Synthesis and Late-Stage Modification of (-)-Doliculide Derivatives Using Matteson's Homologation Approach.

(-)-Doliculide, a marine cyclodepsipeptide derived from the Japanese sea hare, Dolabella auricularia, exhibits potent cytotoxic properties, sparking interest in the field of synthetic chemistry. It is comprised of a peptide segment and a polyketide moiety, rendering it amenable to Matteson's homologation methodology. This technique facilitates the diversification of the distinctive polyketide side chain, thereby permitting the introduction of functional groups in late stages for modifications of the derived compounds and studies on structure-activity relationships.

A poisonous cocktail: interplay of cereulide toxin and its structural isomers in emetic Bacillus cereus.

Food intoxications evoked by emetic Bacillus cereus strains constitute a serious threat to public health, leading to emesis and severe organ failure. The emetic peptide toxin cereulide, assembled by the non-ribosomal peptide synthetase CesNRPS, cannot be eradicated from contaminated food by usual hygienic measures due to its molecular size and structural stability. Next to cereulide, diverse chemical variants have been described recently that are produced concurrently with cereulide by CesNRPS. However, the contribution of these isocereulides to the actual toxicity of emetic B. cereus, which produces a cocktail of these toxins in a certain ratio, is still elusive. Since cereulide isoforms have already been detected in food remnants from foodborne outbreaks, we aimed to gain insights into the composition of isocereulides and their impact on the overall toxicity of emetic B. cereus. The amounts and ratios of cereulide and isocereulides were determined in B. cereus grown under standard laboratory conditions and in a contaminated sample of fried rice balls responsible for one of the most severe food outbreaks caused by emetic B. cereus in recent years. The ratios of variants were determined as robust, produced either under laboratory or natural, food-poisoning conditions. Examination of their actual toxicity in human epithelial HEp2-cells revealed that isocereulides A-N, although accounting for only 10% of the total cereulide toxins, were responsible for about 40% of the total cytotoxicity. An this despite the fact that some of the isocereulides were less cytotoxic than cereulide when tested individually for cytotoxicity. To estimate the additive, synergistic or antagonistic effects of the single variants, each cereulide variant was mixed with cereulide in a 1:9 and 1:1 binary blend, respectively, and tested on human cells. The results showed additive and synergistic impacts of single variants, highlighting the importance of including not only cereulide but also the isocereulides in routine food and clinical diagnostics to achieve a realistic toxicity evaluation of emetic B. cereus in contaminated food as well as in patient samples linked to foodborne outbreaks. Since the individual isoforms confer different cell toxicity both alone and in association with cereulide, further investigations are needed to fully understand their cocktail effect.

PI3K/Akt/FoxO Pathway Mediates Antagonistic Toxicity in HepG2 Cells Coexposed to Deoxynivalenol and Enniatins.

The emerging mycotoxins enniatins (ENNs) and the traditional mycotoxin deoxynivalenol (DON) often co-contaminate various grain raw materials and foods. While the liver is their common target organ, the mechanism of their combined effect remains unclear. In this study, the combined cytotoxic effects of four ENNs (ENA, ENA1, ENB, and ENB1) with DON and their mechanisms were investigated using the HepG2 cell line. Additionally, a population exposure risk assessment of these mycotoxins was performed by using in vitro experiments and computer simulations. The results showed that only ENA at 1/4 IC50 and ENB1 at 1/8 IC50 coexposed with DON showed an additive effect, while ENB showed the strongest antagonism at IC50 (CI = 3.890). Co-incubation of ENNs regulated the signaling molecule levels which were disrupted by DON. Transcriptome analysis showed that ENB (IC50) up-regulated the PI3K/Akt/FoxO signaling pathway and inhibited the expression of apoptotic genes (Bax, P53, Caspase 3, etc.) via phosphorylation of FoxO, thereby reducing the cytotoxic effects caused by DON. Both types of mycotoxins posed serious health risks, and the cumulative risk of coexposure was particularly important for emerging mycotoxins.

Cavomycins A-C, Linear Oligomer Depsipeptides from an Annelid-Associated Streptomyces cavourensis.

Three unique linear oligomeric depsipeptides, designated as cavomycins A-C (1-3), were identified from Streptomyces cavourensis, a gut bacterium associated with the annelid Paraleonnates uschakovi. The structures of these depsipeptides were determined through a combination of spectroscopic methods and chemical derivatization techniques, including methanolysis, the modified Mosher's method, advanced Marfey's methods, and phenylglycine methyl ester derivatization. The unique dipeptidyl residue arrangements in compounds 1-3 indicate that they are not degradation products of valinomycin. Compound 2 and its methylation derivative 2a exhibited antiproliferative activity against PANC-1 pancreatic cancer cells with IC50 values of 1.2 and 1.7 µM, respectively.

