14-Residue peptaibol velutibol A from Trichoderma velutinum: its structural and cytotoxic evaluation.

Velutibol A (1), a new 14-residue peptaibol was isolated from the Himalayan cold habitat fungus Trichoderma velutinum. The structural characterization was carried out by 1D and 2D NMR studies, and tandem mass studies, and Marfey's method aided in determining the stereochemistry of the amino acids. The CD analysis revealed folding of the peptide in a 310-helical conformation. The intramolecular H-bonding was determined by an NMR-VT experiment. Cytotoxic evaluation was carried out against a panel of cancer cell lines. The cell cycle assay was carried out on human myeloid leukaemia (HL-60) cells and revealed the formation of apoptotic bodies and DNA damage in a dose-dependent manner. Three other peptaibols namely velutibol B (2), velutibol C (3), and velutibol D (4) were also isolated in trace amounts from the psychotropic fungus and characterized through tandem mass spectroscopy and Marfey's analysis.

Preclinical development of gastro-protective botanical candidate from Woodfordia fruticosa (Linn.) Kurz: Chemical standardization, efficacy, pharmacokinetics and safety pharmacology.

ETHNOPHARMACOLOGICAL RELEVANCE: Woodfordia fruticosa is traditionally used in the Ayurvedic system of medicine for the treatment of diarrhoea, poisoning, menstrual disorders, ulcers and fertility. In the present study, we report a standardized extract preparation through modern scientific approach for anti-ulcer activity. MATERIALS AND METHODS: The hydro-alcoholic extract of flowers of W. fruticosa was standardized using four chemical markers. The standardized extract was coded as ICB014. HPLC method was developed for identification and quantification of Gallic Acid, Oenothein-C, Quercetin and Kaempferol. Based on the prior published H+, K+-ATPase activity and Anti-bacterial activity against Helicobacter pylori of ICB014, was evaluated for its in-vivo efficacy in gastric ulcers models in rats followed by regulatory safety studies. RESULTS: The extract demonstrated efficacy at 31.25-62.5 mg/kg in gastric ulcer models. The extract was safe by oral route up to 2000 mg/kg in a single dose and NOAEL of 800 mg/kg in 28 days repeat study. Bioequivalent capsule formulation was prepared. CONCLUSIONS: The extract showed anti-ulcer potential and is ready for clinical evaluation.

Synthesis of amides from (E)-3-(1-chloro-3,4-dihydronaphthalen-2-yl)acrylic acid and substituted amino acid esters as NorA efflux pump inhibitors of Staphylococcus aureus.

Inhibitors for NorA efflux pump of Staphylococcus aureus have attracted the attention of many researchers towards the discovery and development of novel efflux pump inhibitors (EPIs). In an attempt to find specific potent inhibitors of NorA efflux pump of S. aureus, a total of 15 amino acid conjugates of 3-(1-chloro-3,4-dihydronaphthalen-2-yl)acrylic acid (4-18) were synthesized using a simple convenient synthetic approach and bioevaluated against NorA efflux pump. Two compounds 7 and 8 (each having MEC of 1.56 µg/mL) were found to restore the activity of ciprofloxacin through reduction of the MIC elucidated by comparing the ethidium bromide efflux in dose dependent manner in addition to ethidium bromide efflux inhibition and accumulation study using NorA overexpressing strain SA-1199B. Most potent compounds among these were able to restore the antibacterial activity of ciprofloxacin completely against SA-1199B. Structure activity relationship (SAR) studies and docking study of potent compounds 7 and 8 could elucidate the structural requirements necessary for interaction with the NorA efflux pumps. On the whole, compounds 7 and 8 have ability to reverse the NorA efflux mediated resistance and could be further optimized for development of potent efflux pump inhibitors.

Boeravinone B, A Novel Dual Inhibitor of NorA Bacterial Efflux Pump of Staphylococcus aureus and Human P-Glycoprotein, Reduces the Biofilm Formation and Intracellular Invasion of Bacteria.

