Fungi are well known to biosynthesize structurally complex secondary metabolites (SMs) with diverse bioactivities. These fungal SMs are frequently produced by biosynthetic gene clusters (BGCs). Linking SMs to their BGCs is key to understanding their chemical and biological functions. Reasoning that structural similarity of SMs arises from similarities in the genes involved in their biosynthesis, we developed an integrative approach that leverages known SM-BGC pairs to infer links between detected SMs and genome-predicted BGC regions in fungi. As proof of concept, we structurally characterized 60 metabolites from metabolomic data of 16 strains of the filamentous fungus Aspergillus fischeri. Our approach assigned 22 to known SM-BGC pairs and proposed specific links to BGCs and genetic pathways for the remaining 38 metabolites. These results suggest that coupling chemical structure similarity and genomic sequence similarity is a straightforward and high-throughput approach for linking fungal SMs to their BGCs.

The growing consumer preference for natural and sustainable products has heightened interest in biopigments across pharmaceutical, cosmetic, and food industries. In this study, we investigate endophytic fungi as a viable and eco-friendly source for the production of bioactive natural pigments. A promising strain, Aspergillus westerdijkiae 17P, was isolated from Betula pendula and assessed for its pigment-producing potential and associated bioactivities. The biomass extract was fractionated, and the resulting components were evaluated for antimicrobial, antioxidant, anticancer, neuroprotective, and peroxisome proliferator-activated receptor gamma (PPAR-γ) agonist activities. Among the fractions, 17P2 exhibited broad-spectrum antimicrobial effects, notable antioxidant activity (83

Perylenequinones (PQs) such as hypocrellins and hypomycins are fungal-derived redox-active metabolites with known roles as photosensitizers in the oxidative stress response and applications in photodynamic therapy (PDT). Here, we report that Shiraia sp., a filamentous fungus, can survive and grow under strictly anaerobic (argon) conditions─an unexpected finding for a multicellular eukaryote. Modulating redox homeostasis through chemical reduction and oxygen limitation promotes the intramolecular cyclization of hypocrellins, enhancing hypomycin biosynthesis. Moisture content further influences these transformations, with high water levels favoring keto-enol tautomerization and dry, reducing environments promoting hydride substitution at the peripheral positions. These findings highlight redox modulation as a key driver of perylenequinone metabolism and suggest that PQs may contribute to maintaining redox balance under anaerobic stress, hinting at a broader role in oxygen-independent adaptation in filamentous fungi. This work offers new insights at the interface of redox biology, chemical signaling, and fungal metabolism, with potential implications for the stability and function of PQ-based PDT agents in hypoxic, reducing conditions such as tumor microenvironments.

Continued growth in global sales of natural products has led to an increased risk of natural product-drug interactions that can compromise drug efficacy and safety. One such natural product, goldenseal, was shown to decrease systemic exposure to a subtherapeutic dose of oral metformin in healthy adults. A follow-up study involving therapeutic metformin doses and adults with type II diabetes demonstrated a metformin dose-dependent pharmacokinetic interaction with goldenseal. These results, along with no change in metformin half-life or renal clearance in both studies, suggested that the goldenseal-metformin interaction occurred in the gut via inhibition of an unidentified saturable intestinal transport process. We used enteroid monolayers derived from the duodenum of 4 healthy human adult donors to recapitulate the goldenseal-metformin interaction in vitro and identify the transporters involved in the observed in vivo interaction. Our results implicate thiamine transporter (ThTr) 2 as the predominant transporter involved in metformin uptake through the apical membrane, accounting for approximately 45% of total metformin intracellular accumulation. Additionally, goldenseal inhibited ThTr-2, but only under subsaturating metformin dosing concentrations. The goldenseal-metformin interaction mediated under therapeutic metformin dose conditions involves a low-affinity basolateral transporter, ThTr-1, which accounts for approximately 50% of inhibitable metformin apical to basolateral flux. However, a substantial fraction of metformin flux appears to involve paracellular transport. These results further elucidate the mechanism underlying the goldenseal-metformin interaction and suggest that enteroid monolayers are a promising model to study intestinal natural product-drug interactions. SIGNIFICANCE STATEMENT: The research presented in this article demonstrates the utility of enteroid monolayers to predict and ascertain the mechanisms of drug-drug and natural product-drug interactions. Using this model, the study was able to identify the transporters (thiamine transporter-1 and thiamine transporter-2) involved in metformin absorption that are inhibited by the natural product, goldenseal, which were previously unidentified.

