Non-replicating Mycobacterium Research Articles

Assessment of the Efficacy of the Antihistamine Drug Rupatadine Used Alone or in Combination against Mycobacteria.

The emergence of drug-resistant mycobacteria has rendered many clinical drugs and regimens ineffective, imposing significant economic and healthcare burden on individuals and society. Repurposing drugs intended for treating other diseases is a time-saving, cost-effective, and efficient approach for identifying excellent antimycobacterial candidates or lead compounds. This study is the first to demonstrate that rupatadine (RTD), a drug used to treat allergic rhinitis, possesses excellent activity against mycobacteria without detectable resistance, particularly Mycobacterium tuberculosis and Mycobacterium marinum, with a minimal inhibitory concentration as low as 3.13 µg/mL. Furthermore, RTD exhibited moderate activity against nonreplicating M. tuberculosis with minimal inhibitory concentrations lower than drugs targeting the cell wall, suggesting that RTD has great potential to be modified and used for the treatment of nonreplicating M. tuberculosis. Additionally, RTD exhibits partial synergistic effects when combined with clofazimine, pretomanid, and TB47 against M. tuberculosis, providing the theoretical foundation for the development of treatment regimens. Transcriptomic profiling leads us to speculate that eight essential genes may be the targets of RTD or may be closely associated with mycobacterial resistance to RTD. In summary, RTD may be a promising hit for further antimycobacterial drug or regimen optimization, especially in the case of nonreplicating mycobacteria.

An O-methylflavone from Artemisia afra kills non-replicating hypoxic Mycobacterium tuberculosis

Ethnopharmacological relevanceAfrican wormwood (Artemisia afra Jacq. ex Willd.) has been used traditionally in southern Africa to treat illnesses causing fever and was recently shown to possess anti-tuberculosis activity. As tuberculosis is an endemic cause of fever in southern Africa, this suggests that the anti-tubercular activity of A. afra may have contributed to its traditional medicinal use. Aim of the studyTuberculosis, caused by Mycobacterium tuberculosis (Mtb), is a deadly and debilitating disease globally affecting millions annually. Emerging drug-resistant Mtb strains endanger the efficacy of the current therapies employed to treat tuberculosis; therefore, there is an urgent need to develop novel drugs to combat this disease. Given the reported activity of A. afra against Mtb, we sought to determine the mechanisms by which A. afra inhibits and kills this bacterium. Materials and methodsWe used transcriptomics to investigate the impact of Artemisia spp. extracts on Mtb physiology. We then used chromatographic fractionation and biochemometric analyses to identify a bioactive fractions of A. afra extracts and identify an active compound. ResultsTranscriptomic analysis revealed that A. afra exerts different effects on Mtb compared to A. annua or artemisinin, suggesting that A. afra possesses other phytochemicals with unique modes of action. A biochemometric study of A. afra resulted in the isolation of an O-methylflavone (1), 5-hydroxy-7-methoxy-2-(4-methoxyphenyl)chromen-4-one, which displayed considerable activity against Mtb strain mc26230 in both log phase growth and metabolically downshifted hypoxic cultures. ConclusionsThe present study demonstrated that an O-methylflavone constituent of Artemisia afra explains part of the activity of this plant against Mtb. This result contributes to a mechanistic understanding of the reported anti-tubercular activity of A. afra and highlights the need for further study of this traditional medicinal plant and its active compounds.

BacPROTACs targeting Clp protease: a promising strategy for anti-mycobacterial drug discovery.

