Trachymyrmex septentrionalis ants promote fungus garden hygiene using Trichoderma-derived metabolite cues.

Fungus-growing ants depend on a fungal mutualist that can fall prey to fungal pathogens. This mutualist is cultivated by these ants in structures called fungus gardens. Ants exhibit weeding behaviors that keep their fungus gardens healthy by physically removing compromised pieces. However, how ants detect diseases of their fungus gardens is unknown. Here, we applied the logic of Koch's postulates using environmental fungal community gene sequencing, fungal isolation, and laboratory infection experiments to establish that Trichoderma spp. can act as previously unrecognized pathogens of Trachymyrmex septentrionalis fungus gardens. Our environmental data showed that Trichoderma are the most abundant noncultivar fungi in wild T. septentrionalis fungus gardens. We further determined that metabolites produced by Trichoderma induce an ant weeding response that mirrors their response to live Trichoderma. Combining ant behavioral experiments with bioactivity-guided fractionation and statistical prioritization of metabolites in Trichoderma extracts demonstrated that T. septentrionalis ants weed in response to peptaibols, a specific class of secondary metabolites known to be produced by Trichoderma fungi. Similar assays conducted using purified peptaibols, including the two previously undescribed peptaibols trichokindins VIII and IX, suggested that weeding is likely induced by peptaibols as a class rather than by a single peptaibol metabolite. In addition to their presence in laboratory experiments, we detected peptaibols in wild fungus gardens. Our combination of environmental data and laboratory infection experiments strongly support that peptaibols act as chemical cues of Trichoderma pathogenesis in T. septentrionalis fungus gardens.

Correlative metabologenomics of 110 fungi reveals metabolite-gene cluster pairs.

Natural products research increasingly applies -omics technologies to guide molecular discovery. While the combined analysis of genomic and metabolomic datasets has proved valuable for identifying natural products and their biosynthetic gene clusters (BGCs) in bacteria, this integrated approach lacks application to fungi. Because fungi are hyper-diverse and underexplored for new chemistry and bioactivities, we created a linked genomics-metabolomics dataset for 110 Ascomycetes, and optimized both gene cluster family (GCF) networking parameters and correlation-based scoring for pairing fungal natural products with their BGCs. Using a network of 3,007 GCFs (organized from 7,020 BGCs), we examined 25 known natural products originating from 16 known BGCs and observed statistically significant associations between 21 of these compounds and their validated BGCs. Furthermore, the scalable platform identified the BGC for the pestalamides, demystifying its biogenesis, and revealed more than 200 high-scoring natural product-GCF linkages to direct future discovery.

Exploring the in vivo anti-cancer potential of Neosetophomone B in leukemic cells using a zebrafish xenograft model.

Neosetophomone B (NSP-B) is a unique meroterpenoid fungal secondary metabolite that has previously demonstrated promising anti-cancer properties against various cancer cell lines in vitro. However, its in vivo anti-cancer potential remaines unexplored. To fill this gap in our knowledge, we tested NSP-B's in vivo anti-cancer activity using a zebrafish model, an organism that has gained significant traction in biomedical research due to its genetic similarities with humans and its transparent nature, allowing real-time tumor growth observation. For our experiments, we employed the K562-injected zebrafish xenograft model. Upon treating these zebrafish with NSP-B, we observed a marked reduction in the size and number of tumor xenografts. Delving deeper, our analyses indicated that NSP-B curtailed tumor growth and proliferation of leukemic grafted xenograft within the zebrafish. These results show that NSP-B possesses potent in vivo anti-cancer properties, making it a potential novel therapeutic agent for addressing hematological malignancies.

Regulation of gliotoxin biosynthesis and protection in Aspergillus species.

