Mycoepoxydiene Research Articles

Rapid discovery of a new antifoulant: From in silico studies targeting barnacle chitin synthase to efficacy against barnacle settlement

Due to the adverse environmental impacts of toxic heavy metal–based antifoulants, the screening of environmentally friendly antifoulants has become important for the development of marine antifouling technology. Compared with the traditional lengthy and costly screening method, computer-aided drug design (CADD) offers a promising and efficient solution that can accelerate the screening process of green antifoulants. In this study, we selected barnacle chitin synthase (CHS, an important enzyme for barnacle settlement and development) as the target protein for docking screening. Three CHS genes were identified in the barnacle Amphibalanus amphitrite, and their encoded proteins were found to share a conserved glycosyltransferase domain. Molecular docking of 31,561 marine natural products with AaCHSs revealed that zoanthamine alkaloids had the best binding affinity (−11.8 to −12.6 kcal/mol) to AaCHSs. Considering that the low abundance of zoanthamine alkaloids in marine organisms would limit their application as antifoulants, a marine fungal–derived natural product, mycoepoxydiene (MED), which has a similar chemical structure to zoanthamine alkaloids and the potential for large-scale production by fermentation, was selected and validated for stable binding to AaCHS2L2 using molecular docking and molecular dynamics simulations. Finally, the efficacy of MED in inhibiting cyprid settlement of A. amphitrite was confirmed by a bioassay that demonstrated an EC50 of 1.97 μg/mL, suggesting its potential as an antifoulant candidate. Our research confirmed the reliability of using AaCHSs as antifouling targets and has provided insights for the efficient discovery of green antifoulants by CADD.

Potent and Selective Inhibitors of Human Monoamine Oxidase A from an Endogenous Lichen Fungus Diaporthe mahothocarpus.

Using 126 endogenous lichen fungus (ELF) extracts, inhibitory activities against monoamine oxidases (MAOs) and cholinesterases (ChEs) were evaluated. Among them, extract ELF29 of the endogenous fungus Diaporthe mahothocarpus of the lichen Cladonia symphycarpia showed the highest inhibitory activity against hMAO-A. Compounds alternariol (AT), 5′-hydroxy-alternariol (HAT), and mycoepoxydiene (MED), isolated from the extract, had potent inhibitory activities against hMAO-A with IC50 values of 0.020, 0.31, and 8.68 µM, respectively. AT, HAT, and MED are reversible competitive inhibitors of hMAO-A with Ki values of 0.0075, 0.116, and 3.76 µM, respectively. The molecular docking studies suggested that AT, HAT, and MED had higher binding affinities for hMAO-A (−9.1, −6.9, and −5.6 kcal/mol, respectively) than for hMAO-B (−6.3, −5.2, and −3.7 kcal/mol, respectively). The relative tight binding might result from a hydrogen bond interaction of the three compounds with a Tyr444 residue in hMAO-A, whereas no hydrogen bond interaction was proposed in hMAO-B. In silico pharmacokinetics, the three compounds showed high gastrointestinal absorption without violating Lipinski’s five rules, but only MED showed high probability to cross the blood–brain barrier. These results suggest that AT, HAT, and MED are candidates for treating neuropsychiatric disorders, such as depression and cardiovascular disease.

Mycoepoxydiene suppresses HeLa cell growth by inhibiting glycolysis and the pentose phosphate pathway

