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

[This corrects the article DOI: 10.1371/journal.pone.0060143.].

Atmospheric Microplastics: Perspectives on Origin, Abundances, Ecological and Health Risks.

Microplastic (MP) pollution has aroused a tremendous amount of public and scientific interest worldwide. MPs are found widely ranging from terrestrial to aquatic ecosystems primarily due to the over-exploitation of plastic products and unscientific disposal of plastic waste. There is a large availability of scientific literature on MP pollution in the terrestrial and aquatic ecosystems, especially the marine environments; however, only recently has greater scientific attention been focused on the presence of MPs in the air and its retrospective health implications. Besides, atmospheric transport has been reported to be an important pathway of transport of MPs to the pristine regions of the world. From a health perspective, existing studies suggest that airborne MPs are priority pollutant vectors, that may penetrate deep into the body through inhalation leading to adverse health impacts such as neurotoxicity, cancer, respiratory problems, cytotoxicity, and many more. However, their effects on indoor and outdoor air quality, and on human health are not yet clearly understood due to the lack of enough research studies on that and the non-availability of established scientific protocols for their characterization. This scientific review entails important information concerning the abundance of atmospheric MPs worldwide within the existing literature. A thorough comparison of existing sampling and analytical protocols has been presented. Besides, this review has unveiled the areas of scientific concern especially air quality monitoring which requires immediate attention, with the information gaps to be filled have been addressed.

Effect of extrusion processing on techno-functional, textural and bioactive properties of whole-grain corn flour-based breakfast cereals sweetened with honey.

The present study investigated the effect of honey and extrusion processing parameters on techno-functional and bioactive properties of whole-grain corn flour breakfast cereals. The central composite rotatable design (CCRD) was used to plan the experiments using feed moisture (FM), extrusion temperature (ET), and honey level as process variables and sectional expansion ratio (SER), bulk density (BD), water absorption index (WAI), water solubility index (WSI), and textural hardness as response variables. The feed containing honey and whole-grain corn flour was extruded through a twin-screw extruder according to CCRD. The data were fit to the appropriate regression models based on model significance and insignificant lack of fit. The selected experiments from CCRD were considered for studying the bioactive properties, and the effect of ET and honey level on bioactive properties was determined. The results of the study indicated that FM and honey adversely affected the SER, BD, and textural hardness, while ET augmented these properties of breakfast cereals. The WAI decreased with an increase in honey level and ET during extrusion. The incremental addition of honey in the mix as well extrusion at elevated temperature led to higher WSI. The whole-grain corn flour added with 10.22% honey and extruded at 16.06% FM, and 138.07°C ET produced optimum quality breakfast cereals with 0.70 desirability. The total phenolic content of breakfast cereals decreased, and antioxidant activity and hydroxymethylfurfural content increased upon extrusion at the higher temperatures. Honey addition lowers the loss of phenolic content during extrusion and improves the antioxidant activity of breakfast cereals.

Wheatgrass powder-enriched functional pasta: Techno-functional, phytochemical, textural, sensory, and structural characterization.

The influence of semolina replacement with wheatgrass powder (WGP; 3%-15%) was evaluated with reference to nutritional, techno-functional, phytochemical, textural, and structural characteristics of functional pasta. Results showed that incorporation of WGP significantly (p < 0.05) decreased the pasting viscosity of flour blends, while it increased the water and oil absorption capacity and water solubility index. Increased levels of WGP significantly decreased the optimum cooking time from 6.00 to 4.22 min but increased the cooking loss (2.83%-4.36%). Enrichment of pasta with WGP significantly (p < 0.05) enhanced the protein (12.16-17.33 g/100 g), fiber (1.21-4.60 g/100 g), antioxidant activities in terms of DPPH, FRAP, and ABTS. The total phenolic and flavonoid content increased from 56.20-253.90 mg GAE/100 g to 47.41-202.90 mg QE/100 g in the functional pasta. Addition of WGP significantly (p < 0.05) decreased the lightness (L*) while the greenness (-a*) of the pasta increased progressively owing to the total chlorophyll pigment. The firmness and toughness of the pasta increased up to 9% WGP level and decreased further, owing to the interaction between WGP protein and fiber with gluten protein matrix as evident from scanning electron microscopy (SEM). Furthermore, the cooking of pasta results in a significant reduction in all the components in comparison to uncooked pasta. Fourier transform infrared (FTIR) spectroscopy further confirmed the presence of phenols, flavonoids, and chlorophyll in WGP-incorporated pasta. Overall acceptability scores of pastas with 9% WGP were found to have the highest (7.57), and with an increase in a further level of WGP, sensory scores decreased (6.55). Moreover, the principal component analysis also compliments the sensory results for 9% WGP-incorporated pasta.

