Antima Gupta

Mycobacterium tuberculosis: Immune evasion, latency and reactivation

One-third of the global human population harbours Mycobacterium tuberculosis in dormant form. This dormant or latent infection presents a major challenge for global efforts to eradicate tuberculosis, because it is a vast reservoir of potential reactivation and transmission. This article explains how the pathogen evades the host immune response to establish a latent infection, and how it emerges from a state of latency to cause reactivation disease. This review highlights the key factors responsible for immune evasion and reactivation. It concludes by identifying interesting candidates for drug or vaccine development, as well as identifying unresolved questions for the future research.

Anti-tubercular screening of natural products from Colombian plants: 3-methoxynordomesticine, an inhibitor of MurE ligase of Mycobacterium tuberculosis

OBJECTIVES New anti-mycobacterial entities with novel mechanisms of action are clinically needed for treating resistant forms of tuberculosis. The purpose of this study was to evaluate anti-tubercular activity and selectivity of seven recently isolated natural products from Colombian plants. METHODS MICs were determined using a liquid medium growth inhibition assay for Mycobacterium tuberculosis H(37)Rv and both solid and liquid media growth inhibition assays for Mycobacterium bovis BCG. Escherichia coli growth inhibition and mammalian macrophage cell toxicity were evaluated to establish the degree of selectivity of the natural product against whole cell organisms. Enzymatic inhibition of ATP-dependent MurE ligase from M. tuberculosis was assayed using a colorimetric phosphate detection method. The most active compound, 3-methoxynordomesticine hydrochloride, was further investigated on M. bovis BCG for its inhibition of sigmoidal growth, acid-fast staining and viability counting analysis. RESULTS Aporphine alkaloids were found to be potent inhibitors of slow-growing mycobacterial pathogens showing favourable selectivity and cytotoxicity. In terms of their endogenous action, the aporphine alkaloids were found inhibitory to M. tuberculosis ATP-dependent MurE ligase at micromolar concentrations. A significantly low MIC was detected for 3-methoxynordomesticine hydrochloride against both M. bovis BCG and M. tuberculosis H(37)Rv. CONCLUSIONS Considering all the data, 3-methoxynordomesticine hydrochloride was found to be a potent anti-tubercular compound with a favourable specificity profile. The alkaloid showed MurE inhibition and is considered an initial hit for exploring related chemical space.

Antitubercular specific activity of ibuprofen and the other 2-arylpropanoic acids using the HT-SPOTi whole-cell phenotypic assay

Objectives Lead antituberculosis (anti-TB) molecules with novel mechanisms of action are urgently required to fuel the anti-TB drug discovery pipeline. The aim of this study was to validate the use of the high-throughput spot culture growth inhibition (HT-SPOTi) assay for screening libraries of compounds against Mycobacterium tuberculosis and to study the inhibitory effect of ibuprofen (IBP) and the other 2-arylpropanoic acids on the growth inhibition of M tuberculosis and other mycobacterial species. Methods The HT-SPOTi method was validated not only with known drugs but also with a library of 47 confirmed anti-TB active compounds published in the ChEMBL database. Three over-the-counter non-steroidal anti-inflammatory drugs were also included in the screening. The 2-arylpropanoic acids, including IBP, were comprehensively evaluated against phenotypically and physiologically different strains of mycobacteria, and their cytotoxicity was determined against murine RAW264.7 macrophages. Furthermore, a comparative bioinformatic analysis was employed to propose a potential mycobacterial target. Results IBP showed antitubercular properties while carprofen was the most potent among the 2-arylpropanoic class. A 3,5-dinitro-IBP derivative was found to be more potent than IBP but equally selective. Other synthetic derivatives of IBP were less active, and the free carboxylic acid of IBP seems to be essential for its anti-TB activity. IBP, carprofen and the 3,5-dinitro-IBP derivative exhibited activity against multidrug-resistant isolates and stationary phase bacilli. On the basis of the human targets of the 2-arylpropanoic analgesics, the protein initiation factor infB (Rv2839c) of M tuberculosis was proposed as a potential molecular target. Conclusions The HT-SPOTi method can be employed reliably and reproducibly to screen the antimicrobial potency of different compounds. IBP demonstrated specific antitubercular activity, while carprofen was the most selective agent among the 2-arylpropanoic class. Activity against stationary phase bacilli and multidrug-resistant isolates permits us to speculate a novel mechanism of antimycobacterial action. Further medicinal chemistry and target elucidation studies could potentially lead to new therapies against TB.