Upregulation of glycolysis and the pentose phosphate pathway (PPP) is a major characteristic of the metabolic reprogramming of cancer and provides cancer cells with energy and vital metabolites to support their rapid proliferation. Targeting glycolysis and the PPP has emerged as a promising antitumor therapeutic strategy. Marine natural products are attractive sources for anticancer therapeutics, as evidenced by the antitumor drug Yondelis. Mycoepoxydiene (MED) is a natural product isolated from a marine fungus that has shown promising inhibitory efficacy against HeLa cells in vitro. We used a proteomic approach with two-dimensional gel electrophoresis (2-DE) coupled with mass spectrometry to explore the cellular targets of MED and to unravel the molecular mechanisms underlying the antitumor activity of MED in HeLa cells. Our proteomic data showed that triosephosphate isomerase (TPI) and 6-phosphogluconolactonase (PGLS), which participate in glycolysis and the PPP, respectively, were significantly downregulated by MED treatment. Functional studies revealed that the expression levels of several other enzymes involved in glycolysis and the PPP, including hexokinase 2 (HK2), phosphofructokinase 1 (PFKM), aldolase A (ALDOA), enolase 1 (ENO1), lactate dehydrogenase A (LDHA), and glucose-6-phosphate dehydrogenase (G6PD), were also reduced in a dose-dependent manner. Moreover, the LDHA and G6PD enzymatic activities in HeLa cells were inhibited by MED, and overexpression of these downregulated enzymes rescued HeLa cells from the growth inhibition induced by MED. Our data suggest that MED suppresses HeLa cell growth by inhibiting glycolysis and the PPP, which provides a mechanistic basis for the development of new therapeutics against cervical cancer.

Inhibitory effects of Mycoepoxydiene on macrophage foam cell formation and atherosclerosis in ApoE-deficient mice

BackgroundMycoepoxydiene (MED) is a polyketide that can be isolated from a marine fungus and is associated with various activities, including antitumor and anti-inflammatory functions. However, its effects on atherosclerosis remain unknown. Macrophage-derived foam cells play crucial roles in the initiation and progression of atherosclerotic plaques. In this study, we investigated the effects of MED on oxidized low-density lipoprotein (ox-LDL)-induced macrophage foam cell formation and activation, and on high fat diet (HFD)-induced atherosclerosis in ApoE-deficient (ApoE−/−) mice.ResultsOur findings show that MED could significantly inhibit ox-LDL-induced macrophage foam cell formation and suppress the expression of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), which is a receptor for ox-LDL. Additionally, MED could significantly inhibit the secretion of proinflammatory cytokines, such as tumor necrosis factor (TNF-α), interleukin (IL)-6, and IL-1β. Mechanistically, MED inhibited NF-κB activation by blocking IκB-α degradation and reducing NF-κB DNA binding activity. Moreover, MED dramatically reduced the occurrence of HFD-induced atherosclerotic lesions in ApoE−/− mice.ConclusionsOur study shows that MED can inhibit macrophage foam cell formation and activation by inhibiting NF-κB activation, thereby protecting ApoE−/− mice from HFD-induced atherosclerosis. Our findings suggest that MED might be a potential lead compound for the development of antiatherosclerotic therapeutics.Electronic supplementary materialThe online version of this article (doi:10.1186/s13578-015-0017-y) contains supplementary material, which is available to authorized users.

Heat shock protein 90 inhibitor mycoepoxydiene modulates kinase signaling in cervical cancer cells and inhibits in-vivo tumor growth.

Heat shock protein 90 (Hsp90) functions within multiple signaling pathways on the basis of its ability to serve as a chaperone for more than 100 client proteins. Thus, inhibition of Hsp90 alone can trigger numerous pathways. Mycoepoxydiene (MED) can inhibit Hsp90 function and induce apoptosis in cervical cancer cells. However, the antitumor efficacy of MED in vivo is still not clear. We examined the efficacy of MED in a mouse xenograft model to further elucidate HeLa cell fate and also assessed the mechanism of altered protein signaling in response to this compound in vitro. Our data showed that Hsp90 inhibition simultaneously triggers signaling that regulates both cell death and cell proliferation, and that HeLa cell death may be a result of the disequilibrium of these signals. MED induces cell death as a result of the destabilization of Akt and IKK, which may promote cell death through a reduction in the activation of Bad and nuclear factor-κB. However, MED also induces the MEK/ERK pathway, which is classically considered to promote cell survival. MEK/ERK activation leads to an increase in p21, a cyclin-dependent kinase inhibitor, and is independent of Raf, but is shown to be mediated by p53. MED also leads to a decrease in several additional G2/M regulatory proteins independent of the MEK/ERK pathway. These results indicate an interesting mechanism of cross-talk between the inhibition of Akt phosphorylation and the activation of the MEK pathway by MED and provide in-vivo evidence for the potential of inhibiting Hsp90 as a candidate anticancer treatment.