Tuberculosis (TB) has claimed more lives over the course of two millennia than any other infectious disease worldwide. In 2021, the World Health Organization (WHO) estimated that 10.6 million people were diagnosed with TB, resulting in the deaths of 1.4 million HIV-negative individuals. The emergence of multidrug-resistant TB (MDR-TB), defined as resistance to at least rifampicin (RIF) and isoniazid (INH), and extensively drug-resistant TB (XDR-TB), poses the primary challenge to overcome in the coming years. We have recently conducted an extensive analysis of investments and research endeavours in the field, with the overarching objective of achieving the established milestone of TB eradication by the year 2030. Over the past several years, there has been notable progress in advancing a multitude of promising compounds, each possessing distinct mechanisms of action, into clinical phases of development. However, it is worth noting that strains of mycobacteria resistant to current antitubercular drugs have already emerged for some of these compounds The exploration of the innovative Proteolytic Target Chimeras (PROTACs) protein degradation approach has emerged as a viable avenue for the discovery of novel antimicrobials. While the ubiquitin system is exclusive to eukaryotic cells, certain bacteria use a similar degradation system that relies on the recognition of phosphorylated arginine residues (pArg) by the ClpC:ClpP (ClpCP) protease, thereby leading to protein degradation. In this opinion article, we have described and analized the advances in the use of PROTACs that leverage bacterial proteolytic machinery (BacPROTACs) to design new antitubercular agents. Scope Statement. The development of novel pharmaceuticals for tuberculosis treatment is deemed urgently necessary due to the emergence of resistant strains. In this context, the introduction of new technologies capable of alleviating the disease and attaining the objectives outlined by the World Health Organization is imperative. Among the innovative strategies, the degradation of proteins that are crucial for the survival of the bacillus holds promise for generating new medications, particularly those that are effective at treating latent (non-replicating) Mycobacterium tuberculosis. Within this perspective, we present the advancements and obstacles encountered in the exploration of new BacPROTAC compounds, with the intention of encouraging research and illuminating challenges associated with the implementation of BacPROTACs to address to the global tuberculosis crisis.

Both Nitro Groups Are Essential for High Antitubercular Activity of 3,5-Dinitrobenzylsulfanyl Tetrazoles and 1,3,4-Oxadiazoles through the Deazaflavin-Dependent Nitroreductase Activation Pathway.

3,5-Dinitrobenzylsulfanyl tetrazoles and 1,3,4-oxadiazoles, previously identified as having high in vitro activities against both replicating and nonreplicating mycobacteria and favorable cytotoxicity and genotoxicity profiles were investigated. First we demonstrated that these compounds act in a deazaflavin-dependent nitroreduction pathway and thus require a nitro group for their activity. Second, we confirmed the necessity of both nitro groups for antimycobacterial activity through extensive structure-activity relationship studies using 32 structural types of analogues, each in a five-membered series. Only the analogues with shifted nitro groups, namely, 2,5-dinitrobenzylsulfanyl oxadiazoles and tetrazoles, maintained high antimycobacterial activity but in this case mainly as a result of DprE1 inhibition. However, these analogues also showed increased toxicity to the mammalian cell line. Thus, both nitro groups in 3,5-dinitrobenzylsulfanyl-containing antimycobacterial agents remain essential for their high efficacy, and further efforts should be directed at finding ways to address the possible toxicity and solubility issues, for example, by targeted delivery.

A dual-targeting succinate dehydrogenase and F1Fo-ATP synthase inhibitor rapidly sterilizes replicating and non-replicating Mycobacterium tuberculosis

Mycobacterial bioenergetics is a validated target space for antitubercular drug development. Here, we identify BB2-50F, a 6-substituted 5-(N,N-hexamethylene)amiloride derivative as a potent, multi-targeting bioenergetic inhibitor of Mycobacterium tuberculosis. We show that BB2-50F rapidly sterilizes both replicating and non-replicating cultures of M.tuberculosis and synergizes with several tuberculosis drugs. Target identification experiments, supported by docking studies, showed that BB2-50F targets the membrane-embedded c-ring of the F1Fo-ATP synthase and the catalytic subunit (substrate-binding site) of succinate dehydrogenase. Biochemical assays and metabolomic profiling showed that BB2-50F inhibits succinate oxidation, decreases the activity of the tricarboxylic acid (TCA) cycle, and results in succinate secretion from M.tuberculosis. Moreover, we show that the lethality of BB2-50F under aerobic conditions involves the accumulation of reactive oxygen species. Overall, this study identifies BB2-50F as an effective inhibitor of M.tuberculosis and highlights that targeting multiple components of the mycobacterial respiratory chain can produce fast-acting antimicrobials.

Dynamic action of an intrinsically disordered protein in DNA compaction that induces mycobacterial dormancy.

