We report here an isatin derivative 45 (ID45) against coxsackievirus B3 (CVB3) replication, which was synthesized based on a high-throughput screen of a unique natural product library. ID45 showed the most potent anti-CVB3 activity among the four synthesized compounds. Treatment of cells with ID45 before or after infection significantly reduced viral particle formation, resulting in protection of cells from virus-induced apoptosis. In addition, ID45 treatment caused remarkable up-regulation of glucose-regulated protein 78 (GRP78), a hallmark of endoplasmic reticulum (ER) stress and an indicator of enhanced cell viability. In identifying the ER stress response pathway induced by ID45, we found that ID45 activated PKR-like ER protein kinase (PERK) but failed to up-regulate eIF2? phosphorylation. Instead ID45 activated transcription factor Nrf2 (NF-E2-related factor-2), which is evidenced by its nuclear translocation and upregulation of its downstream target genes NQO1 (NAD(P)H quinone-oxidoreductase 1) and GCLM (glutamate-cysteine ligase, modifier subunit). This observation was further verified by using siRNAs of GRP78 or Nrf2, which blocked both the translocation of Nrf2 and up-regulation of its target genes, leading to aggressive viral replication and enhanced cell apoptosis. Finally, we found that ID45-induced up-regulation of NQO1 protected eIF4GI, a eukaryotic cap-dependent translation initiation factor, from cleavage by CVB3 protease and degradation by proteasomes. Taken together, our findings established that a novel antiviral mechanism of isatin derivative ID45 inhibits CVB3 replication by promoting cell survival through a PERK/Nrf2-dependent ER stress pathway, which benefits host cap-dependent translation but suppresses CVB3 cap-independent translation.
The high mutation rate of RNA viruses has resulted in limitation of vaccine effectiveness and increased emergence of drug-resistant viruses. New effective antivirals are therefore needed to control of the highly mutative RNA viruses. The n-butanol fraction of the stem bark of Mangifera indica exhibited inhibitory activity against influenza neuraminidase (NA) and coxsackie virus 3C protease. Bioassay guided phytochemical study of M. indica stem bark afforded two new compounds including one benzophenone C-glycoside (4) and one xanthone dimer (7), together with eleven known compounds. The structures of these isolated compounds were elucidated on the basis of spectroscopic evidences and correlated with known compounds. Anti-influenza and anti-coxsackie virus activities were evaluated by determining the inhibition of anti-influenza neuraminidase (NA) from pandemic A/RI/5+/1957 H2N2 influenza A virus and inhibition of coxsackie B3 virus 3C protease, respectively. The highest anti-influenza activity was observed for compounds 8 and 9 with IC50 values of 11.9 and 9.2?M, respectively. Compounds 8 and 9 were even more potent against coxsackie B3 virus 3C protease, with IC50 values of 1.1 and 2.0?M, respectively. Compounds 8 and 9 showed weak cytotoxic effect against human hepatocellular carcinoma and human epithelial carcinoma cell lines through MTT assay.
During the systematic screening of active compounds from marine-derived fungi, the extract of a strain of Aspergillus versicolor MF359 isolated from a marine sponge of Hymeniacidon perleve was identified for detailed chemical investigation. Three new secondary metabolites, named hemiacetal sterigmatocystin (1), acyl-hemiacetal sterigmatocystin (2), and 5-methoxydihydrosterigmatocystin (3), together with a known compound, aversin (4), were characterized. 1 represents a first structure of sterigmatocystin hemiacetal from nature. The antibacterial activities of these identified compounds were evaluated against Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Bacillus subtilis, and Pseudomonas aeruginosa. Compound 3 showed activity against S. aureus and B. subtilis with MIC values of 12.5 and 3.125 ?g/mL, respectively.
