Shichun Lun

The Two-Domain LysX Protein of Mycobacterium tuberculosis Is Required for Production of Lysinylated Phosphatidylglycerol and Resistance to Cationic Antimicrobial Peptides

The well-recognized phospholipids (PLs) of Mycobacterium tuberculosis (Mtb) include several acidic species such as phosphatidylglycerol (PG), cardiolipin, phosphatidylinositol and its mannoside derivatives, in addition to a single basic species, phosphatidylethanolamine. Here we demonstrate that an additional basic PL, lysinylated PG (L-PG), is a component of the PLs of Mtb H37Rv and that the lysX gene encoding the two-domain lysyl-transferase (mprF)-lysyl-tRNA synthetase (lysU) protein is responsible for L-PG production. The Mtb lysX mutant is sensitive to cationic antibiotics and peptides, shows increased association with lysosome-associated membrane protein–positive vesicles, and it exhibits altered membrane potential compared to wild type. A lysX complementing strain expressing the intact lysX gene, but not one expressing mprF alone, restored the production of L-PG and rescued the lysX mutant phenotypes, indicating that the expression of both proteins is required for LysX function. The lysX mutant also showed defective growth in mouse and guinea pig lungs and showed reduced pathology relative to wild type, indicating that LysX activity is required for full virulence. Together, our results suggest that LysX-mediated production of L-PG is necessary for the maintenance of optimal membrane integrity and for survival of the pathogen upon infection.

Indoleamides are active against drug-resistant Mycobacterium tuberculosis

Responsible for nearly two million deaths each year, the infectious disease tuberculosis remains a serious global health challenge. The emergence of multidrug- and extensively drug-resistant strains of Mycobacterium tuberculosis confounds control efforts, and new drugs with novel molecular targets are desperately needed. Here we describe lead compounds, the indoleamides, with potent activity against both drug-susceptible and drug-resistant strains of M. tuberculosis by targeting the mycolic acid transporter MmpL3. We identify a single mutation in mmpL3 which confers high resistance to the indoleamide class while remaining susceptible to currently used first- and second-line tuberculosis drugs, indicating a lack of cross-resistance. Importantly, an indoleamide derivative exhibits dose-dependent anti-mycobacterial activity when orally administered to M. tuberculosis-infected mice. The bioavailability of the indoleamides, combined with their ability to kill tubercle bacilli, indicates great potential for translational developments of this structure class for the treatment of drug-resistant tuberculosis.

Evaluation of gyrase B as a drug target in Mycobacterium tuberculosis

OBJECTIVES New classes of drugs are needed to treat tuberculosis (TB) in order to combat the emergence of resistance to existing agents and shorten the duration of therapy. Targeting DNA gyrase is a clinically validated therapeutic approach using fluoroquinolone antibiotics to target the gyrase subunit A (GyrA) of the heterotetramer. Increasing resistance to fluoroquinolones has driven interest in targeting the gyrase subunit B (GyrB), which has not been targeted for TB. The biological activities of two potent small-molecule inhibitors of GyrB have been characterized to validate its targeting as a therapeutic strategy for treating TB. MATERIALS AND METHODS Novobiocin and aminobenzimidazole 1 (AB-1) were tested for their activity against Mycobacterium tuberculosis (Mtb) H37Rv and other mycobacteria. AB-1 and novobiocin were also evaluated for their interaction with rifampicin and isoniazid as well as their potential for cytotoxicity. Finally, AB-1 was tested for in vivo efficacy in a murine model of TB. RESULTS Novobiocin and AB-1 have both been shown to be active against Mtb with MIC values of 4 and 1 mg/L, respectively. Only AB-1 exhibited time-dependent bactericidal activity against drug-susceptible and drug-resistant mycobacteria, including a fluoroquinolone-resistant strain. AB-1 had potent activity in the low oxygen recovery assay model for non-replicating persistent Mtb. Additionally, AB-1 has no interaction with isoniazid and rifampicin, and has no cross-resistance with fluoroquinolones. In a murine model of TB, AB-1 significantly reduced lung cfu counts in a dose-dependent manner. CONCLUSIONS Aminobenzimidazole inhibitors of GyrB exhibit many of the characteristics required for their consideration as a potential front-line antimycobacterial therapeutic.

