Fungal Secondary Metabolite Exophillic Acid Selectively Inhibits the Entry of Hepatitis B and D Viruses.

Current anti-hepatitis B virus (HBV) drugs are suppressive but not curative for HBV infection, so there is considerable demand for the development of new anti-HBV agents. In this study, we found that fungus-derived exophillic acid inhibits HBV infection with a 50% maximal inhibitory concentration (IC50) of 1.1 µM and a 50% cytotoxic concentration (CC50) of >30 µM in primary human hepatocytes. Exophillic acid inhibited preS1-mediated viral attachment to cells but did not affect intracellular HBV replication. Exophillic acid appears to target the host cells to reduce their susceptibility to viral attachment rather than acting on the viral particles. We found that exophillic acid interacted with the HBV receptor, sodium taurocholate cotransporting polypeptide (NTCP). Exophillic acid impaired the uptake of bile acid, the original function of NTCP. Consistent with our hypothesis that it affects NTCP, exophillic acid inhibited infection with HBV and hepatitis D virus (HDV), but not that of hepatitis C virus. Moreover, exophillic acid showed a pan-genotypic anti-HBV effect. We thus identified the anti-HBV/HDV activity of exophillic acid and revealed its mode of action. Exophillic acid is expected to be a potential new lead compound for the development of antiviral agents.

Mechanism of the natural product moracin-O derived MO-460 and its targeting protein hnRNPA2B1 on HIF-1α inhibition.

Hypoxia-inducible factor-1α (HIF-1α) mediates tumor cell adaptation to hypoxic conditions and is a potentially important anticancer therapeutic target. We previously developed a method for synthesizing a benzofuran-based natural product, (R)-(-)-moracin-O, and obtained a novel potent analog, MO-460 that suppresses the accumulation of HIF-1α in Hep3B cells. However, the molecular target and underlying mechanism of action of MO-460 remained unclear. In the current study, we identified heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1) as a molecular target of MO-460. MO-460 inhibits the initiation of HIF-1α translation by binding to the C-terminal glycine-rich domain of hnRNPA2B1 and inhibiting its subsequent binding to the 3'-untranslated region of HIF-1α mRNA. Moreover, MO-460 suppresses HIF-1α protein synthesis under hypoxic conditions and induces the accumulation of stress granules. The data provided here suggest that hnRNPA2B1 serves as a crucial molecular target in hypoxia-induced tumor survival and thus offer an avenue for the development of novel anticancer therapies.

Pestynol, an Antifungal Compound Discovered Using a Saccharomyces cerevisiae 12geneΔ0HSR-iERG6-Based Assay.

The multidrug-sensitive budding yeast, Saccharomyces cerevisiae 12geneΔ0HSR-iERG6, is very useful in antifungal screens. A novel compound, named pestynol (1), was discovered from a culture of the fungus Pestalotiopsis humus FKI-7473 using the multidrug-sensitive yeast. The structure of 1 was elucidated by NMR studies and modified Mosher's method as (1 R,2 R,3 R,4 R)-( E)-5-(7,11-dimethyl-3-methylenedodeca-6,10-dien-1-yn-1-yl)cyclohex-5-ene-1,2,3,4-tetraol. Compound 1 showed antimicrobial activity against the Gram-positive bacteria, Klebsiella pneumoniae, and S. cerevisiae 12geneΔ0HSR-iERG6 and Mucor racemosus, but displayed only weak cytotoxicity against various human cancer cell lines. Compound 1 displayed antifungal activities against S. cerevisiae 12geneΔ0HSR-iERG6 and Mucor racemosus at 10 µg/disc.

Staurosporine: new lease of life for parent compound of today's novel and highly successful anti-cancer drugs.

Staurosporine, together with such examples as penicillin, aspirin, ivermectin and sildenafil, exemplifies the role that serendipity has in drug discovery and why 'finding things without actually searching for them' retains a prominent role in drug discovery. Hitherto not clinically useful, due to its potency and promiscuity, new delivery technology is opening up new horizons for what was previously just the parent compound of innovative, highly-successful anti-cancer agents.

Marcanine G, a new cytotoxic 1-azaanthraquinone from the stem bark of Goniothalamus marcanii Craib.

The ethanolic extract from the stem bark of Goniothalamus marcanii Craib was shown in preliminary brine shrimp lethality data having good cytotoxic activity. Further bioassay guided isolation was done by means of solvent partition, chromatography and precipitation to provide four isolated compounds: a novel compound 1 with the core structure of 1-azaanthraquinone moiety referred as marcanine G; as well as compounds 2-4 with known aristolactam structures namely, piperolactam C, cepharanone B and taliscanine. These compounds were characterised by spectroscopic techniques. The assessment of cytotoxicity was established on an SRB assay using doxorubicin as a positive control. Marcanine G (1) was considered the most active compound indicating the IC50 values of 14.87 and 15.18 µM against human lung cancer cells (A549) and human breast cancer cells (MCF7), respectively. However, 2 showed mild activity with the IC50 values of 83.72 and 82.32 µM against A549 and MCF7 cells, respectively.