Mycobacteria are the major human pathogens with the capacity to become dormant persisters. Mycobacterial DNA-binding protein 1 (MDP1), an abundant histone-like protein in dormant mycobacteria, induces dormancy phenotypes, e.g. chromosome compaction and growth suppression. For these functions, the polycationic intrinsically disordered region (IDR) is essential. However, the disordered property of IDR stands in the way of clarifying the molecular mechanism. Here we clarified the molecular and structural mechanism of DNA compaction by MDP1. Using high-speed atomic force microscopy, we observed that monomeric MDP1 bundles two adjacent DNA duplexes side-by-side via IDR. Combined with coarse-grained molecular dynamics simulation, we revealed the noveldynamic DNA cross-linking model of MDP1 in which a stretched IDR cross-links two DNA duplexes like double-sided tape. IDR is able to hijack HU function, resulting in the induction of strong mycobacterial growth arrest. This IDR-mediated reversible DNA cross-linking is a reasonable model for MDP1 suppression of the genomic function in the resuscitable non-replicating dormant mycobacteria.

Free energy landscape and thermodynamics properties of novel mutations in PncA of pyrazinamide resistance isolates of Mycobacterium tuberculosis

Pyrazinamide (PZA) is one of the first-line antituberculosis therapy, active against non-replicating Mycobacterium tuberculosis (Mtb). The conversion of PZA into pyrazinoic acid (POA), the active form, required the activity of pncA gene product pyrazinamidase (PZase) activity. Mutations occurred in pncA are the primary cause behind the PZA resistance. However, the resistance mechanism is important to explore using high throughput computational approaches. Here we aimed to explore the mechanism of PZA resistance behind novel P62T, L120R, and V130M mutations in PZase using 200 ns molecular dynamics (MD) simulations. MD simulations were performed to observe the structural changes for these three mutants (MTs) compared to the wild types (WT). Root means square fluctuation, the radius of gyration, free energy landscape, root means square deviation, dynamic cross-correlation motion, and pocket volume were found in variation between WT and MTs, revealing the effects of P62T, L120R, and V130M. The free energy conformational landscape of MTs differs significantly from the WT system, lowering the binding of PZA. The geometric shape complementarity of the drug (PZA) and target protein (PZase) further confirmed that P62T, L120R, and V130M affect the protein structure. These effects on PZase may cause vulnerability to convert PZA into POA. Communicated by Ramaswamy H. Sarma

Benzene Amide Ether Scaffold is Active against Non-replicating and Intracellular Mycobacterium tuberculosis.

New drugs to treat tuberculosis which target intractable bacterial populations are required to develop shorter and more effective treatment regimens. The benzene amide ether scaffold has activity against intracellular Mycobacterium tuberculosis, but low activity against extracellular, actively replicating M. tuberculosis. We determined that these molecules have bactericidal activity against non-replicating M. tuberculosis but not actively replicating bacteria. Exposure to compounds depleted ATP levels in non-replicating bacteria and increased the oxygen consumption rate; a subset of molecules led to the accumulation of intrabacterial reactive oxygen species. A comprehensive screen of M. tuberculosis strains identified a number of under-expressing strains as more sensitive to compounds under replicating conditions including QcrA and QcrB hypomorphs. We determined the global gene expression profile after compound treatment for both replicating and nutrient-starved M. tuberculosis. We saw compound-dependent changes in the expression of genes involved in energy metabolism under both conditions. Taken together, our data suggest that the scaffold targets respiration in M. tuberculosis.

Mycobacterium tuberculosis Inhibitors Based on Arylated Quinoline Carboxylic Acid Backbones with Anti-Mtb Gyrase Activity.

New antitubercular agents with either a novel mode of action or novel mode of inhibition are urgently needed to overcome the threat of drug-resistant tuberculosis (TB). The present study profiles new arylated quinoline carboxylic acids (QCAs) having activity against replicating and non-replicating Mycobacterium tuberculosis (Mtb), the causative agent of TB. Thus, the synthesis, characterization, and in vitro screening (MABA and LORA) of 48 QCAs modified with alkyl, aryl, alkoxy, halogens, and nitro groups in the quinoline ring led to the discovery of two QCA derivatives, 7i and 7m, adorned with C-2 2-(naphthalen-2-yl)/C-6 1-butyl and C-2 22-(phenanthren-3-yl)/C-6 isopropyl, respectively, as the best Mtb inhibitors. DNA gyrase inhibition was shown to be exhibited by both, with QCA 7m illustrating better activity up to a 1 μM test concentration. Finally, a docking model for both compounds with Mtb DNA gyrase was developed, and it showed a good correlation with in vitro results.

