Two antibacterial spiro compounds from the roots of Artemisia pallens wall: evidence from molecular docking.

Bioassay-guided isolation from acetone extract of the roots of Artemisia pallens Wall yielded two spiro compounds (1 and 2). The structures of these compounds were determined on the basis of spectroscopic techniques such as IR, MS, 1 D and 2 D- NMR. The acetone extract, fractions and the isolated two compounds were investigated for their antibacterial activity against two gram negative (E. coli, P. aeruginosa) and two gram positive (S. aureus, B. subtilis) bacterial strains. Compound (2) showed the best spectra of activity with IC50 and MIC values between 2.48-3.08 and 12.78 - 21.77 µM and Compound (1) with 2.57-3.69 and 38.17 - 80.57 µM, respectively, for the four bacterial strains, whereas inactive against Mycobacterium tuberculosis. Molecular docking study could further help in understanding the various interactions between these compounds and DNA gyrase active site in detail and thereby could provide valuable insight into the mechanism of action.

Exploring the Pharmacological Potentials of Biosurfactant Derived from Planococcus maritimus SAMP MCC 3013.

Therapeutic potential of biosurfactant (BS) has been improved in recent years. Our present study deals with production of BS from Planococcus maritimus SAMP MCC 3013 in a mineral salt medium (MSM) supplemented with glucose (1.5% w/v). Further, BS has been purified and partially characterized as glycolipid type through our previous publication. Current research article aimed to evaluate biological potential of BS against Mycobacterium tuberculosis, Plasmodium falciparum and cancerous cell lines. Planococcus derived glycolipid BS was found to be a promising inhibitor of M. tuberculosis (MTB) H37Ra at IC50 64.11 ± 1.64 µg/mL and MIC at 160.8 ± 1.64 µg/mL. BS also showed growth inhibition of P. falciparum at EC50 34.56 ± 0.26 µM. Additionally, BS also displayed the cytotoxicity against HeLa (IC50 41.41 ± 4.21 µg/mL), MCF-7 (IC50 42.79 ± 6.07 µg/mL) and HCT (IC50 31.233 ± 5.08 µg/mL) cell lines. Molecular docking analysis was carried for the most popular glycolipid type BS namely Rhamnolipid (RHL) aiming to interpret the possible binding interaction for anti-tubercular and anti-cancer activity. This analysis revealed the involvement of RHL binding with enoyl reductase (InhA) of M. tuberculosis. Docking studies of RHL with tubulin directed several hydrophobic and Vander Waal interactions to exhibit anti-cancer potential. The present study will be helpful for further development of marine bioactive molecules for therapeutic applications. Their anti-tubercular, anti-plasmodial and cytotoxic activities make BS molecules as a noteworthy candidate to combat several diseases. To the best of our knowledge, this is the first report on projecting the pharmacological potential of Planococcus derived BS.

A high content screening assay for identifying inhibitors against active and dormant state intracellular Mycobacterium tuberculosis.

The antitubercular drug development pipeline could start with an in vitro investigation of several compounds to examine their effect on active and dormant state Mycobacterium tuberculosis (Mtb). However, in vitro screening of dormant state bacilli cannot provide enough information on the simultaneous effect of a compound on the host. Therefore, we developed a live cell fluorescence based screening protocol by utilizing the high content system for determining the effect of inhibitors against active and dormant state intracellular mycobacteria. THP-1 macrophages infected with an actively growing and hypoxia derived dormant Mtb culture were standardized to develop the screening protocol. The signal to noise ratio and the Z' factor of this assay were found to be 7.5-29 and 0.6-0.8, respectively, which confirm the robustness of the protocol. The protocol was then validated with standard inhibitors. This newly developed drug screening assay offers an easy, safe, image based high content screening tool to search for novel antitubercular inhibitors against both active and dormant state intracellular mycobacteria. Therefore, this assay could fill in the gap between in vitro and in vivo latent tuberculosis drug screening programs.

Using diphenyleneiodonium to induce a viable but non-culturable phenotype in Mycobacterium tuberculosis and its metabolomics analysis.