Decatamariic acid, a new mitochondrial respiration inhibitor discovered by pesticidal screening using drug-sensitive Saccharomyces cerevisiae.

A new decalin, decatamariic acid, was isolated from a cultured broth of the fungus Aspergillus tamarii FKI-6817. Its absolute configuration was elucidated by NMR and electronic circular dichroism. Decatamariic acid (10 µM) elicited ~50% inhibition of the ATP production in mitochondria isolated from wild-type Saccharomyces cerevisiae without affecting the activities of respiratory enzymes. The action manner of this compound may be interesting as a possible seed for new pesticides.

Iminimycin A, the new iminium metabolite produced by Streptomyces griseus OS-3601.

A new natural product, designated iminimycin A, was isolated from the cultured broth of a streptomycin-producing microbial strain, Streptomyces griseus OS-3601, via a physicochemical screening method using HP-20, silica gel and ODS column chromatographies and subsequent preparative HPLC. Iminimycin A is an indolizidine alkaloid, containing of an unusual iminium group and a cyclopropane ring with a triene side chain. The absolute configuration of iminimycin A was elucidated by NMR studies and electronic circular dichroism analysis. Iminimycin A shows anti-bacterial activity against Bacillus subtilis, Kocuria rhizophila and Xanthomonas campestris pv. orizae, and cytotoxic activity against HeLa S3 and Jurkat cells with IC50 values of 43 and 36 µM, respectively.

Trichopolyn VI: a new peptaibol insecticidal compound discovered using a recombinant Saccharomyces cerevisiae screening system.

In the course of searching for insecticides from soil microorganisms, we found that a fermentation broth of the fungus, Trichoderma brevicompactum FKI-6324, produced Trichopolyn VI, a new peptaibol, which possessed significant insecticidal potential. Spectroscopic analysis showed the compound to be a new trichopolyn I derivative. This paper describes the isolation, structure elucidation and biological activity of trichopolyn VI.

Illudins C2 and C3 stimulate lipolysis in 3T3-L1 adipocytes and suppress adipogenesis in 3T3-L1 preadipocytes.

The secondary metabolites illudins C2 (1) and C3 (2), obtained from the culture broth of Coprinus atramentarius, have been shown to possess antimicrobial activity. In the present study, we discovered novel biological activities of 1 and 2 in lipolysis of differentiated 3T3-L1 adipocytes and adipogenesis of 3T3-L1 preadipocytes. Compounds 1 and 2 exhibit a dose-dependent increase in glycerol release and thereby reduce intracellular lipid accumulation. The stimulatory effects of 1 and 2 on lipolysis are prevented by cAMP-dependent protein kinase (PKA) and extracellular signal-regulated kinase (ERK) inhibitors. Compounds 1 and 2 down-regulated perilipin and also affected the mRNA and protein levels of adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL). However, 1 and 2 treatment leads to a significant increase in PKA-mediated phosphorylation of HSL at S563 and S660. In addition, 1 and 2 treatment in 3T3-L1 preadipocytes induces down-regulation of the critical transcription factors, CCAAT/enhancer binding protein α and ß (C/EBPα and C/EBPß), and peroxisome proliferator activated receptor γ (PPARγ), which are required for adipogenesis, and accordingly inhibits adipogenesis. These results suggest that 1 and 2 might be useful for treating obesity due to their modulatory effects on fat by affecting adipocyte differentiation and fat mobilization.

Combined treatment with verrucarin A and tumor necrosis factor-α sensitizes apoptosis by overexpression of nuclear factor-kappaB-mediated Fas.

Verrucarin A (VA) is a member of the family of macrocyclic trichothecenes, which exhibit anti-cancer and immune-modulating activities. However, VA has not yet been demonstrated to be involved in the sensitization of tumor necrosis factor-alpha (TNF-α)-mediated apoptosis. In the present study, we found that VA triggers TNF-α-induced apoptosis in human breast cancer MDA-MB-231 and MCF-7 cells. In particular, activation of caspas-3 and caspase-8 as well as release of cytochrome c were significantly enhanced in response to the combined treatment with VA and TNF-α (VA/TNF-α) and the pan-caspase inhibitor z-VAD-fmk completely reversed the apoptosis, suggesting that caspases are the main effector molecules in VA/TNF-α-induced apoptosis via the intrinsic and extrinsic pathway. Moreover, we confirmed that enhanced Fas expression plays a critical role, because the Fas-blocking antibody partially inhibited VA/TNF-α-induced apoptosis. VA also increased specific DNA-binding activity of nuclear factor-kappaB (NF-κB) via nuclear translocation of p50 and p65. In addition, pretreatment with the NF-κB inhibitor MG132 blocked VA/TNF-α-induced apoptosis by suppression of NF-κB-dependent Fas expression. These results indicated that VA enhances TNF-α-induced apoptosis via NF-κB-dependent Fas overexpression.