Aspergillus fumigatus causes a range of human and animal diseases collectively known as aspergillosis. A. fumigatus possesses and expresses a range of genetic determinants of virulence, which facilitate colonisation and disease progression, including the secretion of mycotoxins. Gliotoxin (GT) is the best studied A. fumigatus mycotoxin with a wide range of known toxic effects that impair human immune cell function. GT is also highly toxic to A. fumigatus and this fungus has evolved self-protection mechanisms that include (i) the GT efflux pump GliA, (ii) the GT neutralising enzyme GliT, and (iii) the negative regulation of GT biosynthesis by the bis-thiomethyltransferase GtmA. The transcription factor (TF) RglT is the main regulator of GliT and this GT protection mechanism also occurs in the non-GT producing fungus A. nidulans. However, the A. nidulans genome does not encode GtmA and GliA. This work aimed at analysing the transcriptional response to exogenous GT in A. fumigatus and A. nidulans, two distantly related Aspergillus species, and to identify additional components required for GT protection. RNA-sequencing shows a highly different transcriptional response to exogenous GT with the RglT-dependent regulon also significantly differing between A. fumigatus and A. nidulans. However, we were able to observe homologs whose expression pattern was similar in both species (43 RglT-independent and 11 RglT-dependent). Based on this approach, we identified a novel RglT-dependent methyltranferase, MtrA, involved in GT protection. Taking into consideration the occurrence of RglT-independent modulated genes, we screened an A. fumigatus deletion library of 484 transcription factors (TFs) for sensitivity to GT and identified 15 TFs important for GT self-protection. Of these, the TF KojR, which is essential for kojic acid biosynthesis in Aspergillus oryzae, was also essential for virulence and GT biosynthesis in A. fumigatus, and for GT protection in A. fumigatus, A. nidulans, and A. oryzae. KojR regulates rglT, gliT, gliJ expression and sulfur metabolism in Aspergillus species. Together, this study identified conserved components required for GT protection in Aspergillus species.

Verticillins: fungal epipolythiodioxopiperazine alkaloids with chemotherapeutic potential.

Covering: 1970 through June of 2023Verticillins are epipolythiodioxopiperazine (ETP) alkaloids, many of which possess potent, nanomolar-level cytotoxicity against a variety of cancer cell lines. Over the last decade, their in vivo activity and mode of action have been explored in detail. Notably, recent studies have indicated that these compounds may be selective inhibitors of histone methyltransferases (HMTases) that alter the epigenome and modify targets that play a crucial role in apoptosis, altering immune cell recognition, and generating reactive oxygen species. Verticillin A (1) was the first of 27 analogues reported from fungal cultures since 1970. Subsequent genome sequencing identified the biosynthetic gene cluster responsible for producing verticillins, allowing a putative pathway to be proposed. Further, molecular sequencing played a pivotal role in clarifying the taxonomic characterization of verticillin-producing fungi, suggesting that most producing strains belong to the genus Clonostachys (i.e., Bionectria), Bionectriaceae. Recent studies have explored the total synthesis of these molecules and the generation of analogues via both semisynthetic and precursor-directed biosynthetic approaches. In addition, nanoparticles have been used to deliver these molecules, which, like many natural products, possess challenging solubility profiles. This review summarizes over 50 years of chemical and biological research on this class of fungal metabolites and offers insights and suggestions on future opportunities to push these compounds into pre-clinical and clinical development.

Pharmacokinetic Effects of Different Models of Nonalcoholic Fatty Liver Disease in Transgenic Humanized OATP1B Mice.

Organic anion transporting polypeptide (OATP) 1B1 and OATP1B3 (collectively, OATP1B) transporters encoded by the solute carrier organic anion transporter (SLCO) genes mediate uptake of multiple pharmaceutical compounds. Nonalcoholic steatohepatitis (NASH), a severe form of nonalcoholic fatty liver disease (NAFLD), decreases OATP1B abundance. This research characterized the pathologic and pharmacokinetics effects of three diet- and one chemical-induced NAFLD model in male and female humanized OATP1B mice, which comprises knock-out of rodent Oatp orthologs and insertion of human SLCO1B1 and SLCO1B3. Histopathology scoring demonstrated elevated steatosis and inflammation scores for all NAFLD-treatment groups. Female mice had minor changes in SLCO1B1 expression in two of the four NAFLD treatment groups, and pitavastatin (PIT) area under the concentration-time curve (AUC) increased in female mice in only one of the diet-induced models. OATP1B3 expression decreased in male and female mice in the chemical-induced NAFLD model, with a coinciding increase in PIT AUC, indicating the chemical-induced model may better replicate changes in OATP1B3 expression and OATP substrate disposition observed in NASH patients. This research also tested a reported multifactorial pharmacokinetic interaction between NAFLD and silymarin, an extract from milk thistle seeds with notable OATP-inhibitory effects. Males showed no change in PIT AUC, whereas female PIT AUC increased 1.55-fold from the diet alone and the 1.88-fold from the combination of diet with silymarin, suggesting that female mice are more sensitive to pharmacokinetic changes than male mice. Overall, the humanized OATP1B model should be used with caution for modeling NAFLD and multifactorial pharmacokinetic interactions. SIGNIFICANCE STATEMENT: Advanced stages of NAFLD cause decreased hepatic OATP1B abundance and increase systemic exposure to OATP substrates in human patients. The humanized OATP1B mouse strain may provide a clinically relevant model to recapitulate these observations and predict pharmacokinetic interactions in NAFLD. This research characterized three diet-induced and one drug-induced NAFLD model in a humanized OATP1B mouse model. Additionally, a multifactorial pharmacokinetic interaction was observed between silymarin and NAFLD.

