Epidithiodiketopiperazine Research Articles

Clonostachys rosea ‘omics profiling: identification of putative metabolite-gene associations mediating its in vitro antagonism against Fusarium graminearum

BackgroundClonostachys rosea is an established biocontrol agent. Selected strains have either mycoparasitic activity against known pathogens (e.g. Fusarium species) and/or plant growth promoting activity on various crops. Here we report outcomes from a comparative ‘omics analysis leveraging a temporal variation in the in vitro antagonistic activities of C. rosea strains ACM941 and 88–710, toward understanding the molecular mechanisms underpinning mycoparasitism.ResultsTranscriptomic data highlighted specialized metabolism and membrane transport related genes as being significantly upregulated in ACM941 compared to 88–710 at a time point when the ACM941 strain had higher in vitro antagonistic activity than 88–710. In addition, high molecular weight specialized metabolites were differentially secreted by ACM941, with accumulation patterns of some metabolites matching the growth inhibition differences displayed by the exometabolites of the two strains. In an attempt to identify statistically relevant relationships between upregulated genes and differentially secreted metabolites, transcript and metabolomic abundance data were associated using IntLIM (Integration through Linear Modeling). Of several testable candidate associations, a putative C. rosea epidithiodiketopiperazine (ETP) gene cluster was identified as a prime candidate based on both co-regulation analysis and transcriptomic-metabolomic data association.ConclusionsAlthough remaining to be validated functionally, these results suggest that a data integration approach may be useful for identification of potential biomarkers underlying functional divergence in C. rosea strains.

Dithiodiketopiperazine derivatives from endophytic fungi Trichoderma harzianum and Epicoccum nigrum

A new epidithiodiketopiperazine (ETP), pretrichodermamide G (1), along with three known (epi)dithiodiketopiparazines (2-4) were isolated from cultures of Trichoderma harzianum and Epicoccum nigrum, endophytic fungi associated with medicinal plants Zingiber officinale and Salix sp., respectively. The structure of the new compound (1) was established on the basis of spectroscopic data, including 1D/2D NMR and HRESIMS. The isolated compounds were investigated for their antifungal, antibacterial and cytotoxic potential against a panel of microorganisms and cell lines. Pretrichodermamide A (2) displayed antimicrobial activity towards the plant pathogenic fungus Ustilago maydis and the human pathogenic bacterium Mycobacterium tuberculosis with MIC values of 1 mg/mL (2 mM) and 25 µg/mL (50 µM), respectively. Meanwhile, epicorazine A (3) exhibited strong to moderate cytotoxicity against L5178Y, Ramos, and Jurkat J16 cell lines with IC50 values ranging from 1.3 to 28 µM. Further mechanistic studies indicated that 3 induces apoptotic cell death.

Epidithiodiketopiperazines Inhibit Protein Degradation by Targeting Proteasome Deubiquitinase Rpn11

The 26S proteasome is the major proteolytic machine for breaking down cytosolic and nuclear proteins in eukaryotes. Due to the lack of a suitable assay, it is difficult to measure routinely and quantitatively the breakdown of proteins by the 26S proteasome invitro. In the present study, we developed an assay to monitor proteasome-mediated protein degradation. Using this assay, we discovered that epidithiodiketopiperazine (ETPs) blocked the degradation of our model substrate invitro. Further characterization revealed that ETPs inhibited proteasome function by targeting the essential proteasomaldeubiquitinase Rpn11 (POH1/PSMD14). ETPs also inhibited other JAMM (JAB1/MPN/Mov34 metalloenzyme) proteases such as Csn5 and AMSH. An improved ETP with fewer non-specific effects, SOP11, stabilized a subset of proteasome substrates in cells, induced the unfolded protein response, and led to cell death. SOP11 represents a class of Rpn11 inhibitor and provides an alternative route to develop proteasome inhibitors.

Biosynthesis of the Halogenated Mycotoxin Aspirochlorine in Koji Mold Involves a Cryptic Amino Acid Conversion

AbstractAspirochlorine (1) is an epidithiodiketopiperazine (ETP) toxin produced from koji mold (Aspergillus oryzae), which has been used in the oriental cuisine for over two millennia. Considering its potential risk for food safety, we have elucidated the molecular basis of aspirochlorine biosynthesis. By a combination of genetic and chemical analyses we found the acl gene locus and identified the key role of AclH as a chlorinase. Stable isotope labeling, biotransformation, and mutational experiments, analysis of intermediates and an in vitro adenylation domain assay gave totally unexpected insights into the acl pathway: Instead of one Phe and one Gly, two Phe units are assembled by an iterative non‐ribosomal peptide synthetase (NRPS, AclP), followed by halogenation and an unprecedented Phe to Gly amino acid conversion. Biological assays showed that both amino acid transformations are required to confer cytotoxicity and antifungal activity to the mycotoxin.