A high-throughput screening of a microbial natural product library led to the discovery of two novel compounds named nivetetracyclates A and B (1 and 2), which were produced by Streptomyces niveus designated as LS2151. The backbone of the compounds contains a hydrotetracyclate not previously reported from a natural source. The structures of the compounds were elucidated by spectroscopic methods. The nivetetracyclates exhibited activity against human HeLa cells.
Serial antibacterial furanoditerpenes caesanines A-D (1-4), possessing a cassane-type diterpenoid skeleton with an unusual N bridge between C-19/C-20, were identified from a Chinese herb Caesalpinia sappan Linn. In addition, caesanine D (4) showed the first class of dicassane diterpenoid ethers. Their structures were determined by different spectroscopic methods and ECD calculation. Caesanines A and B exhibited strong activities against MRSA suggesting a promising entry point for the development of anti-infective drugs.
As part of a search for antitubercular substances from natural sources, we screened a library of endophytic microbes (50 strains and 300 crude extracts in total) isolated from traditional Chinese medicines (TCMs) for growth inhibitory activity against Bacillus Calmette-Guérin (BCG). The crude extract of Streptomyces sp. strain Y3111, which was associated with the stems of Heracleum souliei, showed good anti-BCG activity with an MIC value of 12.5 ?g/mL. Bioassay-guided isolation led to four new pluramycin-type compounds, heraclemycins A-D (1-4). Their structures were determined by different spectroscopic techniques including HRMSESI, 1D NMR, and 2D NMR. This is the first report of pluramycin analogues produced by TCM endophytic microbes as well as the first example of BCG-selective pluramycins. Heraclemycin C (3) showed selective antitubercular activity against BCG with a MIC value of 6.25 ?g/mL and a potential new mode of action.
A Gram-positive, endospore-forming, rod-shaped bacterium, designated isolate J2(T) was isolated from a soil sample from Xinjiang Uyghur Autonomous Region, China. The isolate was observed to grow at 16-46 °C and pH 6.5-8.0. Chemotaxonomic analysis showed menaquinone-7 (MK-7) to be the major isoprenoid quinone; diphosphatidylglycerol, phosphatidylglycerol, one aminophospholipid, two phosphoglycolipids and one glycolipid as the major cellular polar lipids; and anteiso-C15:0, iso-C15:0, anteiso-C17:0 and C16:0 as the major fatty acids. Comparative analyses of the 16S rRNA gene sequence showed that strain J2(T) is most closely related to Gracilibacillus ureilyticus (with 98.8 % similarity), Gracilibacillus dipsosauri (97.2 %), Gracilibacillus quinghaiensis (97.1 %) and Gracilibacillus thailandensis (97.0 %). The DNA-DNA reassociation values between strain J2(T) and G. ureilyticus MF38(T), G. dipsosauri DD1(T), G. quinghaiensis YIM-C229(T) and G. thailandensis TP2-8(T) were 29.8 ± 3.7, 23.0 ± 3.5, 15.8 ± 4.9 and 15.9 ± 5.0 %, respectively. The genomic DNA G+C content of strain J2(T) was determined to be 36.5 mol%. Based on these data, strain J2(T) is considered as a novel species of the genus Gracilibacillus, for which the name Gracilibacillus xinjiangensis sp. nov. is proposed. The type species is J2(T) (= CGMCC 1.12449(T) = JCM 18859(T)).
In the course of a screening program for bioactive compounds from a marine natural product library, a newly isolated Actinomycetes strain, designated as MS100061, exhibited strong anti-Mycobacterium bovis Bacillus Calmette-Guérin (BCG) activity. The strain belongs to the genus Streptomyces according to its morphological and 16S rDNA phylogenetic analysis. Bioassay-guided isolation resulted in a new spirotetronate, lobophorin G (1), together with two known compounds, lobophorins A (2) and B (3). The structures were elucidated by extensive spectroscopic methods and comparison with literatures. Compounds 1-3 were subjected to anti-BCG, antituberculosis, and antibacterial screening and exhibited potent anti-BCG activity with minimum inhibitory concentration (MIC) values of 1.56, 1.56, and 0.78 ?g/ml, respectively, and moderate anti-Mycobacterium tuberculosis H37Rv activity with MIC values of 32, 32, and 16 ?g/ml, respectively. The MIC values of compounds 1-3 against Bacillus subtilis were 3.125, 12.5, and 1.56 ?g/ml, respectively, indicating great potential for antibacterial drugs. In addition, this is the first report of the anti-BCG and antituberculosis activities of lobophorins.