Unravelling the flexibility of Mycobacterium tuberculosis: an escape way for the bacilli.

The persistence of Mycobacterium tuberculosis makes it difficult to eradicate the associated infection from the host. The flexible nature of mycobacteria and their ability to adapt to adverse host conditions give rise to different drug-tolerant phenotypes. Granuloma formation restricts nutrient supply, limits oxygen availability and exposes bacteria to a low pH environment, resulting in non-replicating bacteria. These non-replicating mycobacteria, which need high doses and long exposure to anti-tubercular drugs, are the root cause of lengthy chemotherapy. Novel strategies, which are effective against non-replicating mycobacteria, need to be adopted to shorten tuberculosis treatment. This not only will reduce the treatment time but also will help prevent the emergence of multi-drug-resistant strains of mycobacteria.

Stress-induced non-replicating Mycobacterium smegmatis incorporates exogenous fatty acids into glycopeptidolipids.

Nontuberculous mycobacteria (NTM) such as Mycobacterium smegmatis accumulate high levels of glycopeptidolipids (GPLs) on their outer surface. The biosynthesis of GPLs is critically linked to biofilm formation by NTM which also includes opportunistic pathogens such as Mycobacterium abscessus. Although GPLs have been investigated in many earlier studies, the biosynthesis of GPLs using exogenous fatty acids in M. smegmatis subjected to stresses encountered by mycobacteria during infection of the human body has not been studied. Therefore, we subjected M. smegmatis to different combinations of the three stresses of hypoxia, acidic pH and nutrient starvation and report here that the metabolic incorporation of radiolabeled long-chain fatty acids into alkali-stable GPLs was significantly increased under these stress conditions. Endogenously synthesized fatty acids were not preferred for GPL biosynthesis by M. smegmatis subjected to the triple stress combination. Our observations indicate that GPLs may play important roles in cell surface modifications associated with the non-replicating state of M. smegmatis. Our experimental model reported here would be useful in the further study of GPL biosynthesis from exogenous fatty acid sources in M. smegmatis subjected to hypoxia, nutrient starvation and acidic stress conditions and help in the screening of candidate drugs that target this biochemical pathway in pathogenic NTM.

Cholesterol-dependent activity of dapsone against non-replicating persistent mycobacteria.

One-third of the world's population is estimated to be latently infected with Mycobacterium tuberculosis. This reservoir of bacteria is largely resistant to antimicrobial treatment that often only targets actively replicating mycobacteria, with current treatment for latent infection revolving around inhibiting the resuscitation event rather than preventing or treating latent infection. As a result, antimicrobials that target latent infection often have little to no activity in vivo. Here we report a method of in vitro analysis of physiologically relevant non-replicating persistence (NRP) utilizing cholesterol as the sole carbon source, alongside hypoxia as a driver of Mycobacterium bovis BCG into the NRP state. Using the minimal cholesterol media NRP assay, we observed an increased state of in vitro resistance to front-line anti-tubercular compounds. However, following a phenotypic screen of an approved-drug library, we identified dapsone as a bactericidal active molecule against cholesterol-dependent NRP M. bovis BCG. Through an overexpression trial of probable antimicrobial target enzymes, we further identified FolP2, a non-functional dihydropteroate synthase homologue, as the likely target of dapsone under cholesterol-NRP due to a significant increase in bacterial resistance when overexpressed. These results highlight the possible reason for little in vivo activity seen for current front-line anti-NRP drugs, and we introduce a new methodology for future drug screening as well as a potential role for dapsone inclusion within the current treatment regime.