This study elucidated the role of boeravinone B, a NorA multidrug efflux pump inhibitor, in biofilm inhibition. The effects of boeravinone B plus ciprofloxacin, a NorA substrate, were evaluated in NorA-overexpressing, wild-type, and knocked-out Staphylococcus aureus (SA-1199B, SA-1199, and SA-K1758, respectively). The mechanism of action was confirmed using the ethidium bromide accumulation and efflux assay. The role of boeravinone B as a human P-glycoprotein (P-gp) inhibitor was examined in the LS-180 (colon cancer) cell line. Moreover, its role in the inhibition of biofilm formation and intracellular invasion of S. aureus in macrophages was studied. Boeravinone B reduced the minimum inhibitory concentration (MIC) of ciprofloxacin against S. aureus and its methicillin-resistant strains; the effect was stronger in SA-1199B. Furthermore, time-kill kinetics revealed that boeravinone B plus ciprofloxacin, at subinhibitory concentration (0.25 × MIC), is as equipotent as that at the MIC level. This combination also had a reduced mutation prevention concentration. Boeravinone B reduced the efflux of ethidium bromide and increased the accumulation, thus strengthening the role as a NorA inhibitor. Biofilm formation was reduced by four-eightfold of the minimal biofilm inhibitory concentration of ciprofloxacin, effectively preventing bacterial entry into macrophages. Boeravinone B effectively inhibited P-gp with half maximal inhibitory concentration (IC50) of 64.85 µM. The study concluded that boeravinone B not only inhibits the NorA-mediated efflux of fluoroquinolones but also considerably inhibits the biofilm formation of S. aureus. Its P-gp inhibition activity demonstrates its potential as a bioavailability and bioefficacy enhancer.

Identification and characterization of novel small molecule inhibitors of the acetyltransferase activity of Escherichia coli N-acetylglucosamine-1-phosphate-uridyltransferase/glucosamine-1-phosphate-acetyltransferase (GlmU).

This study aims at identifying novel chemical scaffolds as inhibitors specific to the acetyltransferase domain of a bifunctional enzyme, Escherichia coli GlmU, involved in the cell wall biosynthesis of Gram-negative organisms. A two-pronged approach was used to screen a 50,000 small-molecule library. Using the first approach, the library was in silico screened by docking the library against acetyltransferase domain of E. coli GlmU studies. In the second approach, complete library was screened against Escherichia coli ATCC 25922 to identify the whole cell active compounds. Active compounds from both the screens were screened in a colorimetric absorbance-based assay to identify inhibitors of acetyltransferase domain of E. coli GlmU which resulted in the identification of 1 inhibitor out of 56 hits identified by in silico screening and 4 inhibitors out of 35 whole cell active compounds on Gram-negative bacteria with the most potent inhibitor showing IC50 of 1.40 ± 0.69 µM. Mode of inhibition studies revealed these inhibitors to be competitive with AcCoA and uncompetitive with GlcN-1-P. These selected inhibitors were also tested for their antibacterial and cytotoxic activities. Compounds 5175178 and 5215319 exhibited antibacterial activity that co-related with GlmU inhibition. These compounds, therefore, represent novel chemical scaffolds targeting acetyltransferase activity of E. coli GlmU.

Synthesis, antimalarial and antitubercular activities of meridianin derivatives.

Meridianins are marine-derived indole alkaloids, known to possess kinase inhibitory and antimalarial activities. A series of N-aryl and heteroaryl sulfonamide derivatives of meridianins were prepared and screened for antimalarial activity against D6 and W2 strains of Plasmodium falciparum. 2-Nitro-4-trifluoromethyl sulfonamide derivative 14v displayed promising antiplasmodial activity against both strains with IC50 values of 2.56 and 3.41 µM, respectively. These compounds were not cytotoxic to mammalian cell lines including VERO (monkey kidney fibroblasts), LLC-PK1 (pig kidney epithelial cells) and four cancer cell lines; SK-MEL (human malignant, melanoma), KB (human epidermal carcinoma), BT-549 (ductal carcinoma), SK-OV-3 (human ovary carcinoma) up to 25 µg/ml. Furthermore, all sulfonamide derivatives along with acyl, alkyl and C-ring modified derivatives of meridianins were screened for antitubercular activity against a sensitive strain (H37Rv) of Mycobacterium tuberculosis (Mtb), wherein several compounds showed MIC values in the range of 5.2-304.8 µM. Meridianin C (3) and meridianin G (7) showed anti-tubercular activity with MIC values of 111.1 and 304.8 µM, respectively. The C-ring modified analog 12 exhibited potent anti-tubercular activity against H37Rv strain of Mtb with MIC of 5.2 µM. Furthermore, the most potent analogs 11b and 12 were screened against two clinical isolates of M. tuberculosis INH(R) and MDR and one laboratory generated mutant strain Rif(R). These two analogs 11b and 12 displayed promising activity against these resistant strains with MIC values in the range of 5.2-187.7 µM. This is the first report on the anti-tubercular activity of this scaffold.