Characterisation of ATP-Dependent Mur Ligases Involved in the Biogenesis of Cell Wall Peptidoglycan in Mycobacterium tuberculosis

ATP-dependent Mur ligases (Mur synthetases) play essential roles in the biosynthesis of cell wall peptidoglycan (PG) as they catalyze the ligation of key amino acid residues to the stem peptide at the expense of ATP hydrolysis, thus representing potential targets for antibacterial drug discovery. In this study we characterized the division/cell wall (dcw) operon and identified a promoter driving the co-transcription of mur synthetases along with key cell division genes such as ftsQ and ftsW. Furthermore, we have extended our previous investigations of MurE to MurC, MurD and MurF synthetases from Mycobacterium tuberculosis. Functional analyses of the pure recombinant enzymes revealed that the presence of divalent cations is an absolute requirement for their activities. We also observed that higher concentrations of ATP and UDP-sugar substrates were inhibitory for the activities of all Mur synthetases suggesting stringent control of the cytoplasmic steps of the peptidoglycan biosynthetic pathway. In line with the previous findings on the regulation of mycobacterial MurD and corynebacterial MurC synthetases via phosphorylation, we found that all of the Mur synthetases interacted with the Ser/Thr protein kinases, PknA and PknB. In addition, we critically analyzed the interaction network of all of the Mur synthetases with proteins involved in cell division and cell wall PG biosynthesis to re-evaluate the importance of these key enzymes as novel therapeutic targets in anti-tubercular drug discovery.

An integrated surrogate model for screening of drugs against Mycobacterium tuberculosis

OBJECTIVES The intracellularly surviving and slow-growing pathogen, Mycobacterium tuberculosis, adapts the host cell environment for its active and dormant life cycle. It is evident that the lack of appropriate high-throughput screening of inhibitors within host cells is an impediment for the early stages of anti-tubercular drug discovery. We aimed to develop an integrated surrogate model that enhances the screening of large inhibitor libraries. METHODS Different mycobacterial species were compared for their growth, drug susceptibility and intracellular uptake. A 6-well plate solid agar-based spot culture growth inhibition (SPOTi) assay was developed into a higher throughput format. The uptake and intracellular survival of Mycobacterium aurum within mouse macrophage cells (RAW 264.7) were optimized using 24/96-well plate formats. RESULTS Fast-growing, non-pathogenic M. aurum was found to have an antibiotic-susceptibility profile similar to that of M. tuberculosis. The sensitivity to an acidic pH environment and the ability to multiply inside RAW 264.7 macrophages provided additional advantages for employing M. aurum in intracellular drug screening methods. A selection of anti-tubercular drugs inhibited the growth and viability of M. aurum inside the macrophages at different levels. CONCLUSIONS We present a rapid, convenient, high-throughput surrogate model, which provides a comprehensive evaluation platform for new chemical scaffolds against different physiological stages of mycobacteria within the primary cell environment of the host. The results using anti-tubercular drugs validate this model for screening libraries of existing and novel chemical entities.

An antibacterial from Hypericum acmosepalum inhibits ATP-dependent MurE ligase from Mycobacterium tuberculosis.

In a project to characterise new antibacterial chemotypes from plants, hyperenone A and hypercalin B were isolated from the hexane and chloroform extracts of the aerial parts of Hypericum acmosepalum. The structures of both compounds were characterised by extensive one- and two-dimensional nuclear magnetic resonance (NMR) spectroscopy and were confirmed by mass spectrometry. Hyperenone A and hypercalin B exhibited antibacterial activity against multidrug-resistant strains of Staphylococcus aureus, with minimum inhibition concentration ranges of 2–128 mg/L and 0.5–128 mg/L, respectively. Hyperenone A also showed growth-inhibitory activity against Mycobacterium tuberculosis H37Rv and Mycobacterium bovis BCG at 75 mg/L and 100 mg/L. Neither hyperenone A nor hypercalin B inhibited the growth of Escherichia coli and both were non-toxic to cultured mammalian macrophage cells. Both compounds were tested for their ability to inhibit the ATP-dependent MurE ligase of M. tuberculosis, a crucial enzyme in the cytoplasmic steps of peptidoglycan biosynthesis. Hyperenone A inhibited MurE selectively, whereas hypercalin B did not have any effect on enzyme activity.

Fast-growing, non-infectious and intracellularly surviving drug-resistant Mycobacterium aurum: a model for high-throughput antituberculosis drug screening

OBJECTIVES Enoyl acyl-carrier-protein reductase (InhA), the primary endogenous target for isoniazid and ethionamide, is crucial to type-II fatty acid biosynthesis (FAS-II). The objectives of this study were first to generate InhA mutants of Mycobacterium aurum, secondly to characterize InhA-mediated isoniazid and ethionamide resistance mechanisms across those mutants and finally to investigate the interaction of InhA with enzymes in the FAS-II pathway in M. aurum. METHODS Spontaneous mutants were generated by isoniazid overdose and limited broth dilution, while for genetically modified mutants sense-antisense DNA technology was used. Southern hybridization and immunoprecipitation were both used to identify the InhA homologue in M. aurum. The latter method was further used to compare the level of InhA expression in M. aurum with that in corresponding mutants. Isoniazid/ethionamide susceptibility modulation was examined in vitro and ex vivo using a resazurin assay as well as by cfu counting. In addition, circular dichroism and the bacterial two-hybrid system were exploited to investigate the interaction of InhA with other enzymes of the FAS-II pathway. RESULTS A Mycobacterium tuberculosis InhA homologue was detected in M. aurum. Susceptibility to isoniazid/ethionamide was significantly altered in genetically modified mutants and simultaneously InhA was overexpressed in both spontaneous and genetically modified mutants. InhA interacts with other FAS-II enzymes of M. aurum in vivo. CONCLUSION Close resemblance of isoniazid/ethionamide action on InhA between M. tuberculosis and M. aurum further supports the use of fast-growing and intracellularly surviving drug-resistant M. aurum to substitute for highly virulent, extremely slow-growing M. tuberculosis strains in the early stage of antituberculosis inhibitor screening.