Depletion of oxygen levels is a well-accepted model for induction of non-replicating, persistent states in mycobacteria. Increasing the stress levels in mycobacterium bacilli facilitates their entry into a non-cultivable, dormant state. In this study, it was shown that diphenyleneiodonium, an inhibitor of NADH oxidase, induced a viable, but non-culturable state in mycobacteria, having similar features to dormant bacilli, like loss of acid-fastness, upregulation of stress-regulated genes and decreased superoxide levels as compared to actively growing bacilli. Comprehensive, untargeted metabolic profiling also confirmed a decrease in biogenesis of amino acids, NAD, unsaturated fatty acids and nucleotides. Additionally, an increase in the level of lactate, fumarate, succinate and pentose phosphate pathways along with increased mycothiol and sulfate metabolites, similar to dormant bacilli, was observed in the granuloma. These non-cultivable bacilli were resuscitated by supplementation of fetal bovine serum, regaining their culturability in liquid as well as on agar medium. This study focused on the effect of diphenyleneiodonium treatment in causing mycobacteria to rapidly transition from an active state into a viable, but non-cultivable state, and comparing their characteristics with dormant phenotypes.

Design, synthesis, docking studies and biological screening of 2-thiazolyl substituted -2,3-dihydro-1H-naphtho[1,2-e][1,3]oxazines as potent HIV-1 reverse transcriptase inhibitors.

1,3-oxazine nucleus and thiazolyl group features prominently in many biologically important natural products as well as bioactive molecules. A series of novel 2-thiazolyl substituted-2,3-dihydro-1H-naphtho [1,2-e][1,3]oxazine derivatives were designed and synthesized based on their structure-activity relationships (SARs) from 2-naphthol, substituted thiazolyl amines and formalin through ring closure by one-pot three component reaction. These derivatives were first evaluated for their inhibitory effect on HIV-1 Reverse Transcriptase (RT) enzyme activity. Out of 14 compounds, 4 showed potent inhibition of HIV-1 RT activity at significantly low concentration. Docking studies of these molecules revealed their high affinity binding to several amino acids of HIV-1 RT which are less sensitive to point mutations. Furthermore, anti-HIV activity of these molecules was analysed in a CD4+ T cell-line, which indicates that Therapeutic Index (TI) of some of these compounds is better than Zidovudine and Efavirenz, known HIV-1 RT inhibitors. Taken together, our studies report for the first time some novel naphthoxazine derivatives with significant TI, which is through inhibition of HIV-1 RT activity.

Novel red fluorescence protein based microplate assay for drug screening against dormant Mycobacterium tuberculosis by using paraffin.

The hypoxia model of dormancy is widely used in drug screening programs to identify novel inhibitors against latent Mycobacterium tuberculosis disease. In earlier reported microplate assays, hypoxia was maintained by either sealing the microplate or shifting in an anaerobic chamber to develop dormant phenotype. In these assays, inhibitors were added during inoculation, which mainly represents the active stage inhibitors instead of the dormant ones. Herein, the culture was covered with paraffin to develop hypoxia condition and consequently providing the advantage of adding compounds at any stage during incubation of 96-well plate. The stable expression of the red fluorescent protein in the bacilli under both actively growing as well as dormant conditions also facilitate the reliable estimation of growth and inhibition kinetics of bacilli in medium. Furthermore, S/N ratio and Z' factor of this assay were found to be > 27 and 0.91-0.94 respectively, which confirm the robustness of the protocol. This newly developed drug-screening assay offers an easy, inexpensive, safe and high throughput-screening tool to search novel antitubercular inhibitors against both active and dormant bacilli. The red fluorescent H37Ra strain is a suitable surrogate for the more virulent H37Rv strain, and thus this effort will help in combating latent tuberculosis.

Phytogenic silver, gold, and bimetallic nanoparticles as novel antitubercular agents.