Characterization of the MurT/GatD complex in Mycobacterium tuberculosis towards validating a novel anti-tubercular drug target.

OBJECTIVES: Identification and validation of novel therapeutic targets is imperative to tackle the rise of drug resistance in tuberculosis. An essential Mur ligase-like gene (Rv3712), expected to be involved in cell-wall peptidoglycan (PG) biogenesis and conserved across mycobacteria, including the genetically depleted Mycobacterium leprae, was the primary focus of this study. METHODS: Biochemical analysis of Rv3712 was performed using inorganic phosphate release assays. The operon structure was identified using reverse-transcriptase PCR and a transcription/translation fusion vector. In vivo mycobacterial protein fragment complementation assays helped generate the interactome. RESULTS: Rv3712 was found to be an ATPase. Characterization of its operon revealed a mycobacteria-specific promoter driving the co-transcription of Rv3712 and Rv3713. The two gene products were found to interact with each other in vivo. Sequence-based functional assignments reveal that Rv3712 and Rv3713 are likely to be the mycobacterial PG precursor-modifying enzymes MurT and GatD, respectively. An in vivo network involving Mtb-MurT, regulatory proteins and cell division proteins was also identified. CONCLUSIONS: Understanding the role of the enzyme complex in the context of PG metabolism and cell division, and the implications for antimicrobial resistance and host immune responses will facilitate the design of therapeutics that are targeted specifically to M. tuberculosis.

Enhancement of Digestibility of Nutrients (In vitro), Antioxidant Potential and Functional Attributes of Wheat Flour Through Grain Germination.

Wheat grains were germinated at different time (12, 24, 36, and 48 h) and temperature (25, 30, and 35°C) to enhance the functionality of resultant flour. Results revealed that an increase in germination time and temperature enhanced the in vitro digestibility of starch (10.35-42.30 %) and proteins (6.31-44.02 %) owing to their depolymerization by hydrolytic enzymes. Total phenolic and flavonoid content of wheat during germination at variable conditions were enhanced significantly (p < 0.05) from 3.62 to 5.54 mg GAE/g and 32.06 to 54.33 mg QE/100 g, respectively. Germination at elevated temperature (35°C) for a prolonged time (48 h) increased the DPPH RSA by 58.85 %, reducing power by 80.40 % and metal chelating activity by 112.26 % as a result of the structural breakdown of bound phenolics. Increased activity of hydrolytic enzymes also results in a continuous reduction in the viscosity and lightness values of wheat flour. Tailored germination, therefore, can be offered as a tool to increase the nutrient digestibility and bioactive potential of wheat thus resulting in producing the naturally modified flour with enhanced functionality.

3-(5-Nitrofuran-2-yl)prop-2-en-1-one Derivatives, with Potent Antituberculosis Activity, Inhibit A Novel Therapeutic Target, Arylamine N-acetyltransferase, in Mycobacteria.

In this study, the inhibitory potential of 3-(5-nitrofuran-2-yl)prop-2-en-1-one derivatives was evaluated against a panel of bacteria, as well as mammalian cell lines to determine their therapeutic index. In addition, we investigated the mechanism of antibiotic action of the derivatives to identify their therapeutic target. We discovered compound 2 to be an extremely potent inhibitor of Mycobacterium tuberculosis H37Rv growth (MIC: 0.031 mg/L) in vitro, performing better than the currently used first-line antituberculosis drugs such as isoniazid, rifampicin, ethambutol, and pretomanid in vitro. Furthermore, compound 3 was equipotent to pretomanid against a multidrug-resistant M. tuberculosis clinical isolate. The derivatives were selective and bactericidal towards slow-growing mycobacteria. They showed low cytotoxicity towards murine RAW 264.7 and human THP-1 cell lines, with high selectivity indices. Compound 1 effectively eliminated the intracellular mycobacteria in a mycobacteria-infected macrophage model. The derivatives were assessed for their potential to inhibit mycobacterial arylamine N-acetyltransferase (NAT) and were identified as good inhibitors of recombinant mycobacterial NAT, a novel target essential for the intracellular survival of M. tuberculosis. This study provided hits for designing new potent and selective antituberculosis leads, having mycobacterial NAT inhibition as their possible endogenous mechanisms of action.