Coagulase-negative staphylococci are dominant human skin colonizers, producing natural products that shape the community and prevent pathogen colonization. The molecular mechanisms by which these natural products mediate interbacterial competition are not fully understood. Here, we identify a plasmid-borne daptide bacteriocin (hominicin) from a human skin isolate of Staphylococcus hominis, which features an unusual N2-N2-dimethyl-1,2-propanediamine C-terminus. Heterologous expression of the reconstituted biosynthetic loci yields a daptide product of the same molecular mass that exhibits antimicrobial activity against the skin pathogen Staphylococcus aureus, with amino-modified termini being essential for activity. Membrane permeability and voltage-clamp lipid bilayer experiments support a mechanism by which the daptide rapidly dissipates the transmembrane potential by forming peptidic channels. Additionally, we identify a cognate homI gene that confers resistance against membrane damage. Finally, the purified daptide effectively protects mouse skin from S. aureus-induced epicutaneous injury, as evidenced by reduced bacterial burden, inflammation, and transepithelial water loss, highlighting its therapeutic potential for treating bacterial skin infections. Our findings elucidate a mechanism of action, biosynthesis, and resistance for a staphylococcal bacteriocin belonging to a class of natural products called daptides.

Green tea [Camellia sinensis (L.) Kuntze] is widely consumed for its cardiovascular benefits, which may overlap with the use of amiodarone, a critical antiarrhythmic drug. Recent in vitro and clinical data indicate that green tea reduced the intestinal solubility and systemic exposure of raloxifene. Raloxifene and amiodarone are both lipophilic drugs that are dependent on solubility for absorption in the intestine. This study investigated the interaction between green tea catechins and amiodarone, focusing on solubility and dissolution in fasted state simulated intestinal fluid (FaSSIF). In vitro studies found that green tea extract (GTE) equivalent to 1 cup of brewed tea and the flavan-3-gallate catechin, (−)-epigallocatechin-3-gallate (EGCG), at an equimolar concentration to that present in the GTE, both significantly decreased soluble amiodarone by 99% and 50%, respectively. In addition, 1 cup of brewed green tea significantly reduced amiodarone dissolution by 74%. In a pharmacokinetic study in fasted mice, GTE did not significantly affect amiodarone's area under the curve (AUC) and maximum concentration (Cmax), which were 15% and 21% lower than controls, respectively. The flavan-3-ol catechin, (−)-epigallocatechin (EGC), did not affect amiodarone concentrations in FaSSIF or in the mice. A follow-up solubility study found that the vehicle used for liquid gavage in mice contributed to the discrepancy between in vitro and in vivo results. These in vitro findings suggest that green tea consumption may impact the bioavailability of amiodarone, potentially affecting its efficacy, but further research is needed to explore this interaction in vivo in clinical studies.

Informatics-guided approaches involving untargeted mass spectrometry metabolomics to predict active compounds in natural products mixtures have become increasingly common. With such strategies, it is sometimes possible to target active compounds for isolation early in the fractionation process, thereby reducing effort and increasing hit rate success. However, such approaches require follow up studies to address the potential problem of false correlations. To demonstrate this, we employed the botanical Cannabis sativa (hemp) as a test case. A C. sativa extract was fractionated in several stages, and the ability of the fractions to inhibit the growth of Methicillin-resistant Staphylococcus aureus (MRSA) was evaluated in broth microdilution assays. Metabolomics data were collected for the extract and fractions using high performance liquid chromatography coupled to high resolution mass spectrometry on an Orbitrap mass spectrometer, and selectivity ratio analysis was employed as a statistical tool to predict the active compound from two different rounds of fractionation of the C. sativa extract. From the early stage of fractionation, we predicted that the cannabinoids cannabidiol (1, CBD) and cannabidiolic acid (2, CBDA) were major active constituents. These predictions, when verified with follow up minimum inhibitory concentration (MIC) assays of the pure compounds, proved to be accurate. However, in a later stage of fractionation using the same statistical approach, the previously reported C. sativa constituents N-trans-p-cou-maroyltyramine (3, pCT) and N-trans-feruloyltyramine (4, FT) were predicted to be responsible for activity. Follow up assays with these pure compounds revealed that they possess no direct or synergistic antimicrobial activity against MRSA. This study highlights how statistical predictions of active compounds in untargeted metabolomics studies are inherently correlative and emphasizes the need for follow up studies to verify the accuracy of such predictions.

