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.

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 againstMycobacterium tuberculosisand to study the inhibitory effect of ibuprofen (IBP) and the other 2-arylpropanoic acids on the growth inhibition ofM tuberculosisand 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) ofM tuberculosiswas 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.

The number of cases of drug-resistant Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), has risen rapidly in recent years. This has led to the resurgence in repurposing existing drugs, such as non-steroidal anti-inflammatory drugs (NSAIDs), for anti-TB treatment. Evidence from novel drug screening in vitro, in vivo, pharmacokinetic/pharmacodynamics analyses and clinical trials has been used for the preparation of this systematic review of the potential of NSAIDs for use as an adjunct in new TB chemotherapies. Certain NSAIDs have demonstrated inhibitory properties towards actively replicating, dormant and drug-resistant clinical isolates of M. tuberculosis cells. NSAIDs are a diverse class of drugs, which have reported off-target activities, and their endogenous antimicrobial mechanism(s) of action is still unclear. It is essential that clinical trials of NSAIDs continue, in order to assess their suitability for addition to the current TB treatment regimen. Repurposing molecules such as NSAIDs is a vital, low-risk strategy to combat the trend of rapidly increasing antibiotic resistance.

Summary: Biosynthesis of the nucleotide sugar precursor dTDP‐L‐rhamnose is critical for the viability and virulence of many human pathogenic bacteria, including Streptococcus pyogenes (Group A Streptococcus; GAS), Streptococcus mutans and Mycobacterium tuberculosis. Streptococcal pathogens require dTDP‐L‐rhamnose for the production of structurally similar rhamnose polysaccharides in their cell wall. Via heterologous expression in S. mutans, we confirmed that GAS RmlB and RmlC are critical for dTDP‐L‐rhamnose biosynthesis through their action as dTDP‐glucose‐4,6‐dehydratase and dTDP‐4‐keto‐6‐deoxyglucose‐3,5‐epimerase enzymes respectively. Complementation with GAS RmlB and RmlC containing specific point mutations corroborated the conservation of previous identified catalytic residues. Bio‐layer interferometry was used to identify and confirm inhibitory lead compounds that bind to GAS dTDP‐rhamnose biosynthesis enzymes RmlB, RmlC and GacA. One of the identified compounds, Ri03, inhibited growth of GAS, other rhamnose‐dependent streptococcal pathogens as well as M. tuberculosis with an IC₅₀ of 120–410 µM. Importantly, we confirmed that Ri03 inhibited dTDP‐L‐rhamnose formation in a concentration‐dependent manner through a biochemical assay with recombinant rhamnose biosynthesis enzymes. We therefore conclude that inhibitors of dTDP‐L‐rhamnose biosynthesis, such as Ri03, affect streptococcal and mycobacterial viability and can serve as lead compounds for the development of a new class of antibiotics that targets dTDP‐rhamnose biosynthesis in pathogenic bacteria.

New treatment options are needed to maintain and improve therapy for tuberculosis, which caused the death of 1.5 million people in 2013 despite potential for an 86 % treatment success rate. A greater understanding of Mycobacterium tuberculosis (M.tb) bacilli that persist through drug therapy will aid drug development programs. Predictive biomarkers for treatment efficacy are also a research priority. Genome-wide transcriptional profiling was used to map the mRNA signatures of M.tb from the sputa of 15 patients before and 3, 7 and 14 days after the start of standard regimen drug treatment. The mRNA profiles of bacilli through the first 2 weeks of therapy reflected drug activity at 3 days with transcriptional signatures at days 7 and 14 consistent with reduced M.tb metabolic activity similar to the profile of pre-chemotherapy bacilli. These results suggest that a pre-existing drug-tolerant M.tb population dominates sputum before and after early drug treatment, and that the mRNA signature at day 3 marks the killing of a drug-sensitive sub-population of bacilli. Modelling patient indices of disease severity with bacterial gene expression patterns demonstrated that both microbiological and clinical parameters were reflected in the divergent M.tb responses and provided evidence that factors such as bacterial load and disease pathology influence the host-pathogen interplay and the phenotypic state of bacilli. Transcriptional signatures were also defined that predicted measures of early treatment success (rate of decline in bacterial load over 3 days, TB test positivity at 2 months, and bacterial load at 2 months). This study defines the transcriptional signature of M.tb bacilli that have been expectorated in sputum after two weeks of drug therapy, characterizing the phenotypic state of bacilli that persist through treatment. We demonstrate that variability in clinical manifestations of disease are detectable in bacterial sputa signatures, and that the changing M.tb mRNA profiles 0–2 weeks into chemotherapy predict the efficacy of treatment 6 weeks later. These observations advocate assaying dynamic bacterial phenotypes through drug therapy as biomarkers for treatment success.