Preliminary Structure-Activity Relationships and Biological Evaluation of Novel Antitubercular Indolecarboxamide Derivatives Against Drug-Susceptible and Drug-Resistant Mycobacterium tuberculosis Strains

Tuberculosis (TB) remains one of the leading causes of mortality and morbidity worldwide, with approximately one-third of the worlds population infected with latent TB. This is further aggravated by HIV coinfection and the emergence of multidrug- and extensively drug-resistant (MDR and XDR, respectively) TB; hence the quest for highly effective antitubercular drugs with novel modes of action is imperative. We report herein the discovery of an indole-2-carboxamide analogue, 3, as a highly potent antitubercular agent, and the subsequent chemical modifications aimed at establishing a preliminary body of structure-activity relationships (SARs). These efforts led to the identification of three molecules (12-14) possessing an exceptional activity in the low nanomolar range against actively replicating Mycobacterium tuberculosis , with minimum inhibitory concentration (MIC) values lower than those of the most prominent antitubercular agents currently in use. These compounds were also devoid of apparent toxicity to Vero cells. Importantly, compound 12 was found to be active against the tested XDR-TB strains and orally active in the serum inhibition titration assay.

Characterization of a Novel Cell Wall-anchored Protein with Carboxylesterase Activity Required for Virulence in Mycobacterium tuberculosis

Pooled mutant competition assays have shown that the Mycobacterium tuberculosis MT2282 gene (Rv2224c, annotated as encoding a proteinase) is required for bacterial survival in mice. To understand the mechanism of this requirement, we conducted a genetic and biochemical study of the MT2282 gene and its product. MT2282 encodes a member of the microbial esterase/lipase family with active site consensus sequences of G-X-S-X-G, and we have concluded that the MT2282 protein is, in fact, a cell wall-associated carboxylesterase rather than a proteinase, as initially annotated. The MT2282 gene product preferentially hydrolyzes ester bonds of substrates with intermediate carbon chain length. Purified MT2282 is a monomer with enzymatic catalysis properties that fit in the Michaelis-Menten kinetic model. Esterase activity was inhibited by paraoxon and dichlorvos. Replacement of Ser215, Asp450, and His477 by Ala in the consensus motifs completely abolishes esterase activity, suggesting that Ser215-Asp450-His477 forms a catalytic triad with Ser215 as an active site residue. To evaluate the role of the MT2282 in pathogenesis, the gene was deleted from the M. tuberculosis genome. BALB/c mouse aerosol infections showed reduced colony-forming unit loads in lungs and spleens and less lung pathology for the ΔMT2282 mutant. High dose intravenous infection of mice with the mutant resulted in a significantly delayed time to death compared with the wild type or complemented mutant. These results indicate that MT2282 encodes a cell wall-associated carboxylesterase, which is required for full virulence of M. tuberculosis. We propose that MT2282 (Rv2224c) and its adjacent paralogous gene MT2281 (Rv2223c) be named caeA and caeB respectively, for carboxylesterase A and B.

Extrapulmonary Dissemination of Mycobacterium bovis but Not Mycobacterium tuberculosis in a Bronchoscopic Rabbit Model of Cavitary Tuberculosis

ABSTRACT The rabbit model of tuberculosis is attractive because of its pathophysiologic resemblance to the disease in humans. Rabbits are naturally resistant to infection but may manifest cavitary lung lesions. We describe here a novel approach that utilizes presensitization and bronchoscopic inoculation to reliably produce cavities in the rabbit model. With a fixed inoculum of bacilli, we were able to reproducibly generate cavities by using Mycobacterium bovis Ravenel, M. bovis AF2122, M. bovis BCG, M. tuberculosis H37Rv, M. tuberculosis CDC1551, and the M. tuberculosis CDC1551 ΔsigC mutant. M. bovis infections generated cavitary CFU counts of 106 to 109 bacilli, while non-M. bovis species and BCG yielded CFU counts that ranged from 104 to 108 bacilli. Extrapulmonary dissemination was almost exclusively noted among rabbits infected with M. bovis Ravenel and AF2122. Though all of the species yielded secondary lesions at intrapulmonary sites, M. bovis infections led to the most apparent gross pathology. Using the M. tuberculosis icl and dosR gene expression patterns as molecular sentinels, we demonstrated that both the cavity wall and cavity lumen are microenvironments associated with hypoxia, upregulation of the bacterial dormancy program, and the use of host lipids for bacterial catabolism. This unique cavitary model provides a reliable animal model to study cavity pathogenesis and extrapulmonary dissemination.