Structure activity relationship of pyrazinoic acid analogs as potential antimycobacterial agents

Tuberculosis (TB) remains a leading cause of infectious disease-related mortality and morbidity. Pyrazinamide (PZA) is a critical component of the first-line TB treatment regimen because of its sterilizing activity against non-replicating Mycobacterium tuberculosis (Mtb), but its mechanism of action has remained enigmatic. PZA is a prodrug converted by pyrazinamidase encoded by pncA within Mtb to the active moiety, pyrazinoic acid (POA) and PZA resistance is caused by loss-of-function mutations to pyrazinamidase. We have recently shown that POA induces targeted protein degradation of the enzyme PanD, a crucial component of the coenzyme A biosynthetic pathway essential in Mtb. Based on the newly identified mechanism of action of POA, along with the crystal structure of PanD bound to POA, we designed several POA analogs using structure for interpretation to improve potency and overcome PZA resistance. We prepared and tested ring and carboxylic acid bioisosteres as well as 3, 5, 6 substitutions on the ring to study the structure activity relationships of the POA scaffold. All the analogs were evaluated for their whole cell antimycobacterial activity, and a few representative molecules were evaluated for their binding affinity, towards PanD, through isothermal titration calorimetry. We report that analogs with ring and carboxylic acid bioisosteres did not significantly enhance the antimicrobial activity, whereas the alkylamino-group substitutions at the 3 and 5 position of POA were found to be up to 5 to 10–fold more potent than POA. Further development and mechanistic analysis of these analogs may lead to a next generation POA analog for treating TB.

Latent Tuberculosis: A Promising New Compound to Treat Non-Replicating and Intramacrophagic Mycobacteria.

As a biologic reservoir of Mycobacterium tuberculosis (M. tb), one-quarter of the world population is infected with the well-known latent tuberculosis (LTBI). About 5–10% of LTBI patients will progress to active disease in the first years after primary infection and, despite using the recommended treatment, 20% can still reactivate the infection. A new LTBI treatment could minimize adverse effects and antibiotic resistance that can occur when the same drug is used to treat the latent and active disease. New hydrazones were evaluated, and they showed great inhibitory activity against intramacrophagic and non-replicating M. tb, commonly found at this stage of infection, in addition to bactericidal and narrow-spectrum activity. When tested against eukaryotic cells, the hydrazones showed great safety at different exposure times. In vitro, these compounds performed better than isoniazid and could be considered new candidates for LTBI treatment, which may promote greater engagement in its prescription and adherence.

Targeting Non-Replicating Mycobacterium tuberculosis and Latent Infection: Alternatives and Perspectives (Mini-Review).

Latent tuberculosis infection (LTBI) represents a major challenge to curing TB disease. Current guidelines for LTBI management include only three older drugs and their combinations—isoniazid and rifamycins (rifampicin and rifapentine). These available control strategies have little impact on latent TB elimination, and new specific therapeutics are urgently needed. In the present mini-review, we highlight some of the alternatives that may potentially be included in LTBI treatment recommendations and a list of early-stage prospective small molecules that act on drug targets specific for Mycobacterium tuberculosis latency.

Deletion of pknG Abates Reactivation of Latent Mycobacterium tuberculosis in Mice.

Eradication of tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), has been a challenge due to its uncanny ability to survive in a dormant state inside host granulomas for decades. Mtb rewires its metabolic and redox regulatory networks to survive in the hostile hypoxic and nutrient-limiting environment, facilitating the formation of drug-tolerant persisters. Previously, we showed that protein kinase G (PknG), a virulence factor required for lysosomal escape, aids in metabolic adaptation, thereby promoting the survival of nonreplicating mycobacteria. Here, we sought to investigate the therapeutic potential of PknG against latent mycobacterium. We show that inhibition of PknG by AX20017 reduces mycobacterial survival in in vitro latency models such as hypoxia, persisters, and nutrient starvation. Targeting PknG enhances the bactericidal activity of the frontline anti-TB drugs in peritoneal macrophages. Deletion of pknG resulted in 5- to 15-fold-reduced survival of Mtb in chronically infected mice treated with anti-TB drugs. Importantly, in the Cornell mouse model of latent TB, the deletion of pknG drastically attenuated Mtb's ability to resuscitate after antibiotic treatment compared with wild-type and complemented strains. This is the first study to investigate the sterilizing activity of pknG deletion and inhibition for adjunct therapy against latent TB in a preclinical model. Collectively, these results suggest that PknG may be a promising drug target for adjunct therapy to shorten the treatment duration and reduce disease relapse.

Design and Synthesis of Pyrano[3,2-b]indolones Showing Antimycobacterial Activity.