Mycoepoxydiene induces apoptosis and inhibits TPA-induced invasion in human cholangiocarcinoma cells via blocking NF-κB pathway

Human cholangiocarcinoma (CCA) is a chemoresistant bile duct carcinoma with a poor prognosis. Conventional chemotherapy and radiotherapy have not been reported to be effective in improving long-term survival. Mycoepoxydiene (MED), a polyketide isolated from the marine fungal strain Diaporthe sp. HLY-1 associated with mangroves, has been shown to be an agent capable of inducing apoptosis in MCF-7 and Hela cell lines. However, little is known about the effect of MED in CCA. Herein, we investigated the effect of MED on CCA cells proliferation and invasion. The results demonstrated that MED induced apoptosis in CCA cells such as SK-ChA-1 and Mz-ChA-1 through inhibiting the expression of anti-apoptotic proteins such as Bcl-XL and Bcl-2, two targets of NF-κB. In addition, MED significantly inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced CCA cells invasion in a dose-dependent manner by reducing the expression of matrix metalloelastase 9 (MMP-9). Moreover, MED inhibited TPA-induced NF-κB activation via blocking phosphorylation and degradation of IκBα and phosphorylation of IκB kinase (IKK). MED had no effect on the activation of extracellular signal-regulated kinase (ERK), c-jun N-terminal kinase (JNK) and p38, which are also involved in regulating the MMP-9 expression. Collectively, MED significantly suppressed proliferation and invasion of CCA cells such as SK-ChA-1 and Mz-ChA-1, suggesting that MED is a potential lead compound for the development of novel drugs for therapy of CCA.

Mycoepoxydiene inhibits activation of BV2 microglia stimulated by lipopolysaccharide through suppressing NF-κB, ERK 1/2 and toll-like receptor pathways

Mycoepoxydiene (MED) is a polyketide isolated from the marine fungal Diaporthe sp. HLY-1 associated with mangroves. Although MED has been shown to have various biological effects such as antimicrobial, anti-cancer, and anti-inflammatory activities, its activities and cellular mechanisms during microglial activation have yet to be elucidated. In the present study, we assessed the anti-inflammatory effect of MED on the production of inflammatory mediators in lipopolysaccharide (LPS)-stimulated murine BV2 microglia. MED significantly inhibited LPS-induced production of pro-inflammatory mediators such as tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), interferon-γ (INF-γ), and nitric oxide (NO), whereas it increased anti-inflammatory interleukin-10 (IL-10) and transforming growth factor-β1 (TGF-β1) production in BV2 microglia in a concentration-dependent manner without causing cytotoxicity. Moreover, MED suppressed NF-κB activation by blocking IkappaB-α (IκB-α) degradation and inhibited the phosphorylation of ERK 1/2 and toll-like receptor 4 (TLR4) expression, but had no effect on the phosphorylation of JNK, and p38. Our results demonstrate that the inhibitory and promotion effect of MED on LPS-stimulated inflammatory mediators and anti-inflammatory factor production in BV2 microglia is associated with the suppression of the NF-κB, ERK1/2 and TLR signaling pathways. Therefore, MED may have therapeutic potential for neurodegenerative diseases by inhibiting inflammatory mediators and enhancing anti-inflammatory factor production in activated microglia.

Mycoepoxydiene inhibits antigen-stimulated activation of mast cells and suppresses IgE-mediated anaphylaxis in mice

Mycoepoxydiene (MED) is a polyketide isolated from a marine fungus associated with mangrove forest. It has been shown that MED has many kinds of effects such as anti-cancer and anti-inflammatory activities. However, its effects on anaphylaxis are still unknown. Mast cells play a pivotal role in IgE-mediated allergic response. Aggregation of the high affinity IgE receptor (FcεRI) on the surface of mast cell activates a cascade of signaling events leading to the degranulation and cytokine production in mast cells. Our study showed that MED could significantly suppress antigen-stimulated degranulation and cytokine production in mast cells and IgE-mediated passive cutaneous anaphylaxis (PCA) in mice. Furthermore, we found that MED suppressed antigen-induced activation of Syk, and subsequently inhibited the phosphorylation of PLCγ1, Akt, and MAPKs such as extracellular signal-regulated kinase (ERK), c-jun N-terminal kinase (JNK), and p38 in mast cells. Collectively, our study demonstrates that MED can inhibit the activation of mast cells and protect mice from mast cell-mediated allergic response through inhibiting the activation of Syk. These results suggest that MED is a potential compound for developing a promising anti-anaphylaxis drug.