Pyrido[1,2-a]benzimidazole-Based Agents Active Against Tuberculosis (TB), Multidrug-Resistant (MDR) TB and Extensively Drug-Resistant (XDR) TB

The struggle against tuberculosis (TB) is still far from over. TB, caused by Mycobacterium tuberculosis, is one of the deadliest infections worldwide. Co‐infection with human immunodeficiency virus (HIV) and the emergence of multidrug‐resistant tuberculosis (MDR‐TB) and extensively drug‐resistant tuberculosis (XDR‐TB) strains have further increased the burden for this disease. Herein, we report the discovery of 2‐(4‐chlorobenzyl)‐3‐methyl‐1‐oxo‐1H,5H‐pyrido[1,2‐a]benzimidazole‐4‐carbonitrile as an effective antitubercular agent and the structural modifications of this molecule that have led to analogues with improved potency and lower toxicity. A number of these derivatives were also active at sub‐micromolar concentrations against resistant TB strains and devoid of apparent toxicity to Vero cells, thereby underscoring their value as novel scaffolds for the development of new anti‐TB drugs.

Successful Shortening of Tuberculosis Treatment Using Adjuvant Host-Directed Therapy with FDA-Approved Phosphodiesterase Inhibitors in the Mouse Model

Global control of tuberculosis (TB), an infectious disease that claims nearly 2 million lives annually, is hindered by the long duration of chemotherapy required for curative treatment. Lack of adherence to this intense treatment regimen leads to poor patient outcomes, development of new or additional drug resistance, and continued spread of M.tb. within communities. Hence, shortening the duration of TB therapy could increase drug adherence and cure in TB patients. Here, we report that addition of the United Stated Food and Drug Administration-approved phosphodiesterase inhibitors (PDE-Is) cilostazol and sildenafil to the standard TB treatment regimen reduces tissue pathology, leads to faster bacterial clearance and shortens the time to lung sterilization by one month, compared to standard treatment alone, in a murine model of TB. Our data suggest that these PDE-Is could be repurposed for use as adjunctive drugs to shorten TB treatment in humans.

Risk of Tuberculosis Reactivation with Tofacitinib (CP-690550)

Individuals with latent tuberculosis infection (LTBI) live with a risk of reactivation, and several treatments for chronic inflammatory conditions are highly associated with such reactivation. A new Janus kinase inhibitor, tofacitinib (CP-690550), has shown promising results for treatment of inflammatory disorders, thus raising concerns of risk of active tuberculosis. Our goal was to characterize the impact of tofacitinib on LTBI using a mouse model of contained tuberculosis. Our data indicate that tofacitinib reduces host containment of Mycobacterium tuberculosis and promotes bacterial replication in the lungs, suggesting tuberculosis reactivation. Tofacitinib may carry a significant risk for LTBI reactivation in humans.

Indole-2-carboxamide-based MmpL3 Inhibitors Show Exceptional Antitubercular Activity in an Animal Model of Tuberculosis Infection

Our team had previously identified certain indolecarboxamides that represented a new chemical scaffold that showed promising anti-TB activity at both an in vitro and in vivo level. Based on mutational analysis using bacteria found resistant to one of these indolecarboxamides, we identified the trehalose monomycolate transporter MmpL3 as the likely target of these compounds. In the present work, we now further elaborate on the SAR of these compounds, which has led in turn to the identification of a new analog, 4,6-difluoro-N-((1R,2R,3R,5S)-2,6,6-trimethylbicyclo[3.1.1]heptan-3-yl)-1H-indole-2-carboxamide (26), that shows excellent activity against drug-sensitive (MIC = 0.012 μM; SI ≥ 16000), multidrug-resistant (MDR), and extensively drug-resistant (XDR) Mycobacterium tuberculosis strains, has superior ADMET properties, and shows excellent activity in the TB aerosol lung infection model. Compound 26 is also shown to work in synergy with rifampin. Because of these properties, we believe that indolecarboxamide 26 is a possible candidate for advancement to human clinical trials.