Biosynthesis of the Halogenated Mycotoxin Aspirochlorine in Koji Mold Involves a Cryptic Amino Acid Conversion

Aspirochlorine (1) is an epidithiodiketopiperazine (ETP) toxin produced from koji mold (Aspergillus oryzae), which has been used in the oriental cuisine for over two millennia. Considering its potential risk for food safety, we have elucidated the molecular basis of aspirochlorine biosynthesis. By a combination of genetic and chemical analyses we found the acl gene locus and identified the key role of AclH as a chlorinase. Stable isotope labeling, biotransformation, and mutational experiments, analysis of intermediates and an in vitro adenylation domain assay gave totally unexpected insights into the acl pathway: Instead of one Phe and one Gly, two Phe units are assembled by an iterative non-ribosomal peptide synthetase (NRPS, AclP), followed by halogenation and an unprecedented Phe to Gly amino acid conversion. Biological assays showed that both amino acid transformations are required to confer cytotoxicity and antifungal activity to the mycotoxin.

On the Histone Lysine Methyltransferase Activity of Fungal Metabolite Chaetocin

Histone lysine methyltransferases (HKMTs) are an important class of targets for epigenetic therapy. 1 (chaetocin), an epidithiodiketopiperazine (ETP) natural product, has been reported to be a specific inhibitor of the SU(VAR)3-9 class of HKMTs. We have studied the inhibition of the HKMT G9a by 1 and functionally related analogues. Our results reveal that only the structurally unique ETP core is required for inhibition, and such inhibition is time-dependent and irreversible (in the absence of DTT), ultimately resulting in protein denaturation. Mass spectrometric data provide a molecular basis for this effect, demonstrating covalent adduct formation between 1 and the protein. This provides a potential rationale for the selectivity observed in the inhibition of a variety of HKMTs by 1 in vitro and has implications for the activity of ETPs against these important epigenetic targets.

Cloning and Sequencing of the Chaetocin Biosynthetic Gene Cluster

How a fungus does it: The epidithiodiketopiperazine (ETP) fungal alkaloids, which include gliotoxin, have garnered much attention since their initial isolation. The cloning and sequencing of the biosynthetic gene cluster for another ETP, chaetocin A were used to suggest a route towards dimerization and sulfur incorporation in these molecules.

Epidithiodiketopiperazine as a pharmacophore for protein lysine methyltransferase G9a inhibitors: Reducing cytotoxicity by structural simplification

Chaetocin (1), a structurally complex epidithiodiketopiperazine (ETP) alkaloid produced by Chaetomium minutum, is a potent inhibitor of protein lysine methyltransferase G9a, which plays important roles in many biological processes. Here we present our synthetic investigations to identify a simple prototype G9a inhibitor structure based on structure–activity relationship (SAR) studies on chaetocin derivatives. The simple derivative PS-ETP-1 (14) was found to be a potent G9a inhibitor with greatly reduced cytotoxicity.

Abstract 2331: Select epidithiodiketopiperazine (ETP) compounds disrupt the HIF-1α/p300 interaction and HIF-mediated transcription

