Anne J. Lenaerts

Preclinical Testing of the Nitroimidazopyran PA-824 for Activity against Mycobacterium tuberculosis in a Series of In Vitro and In Vivo Models

ABSTRACT This study extends earlier reports regarding the in vitro and in vivo efficacies of the nitroimidazopyran PA-824 against Mycobacterium tuberculosis. PA-824 was tested in vitro against a broad panel of multidrug-resistant clinical isolates and was found to be highly active against all isolates (MIC < 1 μg/ml). The activity of PA-824 against M. tuberculosis was also assessed grown under conditions of oxygen depletion. PA-824 showed significant activity at 2, 10, and 50 μg/ml, similar to that of metronidazole, in a dose-dependent manner. In a short-course mouse infection model, the efficacy of PA-824 at 50, 100, and 300 mg/kg of body weight formulated in methylcellulose or cyclodextrin/lecithin after nine oral treatments was compared with those of isoniazid, rifampin, and moxifloxacin. PA-824 at 100 mg/kg in cyclodextrin/lecithin was as active as moxifloxacin at 100 mg/kg and isoniazid at 25 mg/kg and was slightly more active than rifampin at 20 mg/kg. Long-term treatment with PA-824 at 100 mg/kg in cyclodextrin/lecithin reduced the bacterial load below 500 CFU in the lungs and spleen. No significant differences in activity between PA-824 and the other single drug treatments tested (isoniazid at 25 mg/kg, rifampin at 10 mg/kg, gatifloxacin at 100 mg/kg, and moxifloxacin at 100 mg/kg) could be observed. In summary, its good activity in in vivo models, as well as its activity against multidrug-resistant M. tuberculosis and against M. tuberculosis isolates in a potentially latent state, makes PA-824 an attractive drug candidate for the therapy of tuberculosis. These data indicate that there is significant potential for effective oral delivery of PA-824 for the treatment of tuberculosis.

Discovery of Q203, a potent clinical candidate for the treatment of tuberculosis

New therapeutic strategies are needed to combat the tuberculosis pandemic and the spread of multidrug-resistant (MDR) and extensively drug-resistant (XDR) forms of the disease, which remain a serious public health challenge worldwide. The most urgent clinical need is to discover potent agents capable of reducing the duration of MDR and XDR tuberculosis therapy with a success rate comparable to that of current therapies for drug-susceptible tuberculosis. The last decade has seen the discovery of new agent classes for the management of tuberculosis, several of which are currently in clinical trials. However, given the high attrition rate of drug candidates during clinical development and the emergence of drug resistance, the discovery of additional clinical candidates is clearly needed. Here, we report on a promising class of imidazopyridine amide (IPA) compounds that block Mycobacterium tuberculosis growth by targeting the respiratory cytochrome bc1 complex. The optimized IPA compound Q203 inhibited the growth of MDR and XDR M. tuberculosis clinical isolates in culture broth medium in the low nanomolar range and was efficacious in a mouse model of tuberculosis at a dose less than 1 mg per kg body weight, which highlights the potency of this compound. In addition, Q203 displays pharmacokinetic and safety profiles compatible with once-daily dosing. Together, our data indicate that Q203 is a promising new clinical candidate for the treatment of tuberculosis.

Location of Persisting Mycobacteria in a Guinea Pig Model of Tuberculosis Revealed by R207910