Mechanistic insight into human androgen receptor-mediated endocrine disrupting potential of cyclic depsipeptide mycotoxin, beauvericin, and influencing environmental factors for its biosynthesis in Fusarium oxysporum KFCC 11363P on rice cereal.

In current study, Fusarium mycotoxin, beauvericin (BEA), has endocrine disrupting potential through suppressing the exogenous androgen receptor (AR)-mediated transcriptional activation. BEA was classified as an AR antagonist, with IC30 and IC50 values indicating that it suppressed AR dimerization in the cytosol. BEA suppress the translocation of cytosolic activated ARs to the nucleus via exogenous androgens. Furthermore, we investigated the impact of environmental conditions for BEA production on rice cereal using response surface methodology. The environmental factors affecting the production of BEA, namely temperature, initial moisture content, and growth time were optimized at 20.28 °C, 42.79 % (w/w), and 17.31 days, respectively. To the best of our knowledge, this is the first report showing that BEA has endocrine disrupting potential through suppressing translocation of cytosolic ARs to nucleus, and temperature, initial moisture content, and growth time are important influencing environmental factors for its biosynthesis in Fusarium strains on cereal.

Beauvericin and enniatin B mycotoxins alter aquatic ecosystems: Effects on green algae.

Myxotoxins can contaminate algal-based products and arrive to the food chain to consumers producing chronic toxicity effects. Here, we studied phytotoxicity of mycotoxins, beauvericin (BEA) and ennaitin B (ENN B) in four phytoplankton strains: Acutodesmus sp., Chlamydomonas reinhardtii, Haematococcus pluvialis, and Monoraphidium griffithii, which are all green algae. It was tested the capacity of clearing the media of BEA and ENN B at different concentrations by comparing nominal and measured quantifications. Results revealed that Acutodesmus sp. and C. reinhardtii tended to flow up and down growth rate without reaching values below 50% or 60%, respectively. On the other hand, for H. pluvialis and M. griffith, IC50 values were reached. Regarding the clearance of media, in individual treatment a decrease of the quantified mycotoxin between nominal and measured values was observed; while in binary treatment, differences among both values were higher and more noted for BEA than for ENN B.

Pyroptosis Tuning in Intestinal Cryptosporidiosis via the Natural Histone Deacetylase Inhibitor Romidepsin.

Cryptosporidium is an opportunistic protozoan, with many species of cross-human infectivity. It causes life-threatening diarrhoea in children and CD4-defective patients. Despite its limited efficacy, nitazoxanide remains the primary anti-cryptosporidial drug. Cryptosporidium infects the intestinal brush border (intracellular-extracytoplasmic) and down-regulates pyroptosis to prevent expulsion. Romidepsin is a natural histone deacetylase inhibitor that triggers pyroptosis. Romidepsin's effect on cryptosporidiosis was assessed in immunocompromised mice via gasdermin-D (GSDM-D) immunohistochemical expression, IFN-γ, IL-1ß and IL-18 blood levels by ELISA, and via parasite scanning by modified Ziehl-Neelsen staining and scanning electron microscopy (SEM). Oocyst deformity and local cytokines were also assessed in ex vivo ileal explants. Following intraperitoneal injection of romidepsin, oocyst shedding significantly reduced at the 9th, 12th and 15th d.p.i. compared with infected-control and drug-control (nitazoxanide-treated) mice. H&E staining of intestinal sections from romidepsin-treated mice showed significantly low intestinal scoring with marked reduction in epithelial hyperplasia, villous blunting and cellular infiltrate. SEM revealed marked oocyst blebbing and paucity (in vivo and ex vivo) after romidepsin compared with nitazoxanide. Regarding pyroptosis, romidepsin triggered significantly higher intestinal GSDM-D expression in vivo, and higher serum/culture IFN-γ, IL-1ß and IL-18 levels in romidepsin-treated mice than in the control groups. Collectively, in cryptosporidiosis, romidepsin succeeded in enhancing pyroptosis in the oocysts and infected epithelium, reducing infection and shifting the brush border towards normalisation.

Biosynthesis of a new skyllamycin in Streptomyces nodosus: a cytochrome P450 forms an epoxide in the cinnamoyl chain.

Activation of a silent gene cluster in Streptomyces nodosus leads to synthesis of a cinnamoyl-containing non-ribosomal peptide (CCNP) that is related to skyllamycins. This novel CCNP was isolated and its structure was interrogated using mass spectrometry and nuclear magnetic resonance spectroscopy. The isolated compound is an oxidised skyllamycin A in which an additional oxygen atom is incorporated in the cinnamoyl side-chain in the form of an epoxide. The gene for the epoxide-forming cytochrome P450 was identified by targeted disruption. The enzyme was overproduced in Escherichia coli and a 1.43 Å high-resolution crystal structure was determined. This is the first crystal structure for a P450 that forms an epoxide in a substituted cinnamoyl chain of a lipopeptide. These results confirm the proposed functions of P450s encoded by biosynthetic gene clusters for other epoxidized CCNPs and will assist investigation of how epoxide stereochemistry is determined in these natural products.