Rifampin- and Silymarin-Mediated Pharmacokinetic Interactions of Exogenous and Endogenous Substrates in a Transgenic OATP1B Mouse Model.

Organic anion-transporting polypeptides (OATP) 1B1 and OATP1B3, encoded by the SLCO gene family of the solute carrier superfamily, are involved in the disposition of many exogenous and endogenous compounds. Preclinical rodent models help assess risks of pharmacokinetic interactions, but interspecies differences in transporter orthologs and expression limit direct clinical translation. An OATP1B transgenic mouse model comprising a rodent Slco1a/1b gene cluster knockout and human SLCO1B1 and SLCO1B3 gene insertions provides a potential physiologically relevant preclinical tool to predict pharmacokinetic interactions. Pharmacokinetics of exogenous probe substrates, pitavastatin and pravastatin, and endogenous OATP1B biomarkers, coproporphyrin-I and coproporphyrin-III, were determined in the presence and absence of known OATP/Oatp inhibitors, rifampin or silymarin (an extract of milk thistle [Silybum marianum]), in wild-type FVB mice and humanized OATP1B mice. Rifampin increased exposure of pitavastatin (4.6- and 2.8-fold), pravastatin (3.6- and 2.2-fold), and coproporphyrin-III (1.6- and 2.1-fold) in FVB and OATP1B mice, respectively, but increased coproporphyrin-I AUC0-24h only (1.8-fold) in the OATP1B mice. Silymarin did not significantly affect substrate AUC, likely because the silymarin flavonolignan concentrations were at or below their reported IC50 values for the relevant OATPs/Oatps. Silymarin increased the Cmax of pitavastatin 2.7-fold and pravastatin 1.9-fold in the OATP1B mice. The data of the OATP1B mice were similar to those of the pitavastatin and pravastatin clinical data; however, the FVB mice data more closely recapitulated pitavastatin clinical data than the data of the OATP1B mice, suggesting that the OATP1B mice are a reasonable, though costly, preclinical strain for predicting pharmacokinetic interactions when doses are optimized to achieve clinically relevant plasma concentrations.

Green Tea Catechins Decrease Solubility of Raloxifene In Vitro and Its Systemic Exposure in Mice.

PURPOSE: Green tea is a widely consumed beverage. A recent clinical study reported green tea decreased systemic exposure of raloxifene and its glucuronide metabolites by 34-43%. However, the underlying mechanism(s) remains unknown. This study investigated a change in raloxifene's solubility as the responsible mechanism. METHODS: The effects of green tea extract, (-)-epigallocatechin gallate (EGCG), and (-)-epigallocatechin (EGC) on raloxifene's solubility were assessed in fasted state simulated intestinal fluids (FaSSIF) and fed state simulated intestinal fluids (FeSSIF). EGCG and EGC represent green tea's main bioactive constituents, flavan-3-gallate and flavan-3-ol catechins respectively, and the tested concentrations (mM) match the µg/mg of each compound in the extract. Our mouse study (n = 5/time point) evaluated the effect of green tea extract and EGCG on the systemic exposure of raloxifene. RESULTS: EGCG (1 mM) and EGC (1.27 mM) decreased raloxifene's solubility in FaSSIF by 78% and 13%, respectively. Micelle size in FaSSIF increased with increasing EGCG concentrations (> 1000% at 1 mM), whereas EGC (1.27 mM) did not change micelle size. We observed 3.4-fold higher raloxifene solubility in FeSSIF compared to FaSSIF, and neither green tea extract nor EGCG significantly affected raloxifene solubility or micelle size in FeSSIF. The mice study showed that green tea extract significantly decreased raloxifene Cmax by 44%, whereas EGCG had no effect. Green tea extract and EGCG did not affect the AUC0-24 h of raloxifene or the metabolite-to-parent AUC ratio. CONCLUSIONS: This study demonstrated flavan-3-gallate catechins may decrease solubility of poorly water-soluble drugs such as raloxifene, particularly in the fasted state.