Discovery of 4-acetyl-3-(4-fluorophenyl)-1-(p-tolyl)-5-methylpyrrole as a dual inhibitor of human P-glycoprotein and Staphylococcus aureus Nor A efflux pump.

Polysubstituted pyrrole natural products, lamellarins, are known to overcome multi-drug resistance in cancer via the inhibition of p-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) efflux pumps. Herein, a series of simplified polysubstituted pyrroles, prepared via a one-pot domino protocol, were screened for P-gp inhibition in P-gp overexpressing human adenocarcinoma LS-180 cells using a rhodamine 123 efflux assay. Several compounds showed the significant inhibition of P-gp at 50 µM, as indicated by increase in the intracellular accumulation of Rh123 in LS-180 cells. Furthermore, pyrrole 5i decreased the efflux of digoxin, a FDA approved P-gp substrate in MDCK-MDR1 cells with an IC50 of 11.2 µM. In in vivo studies, following the oral administration of a P-gp substrate drug, rifampicin, along with compound , the Cmax and AUC0-∞ of rifampicin was enhanced by 31% and 46%, respectively. All the compounds were then screened for their ability to potentiate ciprofloxacin activity via the inhibition of Staphylococcus aureus Nor A efflux pump. Pyrrole showed the significant inhibition of S. aureus Nor A efflux pump with 8- and 4-fold reductions in the MIC of ciprofloxacin at 50 and 6.25 µM, respectively. The molecular docking studies of compound with the human P-gp and S. aureus Nor A efflux pump identified its plausible binding site and key interactions. Thus, the results presented herein strongly indicate the potential of this scaffold for its use as multi-drug resistance reversal agent or bioavailability enhancer.

Sulfur rich 2-mercaptobenzothiazole and 1,2,3-triazole conjugates as novel antitubercular agents.

A series of benzfused heterocyclic derivatives such as amide conjugates of 2-(benzo[d]thiazol-2-ylthio)acetic acid with aromatic/aliphatic/cyclic secondary amines (5a-5o & 8a-8m); 1,2,3-triazole conjugates of 2-mercaptobenzothiazoles and amide conjugates of indole-3-glyoxalic acid with cyclic secondary amines (14a-14g) have been synthesized and were screened for their antitubercular activity against Mycobacterium tuberculosis H37Rv strain by broth microdilution assay method. Compounds 8b, 8f, 8g and 8l inhibited the growth of the H37Rv strain at concentrations of 8 µg/mL. These compounds (8b, 8f, 8g and 8l) have been further identified as bactericidal and are completely killing the microbes at 32-64 µg/mL concentrations. Molecular docking studies of the active compounds reveal that these compounds are targeting DprE1 and may act as DprE1 inhibitors.

Antiproliferative and antimicrobial activities of alkylbenzoquinone derivatives from Ardisia kivuensis.