Improving the Potency of N-Aryl-2,5-dimethylpyrroles against Multidrug-Resistant and Intracellular Mycobacteria.

A series of N-phenyl-2,5-dimethylpyrrole derivatives, designed as hybrids of the antitubercular agents BM212 and SQ109, have been synthesized and evaluated against susceptible and drug-resistant mycobacteria strains. Compound 5d, bearing a cyclohexylmethylene side chain, showed high potency against M. tuberculosis including MDR-TB strains at submicromolar concentrations. The new compound shows bacteriostatic activity and low toxicity and proved to be effective against intracellular mycobacteria too, showing an activity profile similar to isoniazid.

Exploration of 5-(5-nitrothiophen-2-yl)-4,5-dihydro-1H-pyrazoles as selective, multitargeted antimycobacterial agents.

We report the biological evaluation of 5-(5-nitrothiophen-2-yl)-4,5-dihydro-1H-pyrazole derivatives against bacteria, eukaryotic cell lines and the assessment of their mechanisms of action to determine their prospects of being developed into potent antituberculosis agents. The compounds were evaluated for their antibacterial property against Mycobacterium tuberculosis H37Rv, multidrug-resistant M. tuberculosis, Mycobacterium bovis BCG, Mycobacterium aurum, Escherichia coli, and Staphylococcus aureus using high-throughput spot-culture growth inhibition assay. They were found to be selective toward slow-growing mycobacteria and Gram-positive bacteria. In M. bovis BCG, they exhibited a bactericidal mode of action. Cytotoxicity was assessed in human THP-1 and murine RAW 264.7 cell lines, and the compounds showed a lower cytotoxicity potential when compared with their antibacterial activity. They were found to be excellent whole-cell efflux pump inhibitors of the mycobacterial surrogate M. aurum, performing better than known efflux pump inhibitor verapamil. The 5-nitrothiophene moiety was identified for the first time as a prospective inhibitor scaffold of mycobacterial arylamine N-acetyltransferase enzyme, which is the key enzyme in metabolizing isoniazid, a first-line antituberculosis drug. The two aforementioned findings make the compounds potential hits in the development of adjunctive tuberculosis therapy.

Synthesis and mycobacterial evaluation of 5-substituted-6-acetyl-2-amino-7-methyl-5,8-dihydropyrido-[2,3-d]pyrimidin-4(3H)-one derivatives.

5-Substituted-6-acetyl-2-amino-7-methyl-5,8-dihydropyrido[2,3-d]pyrimidin-4(3H)-one derivatives were synthesized and evaluated against Mycobacterium tuberculosis H37Rv, Mycobacterium aurum, Escherichia coli, and Staphylococcus aureus as well as a human monocyte-derived macrophage (THP-1), and murine macrophage (RAW 264.7) cell lines to assess their antibacterial and cytotoxic potential, respectively. The compounds showed activity in the range of 1.95-125 µg/ml against M. tuberculosis but showed no activity against M. aurum, E. coli, and S. aureus, indicating selectivity towards slow-growing mycobacterial pathogens. The compounds exhibited very low to no cytotoxicity up to 500 µg/ml concentration against eukaryotic cell lines. The most potent molecule, 2l, showed a minimum inhibitory concentration of 1.95 µg/ml against M. tuberculosis H37Rv and a selectivity index of >250 against both the eukaryotic cell lines. Furthermore, 2l showed moderate inhibition of whole-cell mycobacterial drug-efflux pumps when compared to verapamil, a known potent inhibitor of efflux pumps. Thus, derivative 2l was identified as an antituberculosis hit molecule, which could be used to yield more potent lead molecules.