Intracellular 14-3-3 proteins bind to many proteins, via a specific phosphoserine motif, regulating diverse cellular tasks including cell signalling and disease progression. The 14-3-3? isoform is a molecular chaperone, preventing the stress-induced aggregation of target proteins in a manner comparable with that of the unrelated sHsps (small heat-shock proteins). 1H-NMR spectroscopy revealed the presence of a flexible and unstructured C-terminal extension, 12 amino acids in length, which protrudes from the domain core of 14-3-3? and is similar in structure and length to the C-terminal extension of mammalian sHsps. The extension stabilizes 14-3-3?, but has no direct role in chaperone action. Lys(49) is an important functional residue within the ligand-binding groove of 14-3-3? with K49E 14-3-3? exhibiting markedly reduced binding to phosphorylated and non-phosphorylated ligands. The R18 peptide binds to the binding groove of 14-3-3? with high affinity and also reduces the interaction of 14-3-3? ligands. However, neither the K49E mutation nor the presence of the R18 peptide affected the chaperone activity of 14-3-3?, implying that the C-terminal extension and binding groove of 14-3-3? do not mediate interaction with target proteins during chaperone action. Other region(s) in 14-3-3? are most likely to be involved, i.e. the proteins chaperone and phosphoserine-binding activities are functionally and structurally separated.
Seventeen daucane sesquiterpenoid esters, including a new one (4), were isolated from the root of Ferula hermonis Boiss. The structures of the isolated compounds were elucidated on the basis of spectroscopic evidence and correlated with known compounds. The relative stereochemistry of the new compound was determined using 2D NOESY and the most stable and the lowest energy conformation was determined using molecular modelling. The antimicrobial activity was evaluated by determination of MIC using the broth microdilution method against six bacterial strains and one fungal strain (Pseudomonas aeruginosa PAO1, Escherichia coli, Bacillus subtilis ATCC6633, Mycobacterium bovis BCG Pasteur, Mycobacterium tuberculosis H37Rv, Staphylococcus aureus ATCC6538 and Candida albicans SC5314). There was a significant indication that compounds 15, 16, 17 demonstrated potent activity against Gram +ve (S. aureus, B. subtilis), as well as Mycobacterium strains M. bovis BCG and M. tuberculosis H37Rv. None of the isolated compounds exhibited a significant antifungal activity. In the antioxidant study using the DPPH assay method, the highest radical scavenging activity was observed for compounds 15, 16, 17.
Two new cyclic pentapeptides (1 and 2) and the known blazein (3), ganodesterone (4), ergosterin (5), cerevisterol (6), 24-methylcholesta-4,6,8(14),22-tetraen-3-one (7), 5,8-epidioxyergosta-6,22-dien-3-ol (8), 16-?-d-mannopyranosyloxyisopimar-7-en-19-oic acid (9), and 16-hydroxy isopimar-7-en-19-oic acid (10) have been isolated from the crude extract of an endolichenic Xylaria sp. The structures of 1 and 2 were elucidated primarily by NMR and MS methods. The absolute configurations of 1 and 2 were assigned using Marfeys method on their acid hydrolysate. Compounds 1-10 were evaluated for activity against fungi and for synergistic antifungal activity. Compound 1 showed synergistic antifungal activity against Candida albicans SC5314 with 0.004 ?g/mL ketoconazole.