Analysis of the Valgamicin Biosynthetic Pathway Reveals a General Mechanism for Cyclopropanol Formation across Diverse Natural Product Scaffolds.

Cyclopropanol rings are highly reactive and may function as molecular "warheads" that affect natural product bioactivity. Yet, knowledge on their biosynthesis is limited. Using gene cluster analyses, isotope labeling, and in vitro enzyme assays, we shed first light on the biosynthesis of the cyclopropanol-substituted amino acid cleonine, a residue in the antimicrobial depsipeptide valgamicin C and the cytotoxic glycopeptide cleomycin A2. We decipher the biosynthetic origin of valgamicin C and show that the cleonine cyclopropanol ring is derived from dimethylsulfoniopropionate (DMSP). Furthermore, we demonstrate that part of the biosynthesis is analogous to the formation of malleicyprol polyketides in pathogenic bacteria. By genome mining and metabolic profiling, we identify the potential to produce cyclopropanol rings in other bacterial species. Our results reveal a general mechanism for cyclopropyl alcohol biosynthesis across diverse natural products that may be harnessed for bioengineering and drug discovery.

Enniatin B1 induces damage to Leydig cells via inhibition of the Nrf2/HO-1 and JAK/STAT3 signaling pathways.

Enniatin B1 (ENN B1) is a mycotoxin that can be found in various foods. However, whether ENN B1 is hazardous to the reproductive system is still elusive. Leydig cells are testosterone-generating cells that reside in the interstitial compartment between seminiferous tubules. Dysfunction of Leydig cells could result in male infertility. This study aimed to examine the toxicological effects of ENN B1 against TM3 Leydig cells. ENN B1 significantly inhibited cell viability in a dose-dependent manner. ENN B1 treatment also decreased the expression of functional genes in Leydig cells. Moreover, ENN B1 induced Leydig cells apoptosis and oxidative stress. Mechanistically, ENN B1 leads to the upregulation of Bax and downregulation of Bcl-2 in Leydig cells. In addition, ENN B1 inhibited the Nrf2/HO-1 pathway, which is critical for the induction of oxidative stress. Additionally, ENN B1 treatment repressed the JAK/STAT3 signaling pathway in Leydig cells. Rescue experiments showed that activation of STAT3 resulted in alleviation of ENN B1-induced damage in Leydig cells. Collectively, our study demonstrated that ENN B1 induced Leydig cell dysfunction via multiple mechanisms.

Synthesis, Structure-Activity Relationship Study, Bioactivity, and Nephrotoxicity Evaluation of the Proposed Structure of the Cyclic Lipodepsipeptide Brevicidine B.

The brevicidines represent a novel class of nonribosomal antimicrobial peptides that possess remarkable potency and selectivity toward highly problematic and resistant Gram-negative pathogenic bacteria. A recently discovered member of the brevicidine family, coined brevicidine B (2), comprises a single amino acid substitution (from d-Tyr2 to d-Phe2) in the amino acid sequence of the linear moiety of brevicidine (1) and was reported to exhibit broader antimicrobial activity against both Gram-negative (MIC = 2-4 µgmL-1) and Gram-positive (MIC = 2-8 µgmL-1) pathogens. Encouraged by this, we herein report the first total synthesis of the proposed structure of brevicidine B (2), building on our previously reported synthetic strategy to access brevicidine (1). In agreement with the original isolation paper, pleasingly, synthetic 2 demonstrated antimicrobial activity toward Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae (MIC = 4-8 µgmL-1). Interestingly, however, synthetic 2 was inactive toward all of the tested Gram-positive pathogens, including methicillin-resistant Staphylococcus aureus strains. Substitution of d-Phe2 with its enantiomer, and other hydrophobic residues, yields analogues that were either inactive or only exhibited activity toward Gram-negative strains. The striking difference in the biological activity of our synthetic 2 compared to the reported natural compound warrants the re-evaluation of the original natural product for purity or possible differences in relative configuration. Finally, the evaluation of synthetic 1 and 2 in a human kidney organoid model of nephrotoxicity revealed substantial toxicity of both compounds, although 1 was less toxic than 2 and polymyxin B. These results indicate that modification to position 2 may afford a strategy to mitigate the nephrotoxicity of brevicidine.

Structure and Biosynthesis of Hectoramide B, a Linear Depsipeptide from Marine Cyanobacterium Moorena producens JHB Discovered via Coculture with Candida albicans.