Neosetophomone B induces apoptosis in multiple myeloma cells via targeting of AKT/SKP2 signaling pathway.

Multiple myeloma (MM) is a hematologic malignancy associated with malignant plasma cell proliferation in the bone marrow. Despite the available treatments, drug resistance and adverse side effects pose significant challenges, underscoring the need for alternative therapeutic strategies. Natural products, like the fungal metabolite neosetophomone B (NSP-B), have emerged as potential therapeutic agents due to their bioactive properties. Our study investigated NSP-B's antitumor effects on MM cell lines (U266 and RPMI8226) and the involved molecular mechanisms. NSP-B demonstrated significant growth inhibition and apoptotic induction, triggered by reduced AKT activation and downregulation of the inhibitors of apoptotic proteins and S-phase kinase protein. This was accompanied by an upregulation of p21Kip1 and p27Cip1 and an elevated Bax/BCL2 ratio, culminating in caspase-dependent apoptosis. Interestingly, NSP-B also enhanced the cytotoxicity of bortezomib (BTZ), an existing MM treatment. Overall, our findings demonstrated that NSP-B induces caspase-dependent apoptosis, increases cell damage, and suppresses MM cell proliferation while improving the cytotoxic impact of BTZ. These findings suggest that NSP-B can be used alone or in combination with other medicines to treat MM, highlighting its importance as a promising phytoconstituent in cancer therapy.

Semisynthetic derivatives of the fungal metabolite eupenifeldin via targeting the tropolone hydroxy groups.

Eupenifeldin (1) is a fungal secondary metabolite possessing bis-tropolone moieties that demonstrates nanomolar cytotoxic activity against a number of cancer cell types. As a potential anticancer lead, this meroterpenoid was used to access 29 semisynthetic analogues via functionalization of the reactive hydroxy groups of the bis-tropolones. A series of ester (2-6), carbonate (7-8), sulfonate (9-16), carbamate (17-20), and ether (21-30) analogues of 1 were generated via 22 reactions. Most of these compounds were disubstituted, produced via functionalization of both of the tropolonic hydroxy moieties, although three mono-functionalized analogues (6, 8, and 24) and one tri-functionalized analogue (3) were also obtained. The cytotoxic activities of 1-30 were evaluated against human melanoma and ovarian cancer cell lines (i.e., MDA-MB-435 and OVCAR3, respectively). Ester and carbonate analogues of 1 (i.e., 2-8) maintained cytotoxicity at the nanomolar level, and the greatest improvement in aqueous solubility came from the monosuccinate analogue (6), which was acylated on the secondary hydroxy at the 11 position.

Semisynthesis, Characterization, and Biological Evaluation of Fluorinated Analogues of the Spirobisnaphthalene, Diepoxin-η.

Diepoxin-η (1) is a cytotoxic fungal metabolite belonging to the spirobisnaphthalene structural class. In this study, four mono fluorinated analogues (2-5) of diepoxin-η (1) were semisynthesized in a single-step by selectively fluorinating the naphthalene moiety with Selectfluor. The structures of 2-5 were elucidated using a set of spectroscopic and spectrometric techniques and were further confirmed by means of TDDFT-ECD and isotropic shielding tensors calculations. Compounds 2-5 showed equipotent cytotoxic activity to 1 when tested against OVCAR3 (ovarian) and MDA-MB-435 (melanoma) cancer cell lines with IC50 values that range from 5.7-8.2 µM.

From plant to cancer drug: lessons learned from the discovery of taxol.