Pseudomonas aeruginosa produces a biofilm that provides the bacteria with an effective barrier against antibiotics. Here, we investigated the synergy of various antibiotics with 14-alpha-lipoyl andrographolide (AL-1), focusing upon synthesis of the biofilm. AL-1 also inhibited the production of the exopolysaccharide and pyocyanin components. We propose that AL-1 may potentially serve as a cotherapy to combat P. aeruginosa.
The new secondary metabolites verrucosidinol (1) and its derivative verrucosidinol acetate (2), together with a potent neurotoxin verrucosidin (3), a congener norverrucosidin (4) and a mixture of two known phytotoxic metabolites terrestric acids (5 and 6), were isolated from the marine derived fungus Penicillium aurantiogriseum. Verrucosidinol has a ring-opened ethylene oxide moiety in the polyene ?-pyrone skeleton, and verrucosidinol acetate is its acetate derivative. The chemical structures were determined by comparing with literature data and a combination of spectroscopic techniques, including high resolution mass spectrum and two-dimentional nuclear magnetic resonance spectroscopic analysis.
Marine microorganisms are fascinating resources due to their production of novel natural products with antimicrobial activities. Increases in both the number of new chemical entities found and the substantiation of indigenous marine actinobacteria present a fundamental difficulty in the future discovery of novel antimicrobials, namely dereplication of those compounds already discovered. This review will share our experience on the taxonomic-based construction of a highly diversified and low redundant marine microbial natural product library for high-throughput antibiotic screening. We anticipate that libraries such as these can drive the drug discovery process now and in the future.
A high quality library of marine microbes and their associated natural products is critical for successful drug discovery. In this research a method to assess the quality of our marine microbial natural product library through screening for novel enediyne-like compounds from this library was set up.
Five new polyketide derivatives, 7-O-methylkoninginin D (1) and trichodermaketones A-D (2-5), together with four known compounds, koninginins A, D, E, and F, were isolated from the marine-derived fungus Trichoderma koningii. Trichodermaketones A (2) and B (3) are unprecedented polyketides with a bistetrafuran-containing tricyclic skeleton. The chemical structures and absolute configurations of compounds 1-5 were elucidated by comparing with literature data and extensive spectroscopic methods, including 2D NMR and CD spectroscopic analysis. Compounds 1-5 were evaluated for action against bacteria and fungi and for synergistic antifungal activity. Compound 2 showed synergistic antifungal activity against Candida albicans with 0.05 microg/mL ketoconazole.
Marine microbes are a rich source of bioactive compounds, such as drugs, enzymes, and biosurfactants. To explore the bioactive compounds from our marine natural product library, an oil emulsification assay was applied to discover biosurfactants and bioemulsifiers. A spore-forming bacterial strain from sea mud was found to produce bioemulsifiers with good biosurfactant activity and a broad spectrum of antimicrobial properties. It was identified as Bacillus velezensis H3 using genomic and phenotypic data analysis. This strain was able to produce biosurfactants with an optimum emulsification activity at pH 6.0 and 2% NaCl by using starch as the carbon source and ammonium sulfate as the nitrogen source. The emulsification-guided isolation and purification procedure led to the discovery of the biosurfactant components, which were mainly composed of nC(14)-surfactin and anteisoC(15)-surfactin as determined by NMR and MS spectra. These compounds can reduce the surface tension of phosphate-buffered saline (PBS) from 71.8 to 24.8 mN/m. The critical micelle concentrations (CMCs) of C(14)-surfactin and C(15)-surfactin in 0.1 M PBS (pH 8.0) were determined to be 3.06 x 10(-5) and 2.03 x 10(-5) mol/L, respectively. The surface tension values at CMCs for C(14)-surfactin and C(15)-surfactin were 25.7 and 27.0 mM/m, respectively. In addition, the H3 biosurfactant exhibited antimicrobial activities against Staphyloccocus aureus, Mycobacterium, Klebsiella peneumoniae, Pseudomonas aeruginosa, and Candida albicans. Thus B. velezensis H3 is an alternative surfactin producer with potential application as an industrial strain for the lipopeptide production.