Abstract As solid tumors grow, regions in the tumor become hypoxic because of increased metabolism, heterogeneous and abnormal vasculature delivering oxygen within the tumor, and greater size. In order for the tumor to continue to grow, it depends on survival mechanisms to overcome this hypoxic state, including up-regulation in glycolysis, invasion and metastasis, and angiogenesis, or the growth of new blood vessels. The hypoxia-inducible factor (HIF) transcription factor complex is a major mediator of the cell's response to a hypoxic state and a tumor's survival mechanism under hypoxia. The complex is composed of the hypoxia-dependent HIF-1α subunit and a constitutively expressed HIF-1α subunit, and it requires the co-activator protein p300 to function. In the presence of oxygen, HIF-1α is rapidly degraded. Under hypoxia, HIF-1α forms a complex with HIF-1α and p300 in the nucleus to up-regulate transcription of pro-survival and angiogenic genes. Targeting this transcription factor complex in tumors could circumvent the tumor's hypoxia survival mechanism and lead to growth arrest and cell death. The compounds chetomin, chaetocin, and gliotoxin, members of the epidithiodiketopiperazine (ETP) family of fungal metabolites, have been shown to disrupt the HIF-1α/p300 interaction in an over-expression system. In the following experiments, we sought to validate the disruption of the HIF-1α /p300 transcription factor complex in cells, and to determine the associated downstream cellular effects. By co-immunoprecipitation of the complex from prostate cancer cells, we were able to show that treatment with those select ETPs under hypoxic conditions disrupted the HIF-1α/p300 complex. We performed an enzyme-linked immunosorbent assay (ELISA) to determine the effect of ETPs on levels of secreted vascular endothelial growth factor (VEGF), a major target of HIF transcription. We observed a decrease in VEGF concentration that correlated with ETP treatment under hypoxia. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to examine the transcription levels of other HIF target genes to validate that disruption of the HIF-1α/p300 complex by select ETP treatments indeed affects HIF-mediated transcription. In order to determine the antiangiogenic effects of these agents in vivo, ongoing animal studies in our lab are testing the efficacy of these ETPs in reducing tumor growth and determining feasibility of pre-clinical development. Targeting the HIF complex could circumvent a tumor's survival mechanism under hypoxia, and would be a new treatment strategy in solid tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2331. doi:1538-7445.AM2012-2331

Disruption of the HIF‐1α/p300 interaction as a means of inhibiting angiogenesis

Solid tumors develop regions of hypoxia because of an imbalance in oxygen supply and consumption. Thus, adaptation of cancer cells to hypoxia is critical for tumor survival. The most important mediator of the cell's response to reduced oxygen is the hypoxia inducible factor‐1 (HIF‐1) transcription factor. Due to the involvement of HIF in tumor progression and angiogenesis, inhibition of HIF‐mediated transcription has the potential for treatment of cancer. Since p300 is a crucial coactivator of hypoxia‐inducible transcription, disruption of the HIF‐1α/p300 complex is desirable as a selective mechanism of inhibiting HIF activity. Previous data have shown that several members of the epidithiodiketopiperazine (ETP) family of natural products, namely, chetomin, chaetocin, and gliotoxin, block the interaction between HIF‐1α and p300. We performed a series of rat aortic ring assays and found that ETP treatment had antiangiogenic effects, apparently through disruption of the HIF‐1α/p300 complex, as ETPs blocked co‐immunoprecipitation of HIF‐1α and p300. Aortic rings were immunostained for HIF‐1α to confirm its upregulation. These results show the potential of targeting this interaction in cancer therapeutics. We are currently using mouse xenografts to test the effects of these ETPs on tumor growth, and thus determine the feasibility of moving these compounds forward into pre‐clinical development.

Enantioselective Total Synthesis of (−)-Acetylaranotin, a Dihydrooxepine Epidithiodiketopiperazine

The first total synthesis of the dihydrooxepine-containing epidithiodiketopiperazine (ETP) (-)-acetylaranotin (1) is reported. The key steps of the synthesis include an enantioselective azomethine ylide (1,3)-dipolar cycloaddition reaction to set the absolute and relative stereochemistry, a rhodium-catalyzed cycloisomerization/chloride elimination sequence to generate the dihydrooxepine moiety, and a stereoretentive diketopiperazine sulfenylation to install the epidisulfide. This synthesis provides access to (-)-1 in 18 steps from inexpensive, commercially available starting materials. We anticipate that the approach described herein will serve as a general strategy for the synthesis of additional members of the dihydrooxepine ETP family.

A Dedicated Glutathione S-Transferase Mediates Carbon–Sulfur Bond Formation in Gliotoxin Biosynthesis

Gliotoxin is a virulence factor of the human pathogen Aspergillus fumigatus , the leading cause of invasive aspergillosis. Its toxicity is mediated by the unusual transannular disulfide bridge of the epidithiodiketopiperazine (ETP) scaffold. Here we disclose the critical role of a specialized glutathione S-transferase (GST), GliG, in enzymatic sulfurization. Furthermore, we show that bishydroxylation of the diketopiperazine by the oxygenase GliC is a prerequisite for glutathione adduct formation. This is the first report of the involvement of a GST in enzymatic C-S bond formation in microbial secondary metabolism.