Many researchers in the natural product sciences dream of discovering a successful drug. For almost all of us, this dream will never be realized. Among the heroes of our past, though, there is a team whose efforts led to the discovery of not one but two new drugs. Dr Monroe Wall and Dr Mansukh Wani isolated and solved the structures for taxol and camptothecin, plant-based compounds that continue to play a critical role in cancer therapy today. Since the 1960s and 1970s when Wall, Wani and collaborators did their seminal work, there have been tremendous technological advances in the natural product sciences. With access to most sophisticated technology, it might be expected that the rate of discovery of new drugs from plants and other sources would have sped up. However, this has not come to pass. Why is this? Is it that the promise of new drug candidates from plant-based sources has been exhausted? Has our fascination with new technologies and with the promise of the genomics revolution caused us to stop investing effort and resources in the practices that are proven to yield success? With this Viewpoint, we share the story of taxol's discovery, highlighting critical challenges that were overcome and considering their relevance to botanical natural products drug discovery today. We hope that consideration of lessons learned from the past will help fuel success by researchers currently studying plants with the goal of discovering promising therapeutic leads.

Translating Kratom-Drug Interactions: From Bedside to Bench and Back.

Kratom is a botanical natural product belonging to the coffee family, with stimulant effects at low doses and opioid-like effects at higher doses. During the last two decades, kratom has been purported as a safer alternative to pharmaceutical and illicit drugs to self-manage pain and opioid withdrawal symptoms. Kratom alkaloids, typically mitragynine, have been detected in biologic samples from overdose deaths. These deaths are often observed in combination with other drugs and are suspected to result from polyintoxications. This review focuses on the potential for kratom to precipitate pharmacokinetic interactions with object drugs involved in these reported polyintoxications. The legal status, chemistry, pharmacology, and toxicology are also summarized. The aggregate in vitro and clinical data identified kratom and select kratom alkaloids as modulators of cytochrome P450 (P450) enzyme activity, notably as inhibitors of CYP2D6 and CYP3A, as well as P-glycoprotein-mediated efflux activity. These inhibitory effects could increase the systemic exposure to co-consumed object drugs, which may lead to adverse effects. Collectively, the evidence to date warrants further evaluation of potential kratom-drug interactions using an iterative approach involving additional mechanistic in vitro studies, well designed clinical studies, and physiologically based pharmacokinetic modeling and simulation. This critical information is needed to fill knowledge gaps regarding the safe and effective use of kratom, thereby addressing ongoing public health concerns. SIGNIFICANCE STATEMENT: The botanical kratom is increasingly used to self-manage pain and opioid withdrawal symptoms due to having opioid-like effects. The legal status, chemistry, pharmacology, toxicology, and drug interaction potential of kratom are reviewed. Kratom-associated polyintoxications and in vitro-in vivo extrapolations suggest that kratom can precipitate pharmacokinetic drug interactions by inhibiting CYP2D6, CYP3A, and P-glycoprotein. An iterative approach that includes clinical studies and physiologically based pharmacokinetic modeling and simulation is recommended for further evaluation of potential unwanted kratom-drug interactions.

Goldenseal-Mediated Inhibition of Intestinal Uptake Transporters Decreases Metformin Systemic Exposure in Mice.

Goldenseal is a perennial plant native to eastern North America. A recent clinical study reported goldenseal decreased metformin Cmax and area under the blood concentration versus time curve (AUC) by 27% and 23%, respectively, but half-life and renal clearance were unchanged. These observations suggested goldenseal altered processes involved in metformin absorption. The underlying mechanism(s) remain(s) unknown. One mechanism for the decreased metformin systemic exposure is inhibition by goldenseal of intestinal uptake transporters involved in metformin absorption. Goldenseal extract and three goldenseal alkaloids (berberine, (-)-ß-hydrastine, hydrastinine) were tested as inhibitors of organic cation transporter (OCT) 3, plasma membrane monoamine transporter (PMAT), and thiamine transporter (THTR) 2 using human embryonic kidney 293 cells overexpressing each transporter. The goldenseal extract, normalized to berberine content, was the strongest inhibitor of each transporter (IC50: 4.9, 13.1, and 5.8 µM for OCT3, PMAT, and THTR2, respectively). A pharmacokinetic study in mice compared the effects of berberine, (-)-ß-hydrastine, goldenseal extract, and imatinib (OCT inhibitor) on orally administered metformin. Goldenseal extract and imatinib significantly decreased metformin Cmax by 31% and 25%, respectively, and had no effect on half-life. Berberine and (-)-ß-hydrastine had no effect on metformin pharmacokinetics, indicating neither alkaloid alone precipitated the interaction in vivo. A follow-up murine study involving intravenous metformin and oral inhibitors examined the contributions of basolateral enteric/hepatic uptake transporters to the goldenseal-metformin interaction. Goldenseal extract and imatinib had no effect on metformin AUC and half-life, suggesting lack of inhibition of basolateral enteric/hepatic uptake transporters. Results may have implications for patients taking goldenseal with drugs that are substrates for OCT3 and THTR2. SIGNIFICANCE STATEMENT: Goldenseal is used to self-treat respiratory infections and digestive disorders. We investigated potential mechanisms for the clinical pharmacokinetic interaction observed between goldenseal and metformin, specifically inhibition by goldenseal of intestinal uptake transporters (OCT3, PMAT, THTR2) involved in metformin absorption. Goldenseal extract inhibited all three transporters in vitro and decreased metformin systemic exposure in mice. These data may have broader implications for patients co-consuming goldenseal with other drugs that are substrates for these transporters.