PURPOSE: Multi- and extensively drug-resistant tuberculosis (TB) is a global threat to human health. It requires immediate action to seek new antitubercular compounds and devise alternate strategies. Nanomaterials, in the present scenario, have opened new avenues in medicine, diagnosis, and therapeutics. In view of this, the current study aims to determine the efficacy of phytogenic metal nanoparticles to inhibit mycobacteria. METHODS: Silver (AgNPs), gold (AuNPs), and gold-silver bimetallic (Au-AgNPs) nanoparticles synthesized from medicinal plants, such as Barleria prionitis, Plumbago zeylanica, and Syzygium cumini, were tested against Mycobacterium tuberculosis and M. bovis BCG. In vitro and ex vivo macrophage infection model assays were designed to determine minimum inhibitory concentration (MIC) and half maximal inhibitory concentration of nanoparticles. Microscopic analyses were carried out to demonstrate intracellular uptake of nanoparticles in macrophages. Besides this, biocompatibility, specificity, and selectivity of nanoparticles were also established with respect to human cell lines. RESULTS: Au-AgNPs exhibited highest antitubercular activity, with MIC of <2.56 µg/mL, followed by AgNPs. AuNPs did not show such activity at concentrations of up to 100 µg/mL. In vitro and ex vivo macrophage infection model assays revealed the inhibition of both active and dormant stage mycobacteria on exposure to Au-AgNPs. These nanoparticles were capable of entering macrophage cells and exhibited up to 45% cytotoxicity at 30 µg/mL (ten times MIC concentration) after 48 hours. Among these, Au-AgNPs synthesized from S. cumini were found to be more specific toward mycobacteria, with their selectivity index in the range of 94-108. CONCLUSION: This is the first study to report the antimycobacterial activity of AuNPs, AgNPs, and Au-AgNPs synthesized from medicinal plants. Among these, Au-AgNPs from S. cumini showed profound efficiency, specificity, and selectivity to kill mycobacteria. These should be investigated further to develop novel TB nanoantibiotics.

Isolates of Alpinia officinarum Hance as COX-2 inhibitors: Evidence from anti-inflammatory, antioxidant and molecular docking studies.

BACKGROUND: Inflammation triggered by oxidative stress can cause various ailments, such as cancer, rheumatoid arthritis, asthma, diabetes etc. In the last few years, there has been a renewed interest in studying the antioxidant and anti-inflammatory action of plant constituents such as flavonoids and diarylheptanoids. AIM: To evaluate the antioxidant, anti-inflammatory activity and the total phenolic content of isolated compounds from Alpinia officinarum rhizomes. Furthermore, molecular docking was performed to study the binding mode of these compounds into the active site of cyclooxygenase-2 (COX-2). METHODS: A. officinarum rhizomes were extracted by maceration, using methanol. This extract was further fractionated by partitioning with hexane, chloroform and ethyl acetate and these fractions on further purification resulted in isolation of five pure compounds. Characterization was carried out by using (1)H NMR, (13)C NMR and MS. They were further evaluated for antioxidant and anti-inflammatory activity using carrageenan-induced paw edema model in rats. Molecular docking study was performed using Glide module integrated in Schrodinger molecular modeling software. RESULTS: The compounds were identified as 1,7-diphenylhept-4-en-3-one (1), 5-hydroxy-1,7-diphenyl-3-heptanone (2), 3,5,7-trihydroxyflavone (Galangin, 3), 3,5,7-trihydroxy-4'-methoxyflavone (Kaempferide, 4) and 5-hydroxy-7-(4″-hydroxy-3″-methoxyphenyl)-1-phenyl-3-heptanone (5). The compound-3 and compound-5 (10mg/kg) showed significant (p<0.001) antioxidant and anti-inflammatory potential. Moreover, total phenolic content was detected as 72.96 mg and 51.18 mg gallic acid equivalent respectively. All the five isolates were found to be good binders with COX-2 (average docking score -9.03). CONCLUSIONS: Galangin and 5-hydroxy-7-(4″-hydroxy-3″-methoxyphenyl)-1-phenyl-3-heptanone exhibited anti-inflammatory and in-vitro antioxidant activity which may be due to presence of phenolic content in it. The molecular docking study revealed that these compounds have affinity towards COX-2 active site which can further be explored as selective COX-2 inhibitors. The results obtained in this work justify the use of A. officinarum in the treatment of inflammatory disorders like rheumatoid arthritis and inflammatory bowel diseases.

A new butenolide cinnamate and other biological active chemical constituents from Polygonum glabrum.