Latent Mycobacterium tuberculosis infection presents one of the largest challenges for tuberculosis control and novel antimycobacterial drug development. A series of pyrano[3,2-b]indolone-based compounds was designed and synthesized via an original eight-step scheme. The synthesized compounds were evaluated for their in vitro activity against M. tuberculosis strains H37Rv and streptomycin-starved 18b (SS18b), representing models for replicating and nonreplicating mycobacteria, respectively. Compound 10a exhibited good activity with MIC99 values of 0.3 and 0.4 μg/mL against H37Rv and SS18b, respectively, as well as low toxicity, acceptable intracellular activity, and satisfactory metabolic stability and was selected as the lead compound for further studies. An analysis of 10a-resistant M. bovis mutants disclosed a cross-resistance with pretomanid and altered relative amounts of different forms of cofactor F420 in these strains. Complementation experiments showed that F420-dependent glucose-6-phosphate dehydrogenase and the synthesis of mature F420 were important for 10a activity. Overall these studies revealed 10a to be a prodrug that is activated by an unknown F420-dependent enzyme in mycobacteria.

Dual inhibition of the terminal oxidases eradicates antibiotic-tolerant Mycobacterium tuberculosis.

The approval of bedaquiline has placed energy metabolism in the limelight as an attractive target space for tuberculosis antibiotic development. While bedaquiline inhibits the mycobacterial F1F0 ATP synthase, small molecules targeting other components of the oxidative phosphorylation pathway have been identified. Of particular interest is Telacebec (Q203), a phase 2 drug candidate inhibitor of the cytochrome bcc:aa3 terminal oxidase. A functional redundancy between the cytochrome bcc:aa3 and the cytochrome bd oxidase protects M. tuberculosis from Q203‐induced death, highlighting the attractiveness of the bd‐type terminal oxidase for drug development. Here, we employed a facile whole‐cell screen approach to identify the cytochrome bd inhibitor ND‐011992. Although ND‐011992 is ineffective on its own, it inhibits respiration and ATP homeostasis in combination with Q203. The drug combination was bactericidal against replicating and antibiotic‐tolerant, non‐replicating mycobacteria, and increased efficacy relative to that of a single drug in a mouse model. These findings suggest that a cytochrome bd oxidase inhibitor will add value to a drug combination targeting oxidative phosphorylation for tuberculosis treatment.

InhA inhibitors have activity against non-replicating Mycobacterium tuberculosis.

We previously identified a diazaborine series with potential for development as a new tuberculosis drug. This series has activity in vitro and in vivo and targets cell wall biosynthesis via inhibition of InhA. The overall aim of this study was to determine whether InhA inhibitors have activity against non-replicating Mycobacterium tuberculosis. We tested the ability of two molecules of the diazaborine series to kill non-replicating M. tuberculosis in the nutrient starvation model; both molecules were bactericidal, reducing viability by >3 logs in 21 days. Activity showed similar kill rates to other InhA inhibitors (isoniazid and NITD-916). We conclude that inhibition of InhA is bactericidal against nutrient-starved non-replicating M. tuberculosis.

Activity-Based Protein Profiling Reveals That Cephalosporins Selectively Active on Non-replicating Mycobacterium tuberculosis Bind Multiple Protein Families and Spare Peptidoglycan Transpeptidases.

As β-lactams are reconsidered for the treatment of tuberculosis (TB), their targets are assumed to be peptidoglycan transpeptidases, as verified by adduct formation and kinetic inhibition of Mycobacterium tuberculosis (Mtb) transpeptidases by carbapenems active against replicating Mtb. Here, we investigated the targets of recently described cephalosporins that are selectively active against non-replicating (NR) Mtb. NR-active cephalosporins failed to inhibit recombinant Mtb transpeptidases. Accordingly, we used alkyne analogs of NR-active cephalosporins to pull down potential targets through unbiased activity-based protein profiling and identified over 30 protein binders. None was a transpeptidase. Several of the target candidates are plausibly related to Mtb’s survival in an NR state. However, biochemical tests and studies of loss of function mutants did not identify a unique target that accounts for the bactericidal activity of these beta-lactams against NR Mtb. Instead, NR-active cephalosporins appear to kill Mtb by collective action on multiple targets. These results highlight the ability of these β-lactams to target diverse classes of proteins.