Exploration of Verticillins in High-Grade Serous Ovarian Cancer and Evaluation of Multiple Formulations in Preclinical In Vitro and In Vivo Models.

Verticillins are epipolythiodioxopiperazine alkaloids isolated from a fungus with nanomolar anti-tumor activity in high-grade serous ovarian cancer (HGSOC). HGSOC is the fifth leading cause of death in women, and natural products continue to be an inspiration for new drug entities to help tackle chemoresistance. Verticillin D was recently found in a new fungal strain and compared to verticillin A. Both compounds exhibited nanomolar cytotoxic activity against OVCAR4 and OVCAR8 HGSOC cell lines, significantly reduced 2D foci and 3D spheroids, and induced apoptosis. In addition, verticillin A and verticillin D reduced tumor burden in vivo using OVCAR8 xenografts in the peritoneal space as a model. Unfortunately, mice treated with verticillin D displayed signs of liver toxicity. Tolerability studies to optimize verticillin A formulation for in vivo delivery were performed and compared to a semi-synthetic succinate version of verticillin A to monitor bioavailability in athymic nude females. Formulation of verticillins achieved tolerable drug delivery. Thus, formulation studies are effective at improving tolerability and demonstrating efficacy for verticillins.

Embellicines C-E: Macrocyclic Alkaloids with a Cyclopenta[b]fluorene Ring System from the Fungus Sarocladium sp.

Macrocyclic alkaloids with a cyclopenta[b]fluorene ring system are a relatively young structural class of fungal metabolites, with the first members reported in 2013. Bioassay-guided fractionation of a Sarocladium sp. (fungal strain MSX6737) led to a series of both known and new members of this structural class (1-5), including the known embellicine A (1), three new embellicine analogues (2, 4, and 5), and a semisynthetic acetylated analogue (3). The structures were identified by examining both high-resolution electrospray ionization mass spectrometry data and one-dimensional and two-dimensional NMR spectra. The relative configurations of these molecules were established via 1H-1H coupling constants and nuclear Overhauser effect spectroscopy, while comparisons of the experimental electronic circular dichroism (ECD) spectra with the time-dependent density functional theory ECD calculations were utilized to assign their absolute configurations, which were in good agreement with the literature. These alkaloids (1-5) showed cytotoxic activity against a human breast cancer cell line (MDA-MB-231) that ranged from 0.4 to 4.8 µM. Compounds 1 and 5 were also cytotoxic against human ovarian (OVCAR3) and melanoma (MDA-MB-435) cancer cell lines.

Probing the Cytotoxic Signaling Induced by Eupenifeldin in Ovarian Cancer Models.