Phytochemical investigation of the methanol extract of the aerial parts of Polygonum glabrum afforded one new natural product (-)-2-methoxy-2-butenolide-3-cinnamate (1) along with six known compounds, ß-hydroxyfriedalanol (2), 3-hydroxy-5-methoxystilbene (3), (-) pinocembrin (4), sitosterol-(6'-O-palmitoyl)-3-O-ß-D-glucopyranoside (5), (-) pinocembrin-5-methyl ether (6) and sitosterol-3-O-ß-D-glucopyranoside (7). Compound 1 showed promising in vitro anti-HIV-1 activity against primary isolates HIV-1(UG070) (X4, subtype D) and HIV-1(VB59) (R5, subtype C) assayed using TZM-bl cell line with IC50 in the range of 15.68-22.43 µg/mL. The extract showed TI in the range of 19.19-27.37 with IC50 in the range of 10.90-15.55 µg/mL. Compounds 1, 3 and 4 exhibited in vitro anti-mycobacterium activity against Mycobacterium tuberculosis H37Ra with IC50 values of 1.43, 3.33 and 1.11 µg/mL in dormant phase and 2.27, 3.33 and 1.21 µg/mL in active phase, respectively. Compound 4 was found to be the most active antiproliferative with IC50 values of 1.88-11.00 µg/mL against THP-1, A549, Panc-1, HeLa and MCF7 cell lines.

Antifungal dimeric chalcone derivative kamalachalcone E from Mallotus philippinensis.

From the red coloured extract (Kamala) prepared through acetone extraction of the fresh whole uncrushed fruits of Mallotus philippinensis, one new dimeric chalcone (1) along with three known compounds 1-(5,7-dihydroxy-2,2,6-trimethyl-2H-1-benzopyran-8-yl)-3-phenyl-2-propen-1-one (2), rottlerin (3) and 4'-hydroxyrottlerin (4) were isolated. The structure of compound 1 was elucidated by 1D and 2D NMR analyses that included HSQC, HMBC, COSY and ROESY experiments along with the literature comparison. Compounds 1-4 were evaluated for antifungal activity against different human pathogenic yeasts and filamentous fungi. The antiproliferative activity of the compounds was evaluated against Thp-1 cell lines. Compounds 1 and 2 both exhibited IC50 of 8, 4 and 16 µg/mL against Cryptococcus neoformans PRL518, C. neoformans ATCC32045 and Aspergillus fumigatus, respectively. Compound 4, at 100 µg/mL, showed 54% growth inhibition of Thp-1 cell lines.

A novel screening method based on menadione mediated rapid reduction of tetrazolium salt for testing of anti-mycobacterial agents.

A microplate-based rapid, inexpensive and robust technique is developed by using tetrazolium salt 2,3-bis[2-methyloxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide (XTT) and menadione to determine the viability of Mycobacterium tuberculosis, Mycobacterium bovis BCG and Mycobacterium smegmatis bacilli in microplate format. In general, XTT reduction is an extremely slow process which takes almost 24 h to produce a detectable signal. Menadione could drastically induce this reduction to an almost equal extent within a few minutes in a dose dependent manner. The reduction of XTT is directly proportional to the cell concentration in the presence of menadione. The standardized protocol used 200 µM of XTT and 60 µM of menadione in 250 µl of cell suspension grown either in aerobic or anaerobic conditions. The cell suspension of M. bovis BCG and M. tuberculosis were incubated for 40 min before reading the optical density at 470 nm whereas M. smegmatis was incubated for 20 min. Calculated Signal/Noise (S/N) ratios obtained by applying this protocol were 5.4, 6.4 and 9.4 using M. bovis BCG, M. tuberculosis and M. smegmatis respectively. The calculated Z' factors were >0.8 for all mycobacterium bacilli indicating the robustness of the XTT Reduction Menadione Assay (XRMA) for rapid screening of inhibitors. The assay protocol was validated by applying 10 standard anti-tubercular agents on M. tuberculosis, M. bovis BCG and M. smegmatis. The Minimum Inhibitory Concentration (MIC) values were found to be similar to reported values from Colony Forming Unit (CFU) and REMA (resazurin microplate assay) assays. Altogether, XRMA is providing a novel anti-tubercular screening protocol which could be useful in high throughput screening programs against different physiological stages of the bacilli.