Mycoepoxydiene Inhibits Lipopolysaccharide-Induced Inflammatory Responses through the of TRAF6 Polyubiquitination

Mycoepoxydiene (MED) is a polyketide isolated from a marine fungus associated with mangrove forests. MED has been shown to be able to induce cell cycle arrest and cancer cell apoptosis. However, its effects on inflammatory response are unclear. Herein we showed that MED exhibited inhibitory effect on inflammatory response induced by lipopolysaccharide (LPS). MED significantly inhibited LPS-induced expression of pro-inflammatory mediators such as tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6, and nitric oxide (NO) in macrophages. MED inhibited LPS-induced nuclear translocation of nuclear factor (NF)-κB (NF-κB) p65, IκB degradation, IκB kinase (IKK) phosphorylation, and the activation of extracellular signal-regulated kinase (ERK), c-jun N-terminal kinase (JNK), and p38, suggesting that MED blocks the activation of both NF-κB and mitogen-activated protein kinase (MAPK) pathways. Furthermore, the effects of MED on LPS-induced activation of upstream signaling molecules such as transforming growth factor-β–activated kinase 1 (TAK1), tumor necrosis factor receptor-associated factor 6 (TRAF6) and IL-1 receptor associated kinases1 (IRAK1) were investigated. MED significantly inhibited TAK1 phosphorylation and TRAF6 polyubiquitination, but not IRAK1 phosphorylation and TRAF6 dimerization, indicating that MED inhibits LPS-induced inflammatory responses at least in part through suppression of TRAF6 polyubiquitination. Moreover, MED protected mice from LPS-induced endotoxin shock by reducing serum inflammatory cytokines. These results suggest that MED is a potential lead compound for the development of a novel nonsteroidal anti-inflammatory drug.

Mycoepoxydiene, a fungal polyketide inhibits MCF-7 cells through simultaneously targeting p53 and NF-κB pathways

Mycoepoxydiene (MED) is a cytotoxic polyketide that is isolated from the marine fungal strain Diaporthe sp. HLY-1, which is associated with mangroves; however, the mechanism of action of MED remains unknown. Here, we report the molecular mechanisms of apoptosis activation and growth inhibition induced by MED in MCF-7 cells. The present results show that MED induces DNA damage through the production of reactive oxygen species (ROS), which resulted in the phosphorylation of H2AX and the activation of the Ataxia telangiectasia mutated kinase (ATM) and p53 signaling pathways. In addition, MED increases the accumulation of IκBα and enhances the association between IKKγ and Hsp27 via the activation of Hsp27, which eventually resulted in the inhibition of TNF-α-induced NF-κB transactivation. Therefore, we conclude that MED inhibits MCF-7 cells by simultaneously activating p53 to induce apoptosis and suppressing NF-κB to disrupt cell proliferation. Because small molecules having both of these effects are rare, further exploration of MED as an antitumor lead compound is needed.

Mycoepoxydiene suppresses RANKL-induced osteoclast differentiation and reduces ovariectomy-induced bone loss in mice

Mycoepoxydiene (MED) is a compound isolated from the marine fungal Diaporthe sp. HLY-1 associated with mangroves. MED has various biological effects such as anti-microbial, anti-cancer, and anti-inflammatory activities. However, the effect of MED on the differentiation of osteoclasts, the multinucleated bone-resorbing cells which play a crucial role in bone remodeling, is still unknown. In this study, we showed that MED could inhibit receptor activator of NF-κB ligand (RANKL)-induced osteoclast differentiation and the expression of three well-known osteoclast markers such as tartrate-resistant acid phosphatase, calcitonin receptor, and cathepsin K in bone marrow-derived macrophages. Furthermore, we found that MED inhibited the expression of nuclear factor of activated T cells c1, a key transcriptional factor in osteoclast differentiation, via inhibiting the phosphorylation of TAK1 and then blocking the activation of NF-κB and ERK1/2 pathways. Moreover, MED could prevent bone loss in ovariectomized mice. Taken together, we demonstrate for the first time that MED can suppress RANKL-induced osteoclast differentiation in vitro and ovariectomy-induced osteoporosis in vivo, suggesting that MED is a potential lead compound for the development of novel drugs for osteoporosis treatment.