Abstract 3095: The molecular, cellular, and physiological consequences of disrupting the HIF-1α interaction

Abstract Many cancers contain regions of hypoxia due to rapid cell proliferation and the presence of blood vessels within the tumor that are structurally and functionally abnormal, resulting in the heterogeneity of blood flow. This state of hypoxia is associated with increased risk of treatment failure, metastasis and patient mortality. A principal mechanism by which cancer cells adapt to the hypoxic microenvironment is through the activity of hypoxia-inducible factor 1 (HIF-1), a heterodimeric transcription factor composed of an O2-regulated HIF-1α subunit that dimerizes with a constitutively expressed HIF-1β subunit. HIF-1α expression under hypoxic conditions regulates hundreds of genes including those that play key roles in invasion/metastasis, angiogenesis, tumor cell immortalization, and resistance to chemotherapy and radiation. Therefore, the inhibition of transcription driven by HIF has the potential for cancer treatment. One way to reduce the activity of this protein is by disrupting the complex that HIF forms with p300, an essential transcriptional coactivator. Previous data from our laboratory showed that several members of the epidithiodiketopiperazine (ETP) family of natural products are able to block the interaction between HIF-1α and p300. Here, we extend these studies by examining the comprehensive effect that select ETPs have on the disruption of the HIF/p300 complex at the molecular, cellular, and physiological levels. We began our studies by performing a series of rat aortic ring assays to determine the antiangiogenic effect of selected ETPs, namely chetomin, chaetocin, and gliotoxin. Chetomin and chaetocin concentrations of 50 nM inhibited approximately 90% of outgrowth, while 500 nM of gliotoxin was needed to achieve a similar effect; these compounds had GI50 of 22, 11, and 175 nM, respectively. In ongoing work, we hope to determine if the antiangiogenic effects that were observed in our rat aortic ring assays, are due to disruption of the HIF/p300 complex. To this end we are immunoprecipitating the complex from cells in the absence or presence of the aforementioned ETPs. The molecular consequences of blocking the HIF/p300 interaction will be assayed by qRT-PCR for hypoxia-regulated genes such as VEGF and GLUT1; cell proliferation and migration assays will also be performed to monitor cellular effects. Together, these results will be key in determining the feasibility of targeting this interaction for cancer therapeutics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3095. doi:10.1158/1538-7445.AM2011-3095

Epidithiodiketopiperazines Block the Interaction between Hypoxia-inducible Factor-1α (HIF-1α) and p300 by a Zinc Ejection Mechanism

The hypoxic response in humans is regulated by the hypoxia-inducible transcription factor system; inhibition of hypoxia-inducible factor (HIF) activity has potential for the treatment of cancer. Chetomin, a member of the epidithiodiketopiperazine (ETP) family of natural products, inhibits the interaction between HIF-alpha and the transcriptional coactivator p300. Structure-activity studies employing both natural and synthetic ETP derivatives reveal that only the structurally unique ETP core is required and sufficient to block the interaction of HIF-1alpha and p300. In support of both cell-based and animal work showing that the cytotoxic effect of ETPs is reduced by the addition of Zn(2+) through an unknown mechanism, our mechanistic studies reveal that ETPs react with p300, causing zinc ion ejection. Cell studies with both natural and synthetic ETPs demonstrated a decrease in vascular endothelial growth factor and antiproliferative effects that were abrogated by zinc supplementation. The results have implications for the design of selective ETPs and for the interaction of ETPs with other zinc ion-binding protein targets involved in gene expression.

Direct Inhibition of Hypoxia-Inducible Transcription Factor Complex with Designed Dimeric Epidithiodiketopiperazine

Selective blockade of hypoxia-inducible gene expression by designed small molecules would prove valuable in suppressing tumor angiogenesis, metastasis and altered energy metabolism. We report the design, synthesis, and biological evaluation of a dimeric epidithiodiketopiperazine (ETP) small molecule transcriptional antagonist targeting the interaction of the p300/CBP coactivator with the transcription factor HIF-1alpha. Our results indicate that disrupting this interaction results in rapid downregulation of hypoxia-inducible genes critical for cancer progression. The observed effects are compound-specific and dose-dependent. Controlling gene expression with designed small molecules targeting the transcription factor-coactivator interface may represent a new approach for arresting tumor growth.

A concise approach to the epidithiodiketopiperazine (ETP) core

A novel approach to the epidithiodiketopiperazine nucleus, incorporating two three-component reactions in a four-step synthetic sequence is reported.