High-grade serous ovarian cancer (HGSOC) is the most common and lethal ovarian cancer histotype. Lack of early detection methods, limited therapeutic agents, and low 5-year survival rate reflect the urgent need to develop new therapies. Eupenifeldin, a bistropolone, originally isolated from Eupenicillium brefeldianum, is a cytotoxic fungal metabolite. In three HSGOC cell lines (OVCAR3, OVCAR5, OVCAR8), eupenifeldin was found to have an IC50 value less than 10 nM, while 10 times higher concentrations were required for cytotoxicity in nontumorigenic fallopian tube secretory epithelial cell lines (FTSEC). An in vivo hollow fiber assay showed significant cytotoxicity in OVCAR3. Eupenifeldin significantly increased Annexin V staining in OVCAR3 and -8, but not OVCAR5. Eupenifeldin activated caspases 3/7 in OVCAR3, OVCAR5, and OVCAR8; however, cleaved PARP was only detected in OVCAR3. Quantitative proteomics performed on OVCAR3 implicated ferroptosis as the most enriched cell death pathway. However, validation experiments did not support ferroptosis as part of the cytotoxic mechanism of eupenifeldin. Autophagic flux and LC3B puncta assays found that eupenifeldin displayed weak autophagic induction in OVCAR3. Inhibition of autophagy by cotreatment with bafilomycin reduced the toxicity of eupenifeldin, supporting the idea that induction of autophagy contributes to the cytotoxic mechanism of eupenifeldin.

Fungal-fungal co-culture: a primer for generating chemical diversity.

Covering: 2002 to 2020In their natural environment, fungi must compete for resources. It has been hypothesized that this competition likely induces the biosynthesis of secondary metabolites for defence. In a quest to discover new chemical diversity from fungal cultures, a growing trend has been to recapitulate this competitive environment in the laboratory, essentially growing fungi in co-culture. This review covers fungal-fungal co-culture studies beginning with the first literature report in 2002. Since then, there has been a growing number of new secondary metabolites reported as a result of fungal co-culture studies. Specifically, this review discusses and provides insights into (1) rationale for pairing fungal strains, (2) ways to grow fungi for co-culture, (3) different approaches to screening fungal co-cultures for chemical diversity, (4) determining the secondary metabolite-producing strain, and (5) final thoughts regarding the fungal-fungal co-culture approach. Our goal is to provide a set of practical strategies for fungal co-culture studies to generate unique chemical diversity that the natural products research community can utilize.

Anticancer activity of Neosetophomone B by targeting AKT/SKP2/MTH1 axis in leukemic cells.

Neosetophomone B (NSP-B), a meroterpenoid fungal secondary metabolite, was investigated for its anticancer potential in leukemic cell lines (K562 and U937). NSP-B treatment of leukemic cells suppressed cell viability by triggering apoptotic cell death. Apoptosis induced by NSP-B is triggered by mitochondrial signaling and caspase activation. Additionally, NSP-B treatment of leukemic cells causes AKT's inactivation accompanied by downregulation of SKP2 oncogene and MTH1 with a concomitant increase of p21Cip1and p27Kip1. Furthermore, NSP-B causes suppression of antiapoptotic proteins, including cIAP1, cIAP2, XIAP, survivin and BCl-XL. Overall, NSP-B reduces cell viability by mitochondrial and caspase-dependent apoptosis. The inhibition of AKT and SKP2 axis could be a promising therapeutic target for leukemia treatment.

Structural Diversity of Perylenequinones Is Driven by Their Redox Behavior.

Hypocrellins and hypomycins are two subclasses of fungal perylenequinones with unique structural, biological, and photochemical properties. With the growing interest in these naturally occurring photosensitizers, more studies were warranted to better understand the structural relationships between these two subclasses of perylenequinones. In this study, the long-postulated biosynthetic precursor (7) of class B fungal perylenequinones was isolated and characterized from a Shiraia-like sp. (strain MSX60519). Furthermore, the electrochemical and chemical redox behaviors of hypocrellins and hypomycins were investigated under aerobic and anaerobic conditions. These studies served to define the structural relationship within hypocrellins (1-3), which was further supported by X-ray crystallography, and between hypocrellins and hypomycins (4-6). Chemical reductions of hypocrellins under anaerobic conditions identified the origin of hypomycin A (4), hypomycin C (5), and hypomycin E (6), which in turn served to confirm 4 and revise the absolute configurations of 5 and 6. Hypocrellins were shown to undergo reversible reduction and reoxidation under aerobic conditions, while in an anaerobic environment and longer time scale, the fully reduced form can, to some extent, undergo an intramolecular ring closing metathesis. This may impart a means of reductive pathway for self-protection against these phototoxins and explain the chemical diversity observed in the fungal metabolites.