Perylenequinones, such as hypocrellins and hypomycins, are fungal secondary metabolites with potential for pharmaceutical and industrial applications due to both their physical and biological properties. This study focused on their sustainable production. Additionally, stable isotope labeling was used to probe the biosynthesis of these compounds, demonstrating how sugars are likely incorporated into the perylenequinone scaffold. Shiraia sp. (strain MSX60519; Shiraiaceae, Pleosporales) was cultivated under varying nutrient conditions to evaluate the production of perylenequinones, with sugars serving as primary carbon sources. Five metabolites were isolated (from oatmeal cultures) using environmentally friendly solvent-based techniques, and the process was further optimized to maximize yields. High-performance liquid chromatography (HPLC) and liquid chromatography–high-resolution mass spectrometry (LC–HRMS) were employed to detect, characterize, and quantify the major compounds. Furthermore, feeding experiments were performed using 13C-labeled glucose, with droplet probe mass spectrometry used to monitor stable isotope incorporation in situ. This study yielded three key findings. First, the production of perylenequinones was significantly enhanced by supplementing fermentation media with sugars, and disaccharides significantly enhanced the production of perylenequinones compared to monosaccharides. Optimizing sugar concentrations during the fermentation further influenced the profile of secondary metabolites. Second, stable isotope labeling experiments confirmed that sugars are the primary building blocks of perylenequinones, as noted by tracing 13C-labeling into ent-shiraiachrome A (1). Finally, a green, scalable, and sustainable strategy for producing these compounds on the gram scale was developed by optimizing fermentation conditions, refining purification methods, and improving extraction efficiency. These findings provide critical insights into optimizing fermentation conditions for the scaled and sustainable production of perylenequinones. This approach offers a cost-effective and environmentally friendly pipeline for harnessing these valuable compounds, paving the way for broader pharmaceutical and industrial applications.

Brown tef (Eragrostis tef) grain extracts increase cellular glutathione (GSH) in THP-1 monocytes, however, the molecular mechanisms remained unclear. In this study, we found that the tef extracts induced luciferase activity by 19-fold in HEK293 cells stably expressing the luciferase gene under the control of the nuclear factor erythroid 2-related factor 2 (Nrf2)–antioxidant response element, indicating the activation of the antioxidant pathway. RNA-seq analysis revealed that the tef extract enhanced the expression of 87 genes, including some regulated by the Nrf2 transcription factor. Differentially expressed genes included ferritin heavy chain pseudogenes, tumor necrosis factor (TNF)-α, and solute carrier genes. Using LC-MS, we identified compounds that were differentially enriched in the active tef fraction as compared to the inactive fraction, including vernolic and α-linolenic acid, which were enhanced by over 60-fold. In summary, our findings indicate that tef grains contain phytochemicals that enhance the antioxidant levels through the Nrf2-signaling pathway.

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.

Diepoxin-eta (1) is a cytotoxic fungal metabolite belonging to the spirobisnaphthalene structural class. In this study, four monofluorinated analogues (2-5) of diepoxin-eta (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 to 8.2 mu M.

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 AUC(0-24h) 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 C-max 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 is a widely consumed beverage. A recent clinical study reported green tea decreased systemic exposure of raloxifene and its glucuronide metabolites by 34–43

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, (–)-β-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, (–)-β-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 (–)-β-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.

Due to the emergence of resistance, the World Health Organization considers Gram-negative pathogen Acine-tobacter baumannii a top priority for therapeutic development. Using this priority pathogen and a phenotypic, agar plate-based assay, a unique library of extracts from 2500 diverse fungi was screened for antimicrobial activity against a highly virulent, drug-resistant strain of A. baumannii (AB5075). The most potent hit from this screen was an extract from the fungus Tolypocladium sp., which was found to produce pyridoxatin. Another active extract from the fungus Trichoderma deliquescens was characterized and yielded trichokonin VII and trichokonin VIII. Evaluation of pyridoxatin against A. baumannii (AB5075) in a broth microdilution assay revealed a mini-mum inhibitory concentration (MIC) of 38 mu M, compared to the known antibiotic levofloxacin with MIC of 28 mu M. Mass spectrometry, Marfey's analysis and nuclear magnetic resonance spectroscopy analyses confirmed the structures of trichokonins VII and VIII to be consistent with previous reports. In an in vivo Galleria mellonella model, pyridoxatin tested at 150 mg/kg exhibited minimal toxicity (90% survival) and promising antimicrobial efficacy (50% survival) after 5 days. Trichokonins VII and VIII tested at 150 mg/kg were toxic to G. mellonella , with 20% survival and 40% survival after 5 days, respectively. The findings of this project suggest that pyr-idoxatin may serve as a lead compound for the development of antimicrobials against A. baumannii. They also demonstrate the value of the phenotypic screening approach employed herein.