A simple whole cell based high throughput screening protocol using Mycobacterium bovis BCG for inhibitors against dormant and active tubercle bacilli.

This study aimed at developing a whole cell based high throughput screening protocol to identify inhibitors against both active and dormant tubercle bacilli. A respiratory type of nitrate reductase (NarGHJI), which was induced during dormancy, could reflect the viability of dormant bacilli of Mycobacterium bovis BCG in microplate adopted model of in vitro dormancy. Correlation between reduction in viability and nitrate reductase activity was seen clearly when dormant stage inhibitor metronidazole and itaconic anhydride were applied in this in vitro microplate model. Active replicating stage could also be monitored in the same assay by measuring the A(620) of the culture. MIC values of 0.08, 0.075, 0.3 and 3.0 microg/ml, determined through monitoring A(620) in this assay for rifampin, isoniazid, streptomycin and ethambutol respectively, were well in agreement with previously reported by BACTEC and Bio-Siv assays. S/N ratio and Z' factor for the assay were 8.5 and 0.81 respectively which indicated the robustness of the protocol. Altogether the assay provides an easy, inexpensive, rapid, robust and high content screening tool to search novel antitubercular molecules against both active and dormant bacilli.

High-throughput screening of RNA polymerase inhibitors using a fluorescent UTP analog.

RNA polymerase (RNAP) is a well-validated target for the development of antibacterial and antituberculosis agents. Because the purification of large quantities of native RNA polymerase from pathogenic mycobacteria is hazardous and cumbersome, the primary screening was carried out using Escherichia coli RNAP. The authors have developed a high-throughput screening (HTS) assay to screen for novel inhibitors of RNAP. In this assay, a fluorescent analog of UTP, gamma-amino naphthalene sulfonic acid (gamma-AmNS) UTP, was used as one of the nucleotide substrates. Incorporation of UMP in RNA results in the release of gamma-AmNS-PPi, which has higher intrinsic fluorescence than (gamma-AmNS) UTP. The assay was optimized in a 384-well format and used to screen 670,000 compounds at a concentration of 10 microM. About 0.1% of the compounds showed more than 60% inhibition in the primary HTS. All the primary actives tested for dose response using the same assay had an EC(50) below 100 microM. Eighty percent of the primary HTS actives obtained using E. coli RNAP showed comparable activity against Mycobacterium smegmatis RNAP in the conventional radioactive assay. Activity of hits selected for the hit-to-lead optimization was also confirmed against Mycobacterium bovis RNAP which has >99% sequence identity with Mycobacterium tuberculosis RNAP subunits.

Development of a simple high-throughput screening protocol based on biosynthetic activity of Mycobacterium tuberculosis glutamine synthetase for the identification of novel Inhibitors.

A high-throughput screening protocol has been developed for Mycobacterium tuberculosis glutamine synthetase by quantitative estimation of inorganic phosphate. The K(m) values determined at pH 6.8 are 22 mM for L-glutamic acid, 0.75 mM for NH(4)Cl, 3.25 mM for MgCl(2), and 2.5 mM for adenosine triphosphate. The K(m) value for glutamine is affected significantly by the increase in pH of assay buffer. At the saturating level of the substrate, the enzyme activity at pH 6.8 and 25 degrees C is found to be linear up to 3 h. The reduction of enzyme activity is negligible even in presence of 10% DMSO. The Z' factor and signal-to-noise ratio are found to be 0.75 and 6.18, respectively, when the enzyme is used at 62.5 microg/ml concentration. The IC(50) values obtained at pH 6.8 for both L-methionine S-sulfoximine and DL-phosphothriacin are 500 microM and 30 microM, respectively, which is lowest compared to the values obtained at other pH levels. The Beckman Coulter high-throughput screening platform was found to take 5 h 9 min to complete the screening of 60 plates. For each assay plate, a replica plate is used to normalize the data. Screening of 1164 natural product fractions/extracts and synthetic molecules from an in-house library was able to identify 12 samples as confirmed hits. Altogether, the validation data from screening of a small set of an in-house library coupled with Z' and signal-to-noise values indicate that the protocol is robust for high-throughput screening of a diverse chemical library.