Response surface methodology was employed to optimize the composition of medium for the production of avermectin B1a by Streptomyces avermitilis 14-12A in shaker flask cultivation. Corn starch and yeast extract were found to have significant effects on avermectin B1a production by the Plackett-Burman design. The steepest ascent method was used to access the optimal region of the medium composition, followed by an application of response surface. The analysis revealed that the optimum values of the tested variables were 149.57 g/l corn starch and 8.92 g/l yeast extract. A production of 5128 mg/l, which was in agreement with the prediction, was observed in verification experiment. In comparison to the production of original level (3528 mg/l), 1.45-fold increase had been obtained.
An Aspergillus versicolor isolated from sediment collected from the Bohai Sea, China, yielded the new dimeric diketopiperazine brevianamide S (1), together with three new monomeric cometabolites, brevianamides T (2), U (3), and V (4). Structures were determined by detailed spectroscopic analysis. Brevianamide S exhibited selective antibacterial activity against Bacille Calmette-Guérin (BCG), suggestive of a new mechanism of action that could inform the development of next-generation antitubercular drugs.
Three new alkaloids, including auranomides A and B (1 and 2), a new scaffold containing quinazolin-4-one substituted with a pyrrolidin-2-iminium moiety, and auranomide C (3), as well as two known metabolites auranthine (4) and aurantiomides C (5) were isolated from the marine-derived fungus Penicillium aurantiogriseum. The chemical structures of compounds 1-3 were elucidated by extensive spectroscopic methods, including IR, HRESIMS and 2D NMR spectroscopic analysis. The absolute configurations of compounds 1-3 were suggested from the perspective of a plausible biosynthesis pathway. Compounds 1-3 were subjected to antitumor and antimicrobial screening models. Auranomides A-C exhibited moderate cytotoxic activity against human tumor cells. Auranomides B was the most potent among them with an IC(50) value of 0.097 ?mol/mL against HEPG2 cells.
Multidrug-resistant tuberculosis (MDR-TB) and TB-HIV co-infection have become a great threat to global health. However, the last truly novel drug that was approved for the treatment of TB was discovered 40 years ago. The search for new effective drugs against TB has never been more intensive. Natural products derived from microbes and medicinal plants have been an important source of TB therapeutics. Recent advances have been made to accelerate the discovery rate of novel TB drugs including diversifying strategies for environmental strains, high-throughput screening (HTS) assays, and chemical diversity. This review will discuss the challenges of finding novel natural products with anti-TB activity from marine microbes and plant medicines, including biodiversity- and taxonomy-guided microbial natural products library construction, target- and cell-based HTS, and bioassay-directed isolation of anti-TB substances from traditional medicines.
In Pseudomonas aeruginosa, the quorum-sensing (QS) system is closely related to biofilm formation. We previously demonstrated that 14-alpha-lipoyl andrographolide (AL-1) has synergistic effects on antibiofilm and antivirulence factors (pyocyanin and exopolysaccharide) of P. aeruginosa when combined with conventional antibiotics, while it has little inhibitory effect on its growth. However, its molecular mechanism remains elusive. Here we investigated the effect of AL-1 on QS systems, especially the Las and Rhl systems. This investigation showed that AL-1 can inhibit LasR-3-oxo-C(12)-homoserine lactone (HSL) interactions and repress the transcriptional level of QS-regulated genes. Reverse transcription (RT)-PCR data showed that AL-1 significantly reduced the expression levels of lasR, lasI, rhlR, and rhlI in a dose-dependent manner. AL-1 not only decreased the expression level of Psl, which is positively regulated by the Las system, but also increased the level of secretion of ExoS, which is negatively regulated by the Rhl system, indicating that AL-1 has multiple effects on both the Las and Rhl systems. It is no wonder that AL-1 showed synergistic effects with other antimicrobial agents in the treatment of P. aeruginosa infections.
