demonstrated synergistic lethality in combination with INH and a transcriptional pattern consistent with bactericidality and loss of persisters. Our results also detail a novel dual-binding mechanism for this compound as well as substantial structure-activity relationships (SAR) that may help in lead optimization activities. Together, these results suggest that KasA inhibition, specifically, that shown by the DG167 series, may be developed into a potent therapy that can synergize with existing antituberculars.

Impact of immunopathology on the antituberculous activity of pyrazinamide.

In the 1970s, inclusion of pyrazinamide (PZA) in the drug regimen of tuberculosis (TB) patients for the first 2 mo achieved a drastic reduction of therapy duration. Until now, however, the mechanisms underlying PZA's unique contribution to efficacy have remained controversial, and animal efficacy data vary across species. To understand how PZA kills bacterial populations present in critical lung lesion compartments, we first characterized a rabbit model of active TB, showing striking similarities in lesion types and fates to nonhuman primate models deemed the most appropriate surrogates of human TB. We next employed this model with lesion-centric molecular and bacteriology readouts to demonstrate that PZA exhibits potent activity against Mycobacterium tuberculosis residing in difficult-to-sterilize necrotic lesions. Our data also indicate that PZA is slow acting, suggesting that PZA administration beyond the first 2 mo may accelerate the cure. In conclusion, we provide a pharmacodynamic explanation for PZA's treatment-shortening effect and deliver new tools to dissect the contribution of immune response versus drug at the lesion level.