ABSTRACT The lengthy chemotherapy of tuberculosis reflects the ability of a small subpopulation of Mycobacterium tuberculosis bacteria to persist in infected individuals. To date, the exact location of these persisting bacteria is not known. Lung lesions in guinea pigs infected with M. tuberculosis have striking similarities, such as necrosis, mineralization, and hypoxia, to natural infections in humans. Guinea pigs develop necrotic primary lesions after aerosol infection that differ in their morphology compared to secondary lesions resulting from hematogenous dissemination. In infected guinea pigs conventional therapy for tuberculosis during 6 weeks reduced the bacterial load by 1.7 logs in the lungs and, although this completely reversed lung inflammation associated with secondary lesions, the primary granulomas remained largely unaffected. Treatment of animals with the experimental drug R207910 (TMC207) for 6 weeks was highly effective with almost complete eradication of the bacteria throughout both the primary and the secondary lesions. Most importantly, the few remnants of acid-fast bacilli remaining after R207910 treatment were to be found extracellular, in a microenvironment of residual primary lesion necrosis with incomplete dystrophic calcification. This zone of the primary granuloma is hypoxic and is morphologically similar to what has been described for human lung lesions. These results show that this acellular rim may, therefore, be a primary location of persisting bacilli withstanding drug treatment.

Inhibition of mycolic acid transport across the Mycobacterium tuberculosis plasma membrane

New chemotherapeutics active against multidrug-resistant Mycobacterium tuberculosis (M. tb) are urgently needed. We report on the identification of an adamantyl urea compound displaying potent bactericidal activity against M. tb and a unique mode of action, namely the abolition of the translocation of mycolic acids from the cytoplasm where they are synthesized to the periplasmic side of the plasma membrane where they are transferred onto cell wall arabinogalactan or used in the formation of virulence-associated outer membrane trehalose-containing glycolipids. Whole genome sequencing of spontaneous resistant mutants of M. tb selected in vitro followed by genetic validation experiments revealed that our prototype inhibitor targets the inner membrane transporter, MmpL3. Conditional gene expression of mmpL3 in mycobacteria and analysis of inhibitor-treated cells validate MmpL3 as essential for mycobacterial growth and support the involvement of this transporter in the translocation of trehalose monomycolate across the plasma membrane.

Heterogeneity in tuberculosis pathology, microenvironments and therapeutic responses.

Tuberculosis (TB) lesions are extremely complex and dynamic. Here, we review the multiple types and fates of pulmonary lesions that form following infection by Mycobacterium tuberculosis and the impact of this spatial and temporal heterogeneity on the bacteria they harbor. The diverse immunopathology of granulomas and cavities generates a plethora of microenvironments to which M. tuberculosis bacilli must adapt. This in turn affects the replication, metabolism, and relative density of bacterial subpopulations, and consequently their respective susceptibility to chemotherapy. We outline recent developments that support a paradigm shift in our understanding of lesion progression. The simple model according to which lesions within a single individual react similarly to the systemic immune response no longer prevails. Host‐pathogen interactions within lesions are a dynamic process, driven by subtle and local differences in signaling pathways, resulting in diverging trajectories of lesions within a single host. The spectrum of TB lesions is a continuum with a large overlap in the lesion types found in latently infected and active TB patients. We hope this overview will guide TB researchers in the design, choice of read‐outs, and interpretation of future studies in the search for predictive biomarkers and novel therapies.

Evaluation of a Mouse Model of Necrotic Granuloma Formation Using C3HeB/FeJ Mice for Testing of Drugs against Mycobacterium tuberculosis

ABSTRACT Persistence of Mycobacterium tuberculosis remains a significant challenge for the effective treatment of tuberculosis in humans. In animals that develop necrotic lung lesions following infection with M. tuberculosis, drug-tolerant bacilli are present and persist in an extracellular microenvironment within the necrotic cores. In this study, we examined the efficacy of drug treatment in C3HeB/FeJ (Kramnik) mice that develop lesions with liquefactive necrosis, in comparison to BALB/c mice that develop nonnecrotic lesions following aerosol challenge. To accomplish this, Kramnik and BALB/c mice were infected by aerosol with M. tuberculosis and treated for 7 to 8 weeks with monotherapy using drugs with different modes of action. The efficacy of drug therapy was quantified by enumeration of bacterial load. The progression of disease and location and distribution of bacilli within lesions were visualized using various staining techniques. In the late stages of infection, Kramnik mice developed fibrous encapsulated lung lesions with central liquefactive necrosis containing abundant extracellular bacilli, whereas BALB/c mice formed nonnecrotic lesions with primarily intracellular bacilli. Necrotic lesions in Kramnik mice showed evidence of hypoxia by pimonidazole staining. Kramnik mice were significantly more refractory to drug therapy, especially for pyrazinamide. Metronidazole showed no bactericidal activity in either model. There were significantly higher numbers of drug-resistant colonies isolated from the Kramnik mice compared to BALB/c mice. These results suggest that the Kramnik mouse model will be a valuable model to test antituberculosis drugs, especially against bacilli that persist within necrotic lesions.