Xestospongin C induces monocytic differentiation of HL60 cells through activation of the ERK pathway.

Xestospongin C (XC), which is a group of macrocyclic bis-1-oxaquinolizidines, is a potent inhibitor of sarcoendoplasmic reticulum calcium transport ATPase and IP3 receptor. Nevertheless, very less information is available regarding whether XC induces AML differentiation. We investigated the potential role of XC in the differentiation of human leukemia HL60 cells and mechanisms underlying XC actin. XC treatment inhibited proliferation by inducing G1-phase cell cycle arrest in the HL60 cells. In addition, XC induced differentiation of HL60 cells into the CD14(+) monocytic lineage, which was indicated by morphological changes, nitroblue tetrazolium reduction assay, and expressions of CD11b and CD14 surface antigens. Our results also showed that XC promotes phagocytic activity and granularity in HL60 cells, suggesting that the cells are functionally activated. Furthermore, XC enhanced tumor necrosis factor (TNF)-α-mediated cytotoxic effect by increasing the numbers of TNF receptors. Moreover, we showed that XC activates extracellular signal-regulated kinase (ERK) pathway in the differentiation stages. Inhibition of ERK activation using PD98059 significantly decreased NBT+HL60 cells induced by XC treatment. Taken together, the results show that XC promotes monocytic differentiation of HL60 cells via ERK pathway activation, suggesting that XC could be a candidate for use as a differentiation-inducing agent for AML treatment.

Verrucarin A enhances TRAIL-induced apoptosis via NF-κB-mediated Fas overexpression.

We investigated whether verrucarin A (VA) sensitizes HepG2 hepatoma cells to tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-mediated apoptosis. We found that VA alone induces little apoptosis, but when combined with TRAIL (VA/TRAIL), it triggered significant apoptosis, causing little or no toxicity in normal mouse splenocytes. VA/TRAIL-induced cell death is involved in the loss of mitochondrial transmembrane potential and the consequent activation of caspases. Because nuclear factor (NF)-κB inhibition has been known as a critical target in TRAIL-mediated apoptosis, we also investigated the role of NF-κB in VA/TRAIL treatment. We found that VA upregulated the DNA binding activity of NF-κB, but that the antioxidants glutathione and N-acetyl-l-cysteine, as well as NF-κB inhibitor MG132, and mutant-IκB (m-IκB) transfection, significantly downregulated VA/TRAIL-induced cell death by inhibiting caspase-3 and NF-κB activities. Transfection of mutant-eIF2α also resulted in a decrease in VA/TRAIL-induced cell death by inhibiting of caspase-3, but not NF-κB activity. Although VA/TRAIL treatment led to an increase of DR5 expression, transfection of m-IκB had no influence on the DR5 expressional level. Finally, we showed that NF-κB-mediated Fas expression is critical to VA/TRAIL-induced apoptosis. Taken together, these results indicate that VA/TRAIL sensitizes HepG2 cells to apoptosis via NF-κB-mediated overexpression of Fas.

STK295900, a dual inhibitor of topoisomerase 1 and 2, induces G(2) arrest in the absence of DNA damage.

STK295900, a small synthetic molecule belonging to a class of symmetric bibenzimidazoles, exhibits antiproliferative activity against various human cancer cell lines from different origins. Examining the effect of STK295900 in HeLa cells indicates that it induces G(2) phase arrest without invoking DNA damage. Further analysis shows that STK295900 inhibits DNA relaxation that is mediated by topoisomerase 1 (Top 1) and topoisomerase 2 (Top 2) in vitro. In addition, STK295900 also exhibits protective effect against DNA damage induced by camptothecin. However, STK295900 does not affect etoposide-induced DNA damage. Moreover, STK295900 preferentially exerts cytotoxic effect on cancer cell lines while camptothecin, etoposide, and Hoechst 33342 affected both cancer and normal cells. Therefore, STK295900 has a potential to be developed as an anticancer chemotherapeutic agent.