CONTEXT: Medicinal plants are continuously screened for their pharmacological properties. Despite the diversity and the numerous phytochemicals found in Ardisia (Myrsinaceae) species, its full biological potential has not been fully explored. OBJECTIVE: Four naturally occurring alkylbenzoquinone derivatives, namely ardisiaquinone N (1), ardisiaquinone J (2), ardisiaquinone K (3) and a mixture of ardisiaquinone P (4) and K (3) from Ardisia kivuensis Taton (Myrsinaceae) were investigated in vitro for their cytotoxicity and antimicrobial activity. MATERIALS AND METHODS: Minimal inhibitory concentration (MIC) was determined using the broth micro-dilution assay. Tumor cells growth inhibition was performed by sulphorhodamine B (SRB) assay while sub-diploid DNA fraction was measured by flow cytometry. RESULTS: Compounds 1, 2 and 3 showed significant antimicrobial activity against two Gram-positive bacteria and one fungus (with MICs varying between 3.12 and 6.25 µg/ml). The four compounds exhibited remarkable antiproliferative activity against the leukemia cell line TPH-1 with IC50 inhibition values between 2 and 2.1 µg/ml. Cytotoxic activity was found to be related to apoptosis induction. DISCUSSION AND CONCLUSION: These findings suggest that natural compounds herein studied are interesting potential candidates for the development of new therapeutic agents, especially against leukemia and Gram-positive bacterial infections.

Cytotoxicity and antimicrobial activity of the methanol extract and compounds from Polygonum limbatum.

The present study was designed to investigate the antimicrobial activity and the cytotoxicity of the methanol extract (PLA) as well as fractions (PLA1-4) and compounds [cardamomin (1), (±)-polygohomoisoflavanone (2), (S)-(-)-pinostrobin (3), 2',4'-dihydroxy-3',6'-dimethoxychalcone (4), (2S)-(-)-5-hydroxy-6,7-dimethoxyflavanone (5), and (2S)-(-)-5,7-dimethoxyflavanone (6)] obtained from leaves of Polygonum limbatum. The microbroth dilution was used to determine the minimal inhibitory concentration (MIC) of the samples against 11 microbial strains including Candida albicans, C. krusei, C. tropicalis, Aspergillus fumigatus, Pseudomonas aeruginosa, Escherichia coli, vancomycin-resistant Enterococcus faecalis (VRE), Staphylococcus aureus, methicillin-resistant S. aureus (MRSA), S.epidermidis, and Mycobacterium tuberculosis H37Rv. The sulphorhodamine B cell growth inhibition assay was used to assess the cytotoxicity of the above samples on lung A549 adenocarcinoma, breast carcinoma MCF-7, prostate carcinoma PC-3, cervical carcinoma HeLa, and the acute monocytic leukemia cell line THP-1. The results of the MIC determination indicated that, apart from fraction PLA3, all other fractions as well as PLA and compound 3 were selectively active. MIC values were noted on 100 % of the 11 tested microorganisms for fraction PLA3, 72.7 % for PLA, fraction PLA2, and compound 4, 63.6 % for PLA1, and 54.5 % for fraction PLA4. The results of the cytotoxicity assay revealed that, except for A459 cells, more than 50 % inhibition of the proliferation was obtained with each of the tested samples on at least one of the four other cell lines. IC50 values below 4 µg/mL were obtained with 1 and 4 on THP-1 cells. The overall results of the present study provided baseline information for the possible use of Polygonum limbatum as well as some of the isolated compounds for the control of cancer diseases and mostly leukemia.

Acetyl-11-keto-ß-boswellic acid (AKBA); targeting oral cavity pathogens.

BACKGROUND: Boswellic acids mixture of triterpenic acids obtained from the oleo gum resin of Boswellia serrata and known for its effectiveness in the treatment of chronic inflammatory disease including peritumor edema. Boswellic acids have been extensively studied for a number of activities including anti inflammatory, antitumor, immunomodulatory, and inflammatory bowel diseases. The present study describes the antimicrobial activities of boswellic acid molecules against oral cavity pathogens. Acetyl-11-keto-ß-boswellic acid (AKBA), which exhibited the most potent antibacterial activity, was further evaluated in time kill studies, mutation prevention frequency, postantibiotic effect (PAE) and biofilm susceptibility assay against oral cavity pathogens. FINDINGS: AKBA exhibited an inhibitory effect on all the oral cavity pathogens tested (MIC of 2-4 µg/ml). It exhibited concentration dependent killing of Streptococcus mutans ATCC 25175 up to 8 × MIC and also prevented the emergence of mutants of S.mutans ATCC 25175 at 8× MIC. AKBA demonstrated postantibiotic effect (PAE) of 5.7 ± 0.1 h at 2 × MIC. Furthermore, AKBA inhibited the formation of biofilms generated by S.mutans and Actinomyces viscosus and also reduced the preformed biofilms by these bacteria. CONCLUSIONS: AKBA can be useful compound for the development of antibacterial agent against oral pathogens and it has great potential for use in mouthwash for preventing and treating oral infections.