Comprehensive analysis of methods used for the evaluation of compounds against Mycobacterium tuberculosis

In drug development, there are typically a series of preclinical studies that must be completed with new compounds or regimens before use in humans. A sequence of in vitro assays followed by in vivo testing in validated animal models to assess the activity against Mycobacterium tuberculosis, pharmacology and toxicity is generally used for advancing compounds against tuberculosis in a preclinical stage. A plethora of different assay systems and conditions are used to study the effect of drug candidates on the growth of M. tuberculosis, making it difficult to compare data from one laboratory to another. The Bill and Melinda Gates Foundation recognized the scientific gap to delineate the spectrum of variables in experimental protocols, identify which of these are biologically significant, and converge towards a rationally derived standard set of optimized assays for evaluating compounds. The goals of this document are to recommend protocols and hence accelerate the process of TB drug discovery and testing. Data gathered from preclinical in vitro and in vivo assays during personal visits to laboratories and an electronic survey of methodologies sent to investigators is reported. Comments, opinions, experiences as well as final recommendations from those currently engaged in such preclinical studies for TB drug testing are being presented. Certain in vitro assays and mouse efficacy models were re-evaluated in the laboratory as head-to-head experiments and a summary is provided on the results obtained. It is our hope that this information will be a valuable resource for investigators in the field to move forward in an efficient way and that key variables of assays are included to ensure accuracy of results which can then be used for designing human clinical trials. This document then concludes with remaining questions and critical gaps that are in need of further validation and experimentation.

Rapid In Vivo Screening of Experimental Drugs for Tuberculosis Using Gamma Interferon Gene-Disrupted Mice

ABSTRACT We have developed a rapid new in vivo method for screening experimental drugs for their activity against Mycobacterium tuberculosis by using the gamma interferon gene-disrupted (GKO) C57BL/6 mouse. Due to the rapid growth of the infection, statistical differences indicating positive efficacy of active compounds can be seen after only 8 days of treatment. To validate this model, several fluoroquinolones, including ciprofloxacin, levofloxacin, moxifloxacin, and gatifloxacin, were tested in parallel.

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

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

Spectinamides: a new class of semisynthetic antituberculosis agents that overcome native drug efflux

Although the classical antibiotic spectinomycin is a potent bacterial protein synthesis inhibitor, poor antimycobacterial activity limits its clinical application for treating tuberculosis. Using structure-based design, we generated a new semisynthetic series of spectinomycin analogs with selective ribosomal inhibition and excellent narrow-spectrum antitubercular activity. In multiple murine infection models, these spectinamides were well tolerated, significantly reduced lung mycobacterial burden and increased survival. In vitro studies demonstrated a lack of cross resistance with existing tuberculosis therapeutics, activity against multidrug-resistant (MDR) and extensively drug-resistant tuberculosis and an excellent pharmacological profile. Key to their potent antitubercular properties was their structural modification to evade the Rv1258c efflux pump, which is upregulated in MDR strains and is implicated in macrophage-induced drug tolerance. The antitubercular efficacy of spectinamides demonstrates that synthetic modifications to classical antibiotics can overcome the challenge of intrinsic efflux pump-mediated resistance and expands opportunities for target-based tuberculosis drug discovery.