Verrucarin A sensitizes TRAIL-induced apoptosis via the upregulation of DR5 in an eIF2α/CHOP-dependent manner.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is one of the most promising candidates for new cancer therapeutics. However, resistance to TRAIL in some cancers remains a current problem in recent. The protein-folding compartment of the endoplasmic reticulum (ER) is particularly sensitive to disturbances, which, if severe, may trigger apoptosis. Therefore, we examined whether verrucarin A (VA) sensitize TRAIL-induced apoptosis in cancer cells by induction of ER stress. We first found that VA induces a major molecule of ER stress, CCAAT/enhancer binding protein homologous protein (CHOP)-dependent DR5 induction and subsequently increases TRAIL-induced cleavage of caspases and PARP in TRAIL-resistant Hep3B cells. Importantly, the transient knockdown using siRNA for CHOP abrogated VA-induced DR5 expression and attenuated TRAIL-induced apoptosis. Treatment with VA also increased the levels of phosphorylation of eukaryotic translation initiation factor-2α (eIF2α), which is a common cellular response of ER stress. Furthermore, salubrinal, a specific eIF2α phosphorylation-inducing agent, increased CHOP and DR5 expression in the presence of VA. In contrast, transfection of mutant-eIF2α significantly reversed VA-induced apoptosis with downregulation of CHOP-dependent DR5 expression. Therefore, VA-induced eIF2α phosphorylation seemed to be important for CHOP and DR5 upregulation and TRAIL-induced apoptosis. In addition, generation of reactive oxygen species (ROS) is an effector molecular in sensitization of VA-induced ER stress. We concluded that VA triggers TRAIL-induced apoptosis by eIF2α/CHOP-dependent DR5 induction via ROS generation.

Violaceols function as actin inhibitors inducing cell shape elongation in fibroblast cells.

Violaceol-I and -II were isolated from a fractionated library of marine-derived fungal metabolites. These compounds increased the calcium ion concentration inside the cell and caused F-actin aggregation in rat fibroblast 3Y1 cells within 3 h resulting in cell shape elongation. Calcium chelator BAPTA-AM (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxymethyl ester) inhibited violaceol-I and -II induced F-actin aggregation in 3Y1 cells, and hence violaceol-I and -II act in a calcium dependent manner. Violaceol-I and -II inhibited G-actin polymerization in vitro in a dose-dependent manner and strongly associated with G-actin, at dissociation equilibrium constants of 1.44 × 10(-8) M and 2.52 × 10(-9) M respectively. Here we report the identification of a novel function of violaceol-I and -II as actin inhibitors. Violaceol-I and -II induced cell shape elongation through F-actin aggregation in 3Y1 fibroblasts. These compounds may give researchers new insights into the role of actin in tumorigenesis and lead to the development of additional anti-tumor drugs.

Protuboxepin A, a marine fungal metabolite, inducing metaphase arrest and chromosomal misalignment in tumor cells.

Previously we reported the identification of a new oxepin-containing diketopiperazine-type marine fungal metabolite, named protuboxepin A which showed antiproliferative activity in several cancer cell lines. In this study we elucidated the mechanism by which protuboxepin A induces cancer cell growth inhibition. Here we report that protuboxepin A induced round-up morphology, M phase arrest, and an increase in the subG(1) population in tumor cells in a dose dependent manner. Our investigations revealed that protuboxepin A directly binds to α,ß-tubulin and stabilizes tubulin polymerization thus disrupting microtubule dynamics. This disruption leads to chromosome misalignment and metaphase arrest which induces apoptosis in cancer. Overall, we identified protuboxepin A as a microtubule-stabilizing agent which has a distinctly different chemical structure from previously reported microtubule inhibitors. These results indicate that protuboxepin A has a potential of being a new and effective anti-cancer drug.

Fusarisetin A, an acinar morphogenesis inhibitor from a soil fungus, Fusarium sp. FN080326.

An acinar morphogenesis inhibitor named fusarisetin A (1) that possesses both an unprecedented carbon skeleton and a new pentacyclic ring system has been identified from an in-house fractionated fungal library using a three-dimensional matrigel-induced acinar morphogenesis assay system. The structure of 1 was determined in detail by NMR and circular dichroism spectroscopy, X-ray analysis, and chemical reaction experiments.

Protuboxepins A and B and protubonines A and B from the marine-derived fungus Aspergillus sp. SF-5044.

Two new oxepin-containing (1 and 2) and two diketopiperazine-type alkaloids (3 and 4) have been isolated from an EtOAc extract of the marine-derived fungus Aspergillus sp. SF-5044. The structures of these metabolites were determined through analysis of NMR and MS data, along with Marfey's method. Compound 1 showed weak growth inhibitory activity against a small panel of cell lines.