AbstractCyclopeptide alkaloids are an abundant class of plant cyclopeptides with over 200 analogs described and bioactivities ranging from analgesic to antiviral. While these natural products have been known for decades, their biosynthetic basis remains unclear. Using a transcriptome‐mining approach, we link the cyclopeptide alkaloids from Ceanothus americanus to dedicated RiPP precursor peptides and identify new, widely distributed split BURP peptide cyclase containing gene clusters. Guided by our bioinformatic analysis, we identify and isolate new cyclopeptides from Coffea arabica, which we named arabipeptins. Reconstitution of the enzyme activity for the BURP found in the biosynthesis of arabipeptin A validates the activity of the newly discovered split BURP peptide cyclases. These results expand our understanding of the biosynthetic pathways responsible for diverse cyclic plant peptides and suggest that these side chain cross‐link modifications are widely distributed in eudicots.

Supplementary Figure Legends from Milk Thistle and Prostate Cancer: Differential Effects of Pure Flavonolignans from Silybum marianum on Antiproliferative End Points in Human Prostate Carcinoma Cells

Tef (Eragrostis tef) is an orphan crop that is widely grown in East Africa, primarily in Ethiopia as a staple crop. It is becoming popular in the Western world owing to its nutritious and gluten-free grains and the forage quality of its biomass. Tef is also considered to have a high antioxidant capacity based on cell-free studies. However, the antioxidant activity of tef has never been validated using a physiologically relevant cell model. The purpose of this study was to investigate the antioxidant capacity of tef grain extracts using a mammalian cell model. We hypothesized that the tef grain extracts are capable of modulating the cellular antioxidant response via the modulation of glutathione (GSH) biosynthetic pathways. Therefore, we evaluated the antioxidant activity of purified tef grain extracts in the human acute monocytic leukemia (THP-1) cell line. Our findings revealed that the organic fraction of grain extracts increased the cellular GSH level, which was more evident for brown-colored tef than the ivory variety. Moreover, a brown-tef fraction increased the expressions of GSH-pathway genes, including γ-glutamate cysteine ligase catalytic (GCLC) and modifier (GCLM) subunits and glutathione reductase (GR), an enzyme that plays a key role in GSH biosynthesis, suggesting that tef extracts may modulate GSH metabolism. Several compounds were uniquely identified via mass spectrometry (MS) in GSH-modulating brown-tef samples, including 4-oxo-β-apo-13-carotenone, γ-linolenic acid (methyl ester), 4,4′-(2,3-dimethyl-1,4-butanediyl)bis-phenol (also referred to as 8,8′-lignan-4,4′-diol), and (3β)-3-[[2-[4-(Acetylamino)phenoxy]acetyl]oxy]olean-12-en-28-oic acid. Tef possesses antioxidant activity due to the presence of phytochemicals that can act as direct antioxidants, as well as modulators of antioxidant-response genes, indicating its potential role in alleviating diseases triggered by oxidative stresses. To the best of our knowledge, this is the first report revealing the antioxidant ability of tef extracts in a physiologically relevant human cell model.

Green tea is a popular botanical natural product, and its consumption has been associated with pharmacokinetic natural product-drug interactions (NP-DIs). Unpublished clinical data show that acute and chronic oral administration of green tea decreased the systemic exposure of raloxifene in healthy subjects. These clinical data indicate an intestinal interaction, although the mechanism for reduced absorption and systemic exposure is not known. NP-mediated modulation of drug solubility in the intestinal lumen is an area of increasing interest and concern. Intestinal bile acids can enhance the absorption of hydrophobic compounds by micellar solubilization, and raloxifene is a Biopharmaceutic Drug Disposition Classification System (BDDCS) class 2 drug with poor water solubility. We hypothesize that green tea reduces intestinal absorption of raloxifene through decreased micellar solubility. To investigate this interaction, bile acid mixed micelles were prepared and incubated with raloxifene, green tea extract (GTE), (-)-epigallocatechin gallate EGCG, and a combination of raloxifene with GTE or EGCG. The size of the micelles was determined by dynamic light scattering and raloxifene quantification was determined by UPLC-MS/MS. Raloxifene did not affect micelle diameter (micelle alone: 63.2 ± 5.6 nm, micelle with raloxifene 76.0 ± 5.8). In contrast, micelle diameter increased after incubation with GTE (416 ± 150.3 nm), EGCG (300.9 ± 173 nm), raloxifene + GTE (451 ± 164.2 nm), and raloxifene + EGCG (294.8 ± 303 nm). The micellar solubility of raloxifene was decreased by ~80% after incubation with GTE, whereas EGCG produced inconsistent results. These data suggest GTE decreased the micellar solubility of raloxifene and may play a role in the observed green tea-raloxifene intestinal interaction.