Hsp90 interacts with AMPK and mediates acetyl-CoA carboxylase phosphorylation

Heat shock protein 90 (Hsp90) serves to stabilise and correctly fold multiple significant client proteins associated with cell proliferation and cell survival. However, little is known about the Hsp90 client proteins that regulate cell metabolism. Here, we describe a unique ability of Hsp90 to regulate the stability and activity of AMP-activated kinase (AMPK), a key sensor of cellular energy status. Hsp90 is found to interact with AMPK and to maintain its AMP-activated kinase activity, which in turn is required for the phosphorylation of its substrate, acetyl-CoA carboxylase (ACC), the key enzyme in fatty acid metabolism. Our binding analysis reveals that both the γ subunit and the α subunit of AMPK bind to Hsp90 with a high affinity. We demonstrate that Hsp90 inhibitors, including geldanamycin (GA) and mycoepoxydiene (MED), can induce the dissociation of AMPK from Hsp90, and cause a significant decrease in phosphorylation of AMPK and ACC. Furthermore, we demonstrate that shRNAs of Hsp90 can efficiently suppress the activation of AMPK. These findings not only establish a novel interaction between Hsp90 and AMPK but also suggest a new mechanism for regulating tumour cell fatty acid metabolism.

Transformation of Fusarium verticillioides with a polyketide gene cluster isolated from a fungal endophyte activates the biosynthesis of fusaric acid

A large number of bioactive natural products have been isolated from plant endophytic fungi. However, molecular mechanisms for the biosynthesis of these metabolites have lagged behind because genetic and biochemical studies are difficult to perform within many of the endophytes. In this work, we describe our attempt to express a putative mycoepoxydiene (MED) biosynthetic gene cluster in Fusarium verticillioides, which has a well-developed genetic system for the study fungal polyketide biosynthesis. MED was isolated from Phomopsis sp. A123, a fungal endophyte of the mangrove plant, Kandelia candel. It has several unusual structural features and interesting biological activities. Integration of this Phomopsis gene cluster into the F. verticillioides genome led to the biosynthesis of multiple metabolites. The most highly activated metabolite was isolated and its structure was shown by 1D- and 2D-NMR to be fusaric acid, which is a mycotoxin in Fusarium species and is implicated in fungal pathogenesis. Although fusaric acid was isolated more than 70 years ago, its biosynthetic mechanism remains unclear. These transformants produced 30–35 mg fusaric acid per 100 ml culture. The high level production of fusaric acid will greatly facilitate the genetic and biochemical study of its biosynthetic mechanism. Although we have not detected MED or its analogs from the heterologous host, this work represents the first attempt to express a fungal endophytic gene cluster in a Fusarium species.

Mycoepoxydiene, a fungal polyketide, induces cell cycle arrest at the G2/M phase and apoptosis in HeLa cells

Mycoepoxydiene (MED) is a polyketide isolated from a marine fungus associated with mangrove forests. It contains an oxygen-bridged cyclooctadiene core and an α,β-unsaturated δ-lactone moiety. MED induced the reorganization of cytoskeleton in actively growing HeLa cells by promoting formation of actin stress fiber and inhibiting polymerization of tubulin. MED could induce cell cycle arrest at G2/M in HeLa cells. MED-associated apoptosis was characterized by the formation of fragmented nuclei, PARP cleavage, cytochrome c release, activation of caspase-3, and an increased proportion of sub-G1 cells. Additionally, MED activated MAPK pathways. Interestingly, the time of JNK, p38, and Bcl-2 activation did not correlate with the release of cytochrome c. This study is the first report demonstrating the action mechanism of MED against tumor cell growth. These results provide the potential of MED as a novel low toxic antitumor agent.