Antistaphylococcal and biofilm inhibitory activities of acetyl-11-keto-ß-boswellic acid from Boswellia serrata.

BACKGROUND: Boswellic acids are pentacyclic triterpenes, which are produced in plants belonging to the genus Boswellia. Boswellic acids appear in the resin exudates of the plant and it makes up 25-35% of the resin. ß-boswellic acid, 11-keto-ß-boswellic acid and acetyl-11-keto-ß-boswellic acid have been implicated in apoptosis of cancer cells, particularly that of brain tumors and cells affected by leukemia or colon cancer. These molecules are also associated with potent antimicrobial activities. The present study describes the antimicrobial activities of boswellic acid molecules against 112 pathogenic bacterial isolates including ATCC strains. Acetyl-11-keto-ß-boswellic acid (AKBA), which exhibited the most potent antibacterial activity, was further evaluated in time kill studies, postantibiotic effect (PAE) and biofilm susceptibility assay. The mechanism of action of AKBA was investigated by propidium iodide uptake, leakage of 260 and 280 nm absorbing material assays. RESULTS: AKBA was found to be the most active compound showing an MIC range of 2-8 µg/ml against the entire gram positive bacterial pathogens tested. It exhibited concentration dependent killing of Staphylococcus aureus ATCC 29213 up to 8 × MIC and also demonstrated postantibiotic effect (PAE) of 4.8 h at 2 × MIC. Furthermore, AKBA inhibited the formation of biofilms generated by S. aureus and Staphylococcus epidermidis and also reduced the preformed biofilms by these bacteria. Increased uptake of propidium iodide and leakage of 260 and 280 nm absorbing material by AKBA treated cells of S aureus indicating that the antibacterial mode of action of AKBA probably occurred via disruption of microbial membrane structure. CONCLUSIONS: This study supported the potential use of AKBA in treating S. aureus infections. AKBA can be further exploited to evolve potential lead compounds in the discovery of new anti-Gram-positive and anti-biofilm agents.

Identification of Mycobacterium tuberculosis genes preferentially expressed during human infection.

The identification of Mycobacterium tuberculosis genes, specifically expressed during infection is a key step in understanding molecular mechanism of mycobacterial pathogenesis. Such genes likely encode proteins required for mycobacterium's survival and progressive infection within the host. In this study, we applied in-vivo-induced antigen technology (IVIAT) to M. tuberculosis and identified 11 putative in-vivo induced genes encoding for immunogenic proteins of diverse functions; these included transcriptional regulators (Rv1460 and Rv2565), biosynthesis and macromolecule metabolism (leuD, guaB1, plcC, hupB and glyS), polyketide synthases (pks6 and pks9), cell processes (ctpA) and one with unknown function (Rv3701c). Quantitative real time-PCR analysis of these genes in the specimens obtained from TB patients demonstrated induced expression of eight genes as compared with bacteria grown in-vitro. In addition, distribution of these genes in different strains of M. tuberculosis was analyzed using PCR and their nucleotide sequence alignments and they were found to be widely distributed among M. tuberculosis isolates including multiple-drug resistant (MDR) and extensively-drug resistant (XDR). This study identified several antigenic determinants of M. tuberculosis expressed during infection, which might help pathogens adapt to or counter hostile environments and suggesting their role during disease process.

Substituted dihydronaphthalenes as efflux pump inhibitors of Staphylococcus aureus.

A new series of 3-(substituted-3,4-dihydronaphthyl)-2-propenoic acid amides has been prepared through convergent synthetic strategies and tested in combination with ciprofloxacin against NorA overexpressing Staphylococcus aureus 1199B as test strain for potentiating of the drug activity. Out of 24 compounds evaluated, 12 compounds potentiated the activity of ciprofloxacin and resulted in 2-16 fold reduction in the MIC (4-0.5 microg/mL) of the drug. The failure of these efflux pump inhibitors (EPIs) to potentiate the activity of ciprofloxacin when tested against NorA knock out S. aureus SA-K1758 established their identity as NorA inhibitors. The structure of all these newly synthesised compounds was confirmed by spectral data. The present communication describes the synthesis, bioevaluation, structure activity relationship and mechanism of action of these EPIs.