Antimycobacterials from natural sources: ancient times, antibiotic era and novel scaffolds.

Mycobacteria are a group of aerobic, non-motile, acid fast bacteria that have a characteristic cell wall composed of a mycolyl-arabinogalactan-peptidoglycan complex. They display different phenotypic attributes in their growth, color and biochemistry. Tuberculosis (TB) is defined as the infection with Mycobacterium tuberculosis complex and was declared a global health emergency principally because of the appearance of multidrug-resistant strains and the associated risk of infection in immune-compromised population. There is an urgent clinical need for novel, potent and safe anti-TB drugs. Natural products have been used since antiquity for treating diverse complaints and novel pharmacophores are discovered every year. Two of the most potent used antimycobacterials, the rifamycins and streptomycin, were first detected in Streptomyces bacteria. Plants are also the source of an exquisite variety of antimicrobials that can lead to useful therapeutics in the future. In this review, natural preparations used since antiquity for treating tuberculosis are described, together with a rapid view of the 20th century antibiotic development against TB. Finally a summary of the most potent recent natural antimycobacterials is displayed.

Interaction of N-methyl-2-alkenyl-4-quinolones with ATP-dependent MurE ligase of Mycobacterium tuberculosis: antibacterial activity, molecular docking and inhibition kinetics

Objectives The aim of this study was to comprehensively evaluate the antibacterial activity and MurE inhibition of a set of N-methyl-2-alkenyl-4-quinolones found to inhibit the growth of fast-growing mycobacteria. Methods Using the spot culture growth inhibition assay, MICs were determined for Mycobacterium tuberculosis H37Rv, Mycobacterium bovis BCG and Mycobacterium smegmatis mc2155. MICs were determined for Mycobacterium fortuitum, Mycobacterium phlei, methicillin-resistant Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa using microplate dilution assays. Inhibition of M. tuberculosis MurE ligase activity was determined both by colorimetric and HPLC methods. Computational modelling and binding prediction of the quinolones in the MurE structure was performed using Glide. Kinetic experiments were conducted for understanding possible competitive relations of the quinolones with the endogenous substrates of MurE ligase. Results The novel synthetic N-methyl-2-alkenyl-4-quinolones were found to be growth inhibitors of M. tuberculosis and rapid-growing mycobacteria as well as methicillin-resistant S. aureus, while showing no inhibition for E. coli and P. aeruginosa. The quinolones were found to be inhibitory to MurE ligase of M. tuberculosis in the micromolar range (IC50 ∼40–200 μM) when assayed either spectroscopically or by HPLC. Computational docking of the quinolones on the published M. tuberculosis MurE crystal structure suggested that the uracil recognition site is a probable binding site for the quinolones. Conclusions N-methyl-2-alkenyl-4-quinolones are inhibitors of mycobacterial and staphylococcal growth, and show MurE ligase inhibition. Therefore, they are considered as a starting point for the development of increased affinity MurE activity disruptors.

Antimycobacterials from Lovage Root (Ligusticum officinale Koch)

The n‐hexane extract of Lovage root was found to significantly inhibit the growth of both Mycobacterium smegmatis mc2155 and Mycobacterium bovis BCG, and therefore a bioassay‐guided isolation strategy was undertaken. (Z)‐Ligustilide, (Z)‐3‐butylidenephthalide, (E)‐3‐butylidenephthalide, 3‐butylphthalide, α‐prethapsenol, falcarindiol, levistolide A, psoralen and bergapten were isolated by chromatographic techniques, characterized by NMR spectroscopy and MS, and evaluated for their growth inhibition activity against Mycobacterium tuberculosis H37Rv using the whole‐cell phenotypic spot culture growth inhibition assay (SPOTi). Cytotoxicity against RAW 264.7 murine macrophage cells was employed for assessing their degree of selectivity. Falcarindiol was the most potent compound with a minimum inhibitory concentration (MIC) value of 20 mg/L against the virulent H37Rv strain; however, it was found to be cytotoxic with a half‐growth inhibitory concentration (GIC50) in the same order of magnitude (SI < 1). Interestingly the sesquiterpene alcohol α‐prethapsenol was found to inhibit the growth of the pathogenic mycobacteria with an MIC value of 60 mg/L, being more specific towards mycobacteria than mammalian cells (SI ~ 2). Colony forming unit analysis at different concentrations of this phytochemical showed mycobacteriostatic mode of action. Copyright