Two of our long term efforts are to discover compounds with synergistic antifungal activity from metabolites of marine derived microbes and to optimize the production of the interesting compounds produced by microorganisms. In this respect, new applications or mechanisms of already known compounds with a high production yield could be continually identified. Surfactin is a well-known lipopeptide biosurfactant with a broad spectrum of antimicrobial and antiviral activity; however, there is less knowledge on surfactins antifungal activity. In this study, we investigated the synergistic antifungal activity of C(15)-surfactin and the optimization of its production by the response surface method.
Despite the urgent need for new antitubercular drugs, few are on the horizon. To combat the problem of emerging drug resistance, structurally unique chemical entities that inhibit new targets will be required. Here we describe our investigations using whole cell screening of a diverse collection of small molecules as a methodology for identifying novel inhibitors that target new pathways for Mycobacterium tuberculosis drug discovery. We find that conducting primary screens using model mycobacterial species may limit the potential for identifying new inhibitors with efficacy against M. tuberculosis. In addition, we confirm the importance of developing in vitro assay conditions that are reflective of in vivo biology for maximizing the proportion of hits from whole cell screening that are likely to have activity in vivo. Finally, we describe the identification and characterization of two novel inhibitors that target steps in M. tuberculosis cell wall biosynthesis. The first is a novel benzimidazole that targets mycobacterial membrane protein large 3 (MmpL3), a proposed transporter for cell wall mycolic acids. The second is a nitro-triazole that inhibits decaprenylphosphoryl-?-D-ribose 2-epimerase (DprE1), an epimerase required for cell wall biosynthesis. These proteins are both among the small number of new targets that have been identified by forward chemical genetics using resistance generation coupled with genome sequencing. This suggests that methodologies currently employed for screening and target identification may lead to a bias in target discovery and that alternative methods should be explored.
In the course of our screening program for anti-Mycobacterium bovis bacillus Calmette-Guérin (BCG) and anti-Mycobacterium tuberculosis H37Rv (MTB H37Rv) agents from our marine natural product library, a newly isolated actinomycete strain, designated as MS449, was picked out for further investigation. The strain MS449, isolated from a sediment sample collected from South China Sea, produced actinomycin X(2) and actinomycin D in substantial quantities, which showed strong inhibition of BCG and MTB H37Rv. The structures of actinomycins were elucidated by nuclear magnetic resonance and mass spectrometric analysis. The strain MS449 was taxonomically characterized on the basis of morphological and phenotypic characteristics, genotypic data, and phylogenetic analysis. The 16S rRNA gene sequence of the strain was determined and a database search indicated that the strain was closely associated with the type strain of Streptomyces avermitilis (99.7 % 16S rRNA gene similarity). S. avermitilis has not been previously reported to produce actinomycins. The marine-derived strain of Streptomyces sp. MS449 produced notably higher quantities of actinomycin X(2) (1.92 mg/ml) and actinomycin D (1.77 mg/ml) than previously reported actinomycins producing strains. Thus, MS449 was considered of great potential as a new industrial producing strain of actinomycin X(2) and actinomycin D.
Herein we outline the antibacterial activity of amino acid containing thiazolidinediones and rhodanines against Gram-positive bacteria Staphylococcus aureus ATCC 31890, Staphylococcus epidermidis and Bacillus subtilis ATCC 6633. The rhodanine derivatives were generally more active than the analogous thiazolidinediones. Compounds of series 5 showed some selectivity for Bacillus subtilis ATCC 6633, the extent of which is enhanced by the inclusion of a non-polar amino acid at the 5-position of the core thiazolidinediones and rhodanines scaffolds. SAR data of series 8 demonstrated improved activity against the clinically more significant Staphylococci with selectivity over Bacillus subtilis ATCC 6633 induced by introduction of a bulky aryl substituent at the 5-position of the core scaffolds.
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