Novel bisstyryl derivatives of bakuchiol: targeting oral cavity pathogens.

Novel bisstyryl derivatives of bakuchiol using Heck coupling reaction as the key step were synthesized and screened against a panel of six oral cavity pathogens for their antimicrobial activity. Four compounds (9-12) showed two to fourfold and four to eightfold better activity (MIC 0.25-16 microg/ml) than bakuchiol and triclosan respectively. These compounds effectively inhibit the biofilm formation of single and multiple species at 2 - 8 x MICs. 4- and 4'-Hydroxy/methoxy styryl moieties of the bakuchiol derivatives play a pivotal role towards the activity as established in the SAR studies. Mechanism of action studies revealed microbial membrane structure disruption as the probable mode of action of these compounds.

In vitro antifungal activity of hydroxychavicol isolated from Piper betle L.

BACKGROUND: Hydroxychavicol, isolated from the chloroform extraction of the aqueous leaf extract of Piper betle L., (Piperaceae) was investigated for its antifungal activity against 124 strains of selected fungi. The leaves of this plant have been long in use tropical countries for the preparation of traditional herbal remedies. METHODS: The minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of hydroxychavicol were determined by using broth microdilution method following CLSI guidelines. Time kill curve studies, post-antifungal effects and mutation prevention concentrations were determined against Candida species and Aspergillus species "respectively". Hydroxychavicol was also tested for its potential to inhibit and reduce the formation of Candida albicans biofilms. The membrane permeability was measured by the uptake of propidium iodide. RESULTS: Hydroxychavicol exhibited inhibitory effect on fungal species of clinical significance, with the MICs ranging from 15.62 to 500 microg/ml for yeasts, 125 to 500 microg/ml for Aspergillus species, and 7.81 to 62.5 microg/ml for dermatophytes where as the MFCs were found to be similar or two fold greater than the MICs. There was concentration-dependent killing of Candida albicans and Candida glabrata up to 8 x MIC. Hydroxychavicol also exhibited an extended post antifungal effect of 6.25 to 8.70 h at 4 x MIC for Candida species and suppressed the emergence of mutants of the fungal species tested at 2 x to 8 x MIC concentration. Furthermore, it also inhibited the growth of biofilm generated by C. albicans and reduced the preformed biofilms. There was increased uptake of propidium iodide by C. albicans cells when exposed to hydroxychavicol thus indicating that the membrane disruption could be the probable mode of action of hydroxychavicol. CONCLUSIONS: The antifungal activity exhibited by this compound warrants its use as an antifungal agent particularly for treating topical infections, as well as gargle mouthwash against oral Candida infections.

Piperine analogs as potent Staphylococcus aureus NorA efflux pump inhibitors.

Based on our recent findings that piperine is a potent Staphylococcus aureus NorA efflux pump inhibitor (EPI), 38 piperine analogs were synthesized and bioevaluated for their EPI activity. Twenty-five of them were found active with potentiating activity equivalent or more than known EPIs like reserpine, carsonic acid and verapamil. The inhibitory mechanism of the compounds was confirmed by efflux inhibition assay using ethidium bromide as NorA substrate. The present communication describes the synthesis, bioevaluation and structure related activity of these efflux pump inhibitors.

Citral derived amides as potent bacterial NorA efflux pump inhibitors.

Monoterpene citral and citronellal have been used as starting material for the preparation of 5,9-dimethyl-deca-2,4,8-trienoic acid amides and 9-formyl-5-methyl-deca-2,4,8-trienoic acid amides. The amides on bioevaluation as efflux pump inhibitors (EPIs) against Staphylococcus aureus 1199 and NorA overexpressing S. aureus 1199B bacteria resulted in the identification of several of these as potent EPIs. Many of these amides have been shown to possess potency higher or equivalent to known EPIs such as reserpine, verapamil, carsonic acid, and piperine. In this communication, we report a convenient synthesis of alkenyl amides, their bioevaluation and identification as efflux pump inhibitors against S. aureus.