Novel indole-thiazolidinone conjugates: Design, synthesis and whole-cell phenotypic evaluation as a novel class of antimicrobial agents.

In connection with our research program on the development of novel anti-tubercular candidates, herein we report the design and synthesis of two different sets of indole-thiazolidinone conjugates (8a,b; 11a-d) and (14a-k; 15a-h). The target compounds were evaluated for their in vitro antibacterial and antifungal activities against selected human pathogens viz. Staphylococcus aureus (Gram positiveve), Pseudomonas aeruginosa, Escherichia coli (Gram negative), Mycobacterium tuberculosis (Acid-fast bacteria), Aspergillus fumigates and Candida albicans (fungi). Moreover, eukaryotic cell-toxicity was tested via an integrated ex vivo drug screening model in order to evaluate the selective therapeutic index (SI) towards antimicrobial activity when microbes are growing inside primary immune cells. Also, the cytotoxicity towards a panel of cancer cell lines and human lung fibroblast normal cell line, WI-38 cells, was explored to assure their safety. Compound 15b emerged as a hit in this study with potent broad spectrum antibacterial (MIC: 0.39-0.98 µg/mL) and antifungal (MIC: 0.49-0.98 µg/mL) activities, in addition to its ability to kill mycobacteria M. aurum inside an infected macrophage model with good therapeutic window. Moreover, compound 15b displayed promising activity towards resistant bacteria strains MRSA and VRE with MIC values equal 3.90 and 7.81 µg/mL, respectively. These results suggest compound 15b as a new therapeutic lead with good selectivity for further optimization and development.

The draft genome of Mycobacterium aurum, a potential model organism for investigating drugs against Mycobacterium tuberculosis and Mycobacterium leprae.

Mycobacterium aurum (M. aurum) is an environmental mycobacteria that has previously been used in studies of anti-mycobacterial drugs due to its fast growth rate and low pathogenicity. The M. aurum genome has been sequenced and assembled into 46 contigs, with a total length of 6.02Mb containing 5684 annotated protein-coding genes. A phylogenetic analysis using whole genome alignments positioned M. aurum close to Mycobacterium vaccae and Mycobacterium vanbaalenii, within a clade related to fast-growing mycobacteria. Large-scale genomic rearrangements were identified by comparing the M. aurum genome to those of Mycobacterium tuberculosis and Mycobacterium leprae. M. aurum orthologous genes implicated in resistance to anti-tuberculosis drugs in M. tuberculosis were observed. The sequence identity at the DNA level varied from 68.6% for pncA (pyrazinamide drug-related) to 96.2% for rrs (streptomycin, capreomycin). We observed two homologous genes encoding the catalase-peroxidase enzyme (katG) that is associated with resistance to isoniazid. Similarly, two embB homologues were identified in the M. aurum genome. In addition to describing for the first time the genome of M. aurum, this work provides a resource to aid the use of M. aurum in studies to develop improved drugs for the pathogenic mycobacteria M. tuberculosis and M. leprae.

Characterisation of a putative AraC transcriptional regulator from Mycobacterium smegmatis.

MSMEG_0307 is annotated as a transcriptional regulator belonging to the AraC protein family and is located adjacent to the arylamine N-acetyltransferase (nat) gene in Mycobacterium smegmatis, in a gene cluster, conserved in most environmental mycobacterial species. In order to elucidate the function of the AraC protein from the nat operon in M. smegmatis, two conserved palindromic DNA motifs were identified using bioinformatics and tested for protein binding using electrophoretic mobility shift assays with a recombinant form of the AraC protein. We identified the formation of a DNA:AraC protein complex with one of the motifs as well as the presence of this motif in 20 loci across the whole genome of M. smegmatis, supporting the existence of an AraC controlled regulon. To characterise the effects of AraC in the regulation of the nat operon genes, as well as to gain further insight into its function, we generated a ΔaraC mutant strain where the araC gene was replaced by a hygromycin resistance marker. The level of expression of the nat and MSMEG_0308 genes was down-regulated in the ΔaraC strain when compared to the wild type strain indicating an activator effect of the AraC protein on the expression of the nat operon genes.

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.

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.

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.

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.

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.

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 100mg/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.