The tropical marine cyanobacterium Moorena producens JHB is a prolific source of secondary metabolites with potential biomedical utility. Previous studies on this strain led to the discovery of several novel compounds such as hectochlorins and jamaicamides. However, bioinformatic analyses of its genome indicate the presence of numerous cryptic biosynthetic gene clusters that have yet to be characterized. To potentially stimulate the production of novel compounds from this strain, it was cocultured with Candida albicans. From this experiment, we observed the increased production of a new compound that we characterize here as hectoramide B. Bioinformatic analysis of the M. producens JHB genome enabled the identification of a putative biosynthetic gene cluster responsible for hectoramide B biosynthesis. This work demonstrates that coculture competition experiments can be a valuable method to facilitate the discovery of novel natural products from cyanobacteria.

Plitidepsin as an Immunomodulator against Respiratory Viral Infections.

Plitidepsin is a host-targeted compound known for inducing a strong anti-SARS-CoV-2 activity, as well as for having the capacity of reducing lung inflammation. Because IL-6 is one of the main cytokines involved in acute respiratory distress syndrome, the effect of plitidepsin in IL-6 secretion in different in vitro and in vivo experimental models was studied. A strong plitidepsin-mediated reduction of IL-6 was found in human monocyte-derived macrophages exposed to nonproductive SARS-CoV-2. In resiquimod (a ligand of TLR7/8)-stimulated THP1 human monocytes, plitidepsin-mediated reductions of IL-6 mRNA and IL-6 levels were also noticed. Additionally, although resiquimod-induced binding to DNA of NF-κB family members was unaffected by plitidepsin, a decrease in the regulated transcription by NF-κB (a key transcription factor involved in the inflammatory cascade) was observed. Furthermore, the phosphorylation of p65 that is required for full transcriptional NF-κB activity was significantly reduced by plitidepsin. Moreover, decreases of IL-6 levels and other proinflammatory cytokines were also seen in either SARS-CoV-2 or H1N1 influenza virus-infected mice, which were treated at low enough plitidepsin doses to not induce antiviral effects. In summary, plitidepsin is a promising therapeutic agent for the treatment of viral infections, not only because of its host-targeted antiviral effect, but also for its immunomodulatory effect, both of which were evidenced in vitro and in vivo by the decrease of proinflammatory cytokines.

Beauvericin, produced by Fusarium oxysporum inhibits bisphenol A-induced proliferation of human breast cancer cell line by regulating ERα/p38 pathway.

Beauvericin (BEA) is a cyclic depsipeptide secondary metabolite of Fusarium species. It causes chemical hazards in food products and exists in an environment containing soil and various food types. On the other hand, the purified BEA has various biological activities and is regarded as a potential candidate for pharmaceutical research. This study was performed to assess the anti-proliferation activity of BEA against human breast cancer cells by regulating the estrogen receptor-alpha (ERα)/p38 pathway. TA and BA assays verified that BEA is a completed ER antagonist. Additionally, BEA suppressed cell proliferation in the anti-proliferation assay involving ER-positive human breast cancer cells co-treated with BPA and BEA. In respect to an anti-proliferation activity, the BPA-induced phosphorylation of p38 protein was inhibited in the presence of BEA. These results suggested that BEA exerts inhibitory potentials on endocrine disrupting effect and possibly acts as a natural therapeutic material for human estrogen hormonal health.

Biosynthesis of Dolastatin 10 in Marine Cyanobacteria, a Prototype for Multiple Approved Cancer Drugs.

Dolastatin 10, a potent tubulin-targeting marine anticancer natural product, provided the basis for the development of six FDA-approved antibody-drug conjugates. Through the screening of cyanobacterial Caldora penicillata environmental DNA libraries and metagenome sequencing, we identified its biosynthetic gene cluster. Functional prediction of 10 enzymes encoded in the 39 kb cluster supports the dolastatin 10 biosynthesis. The nonheme diiron monooxygenase DolJ was biochemically characterized to mediate the terminal thiazole formation in dolastatin 10.

Unearthing a Cryptic Biosynthetic Gene Cluster for the Piperazic Acid-Bearing Depsipeptide Diperamycin in the Ant-Dweller Streptomyces sp. CS113.

Piperazic acid is a cyclic nonproteinogenic amino acid that contains a hydrazine N-N bond formed by a piperazate synthase (KtzT-like). This amino acid, found in bioactive natural products synthesized by non-ribosomal peptide synthetases (NRPSs), confers conformational constraint to peptides, an important feature for their biological activities. Genome mining of Streptomyces strains has been revealed as a strategy to identify biosynthetic gene clusters (BGCs) for potentially active compounds. Moreover, the isolation of new strains from underexplored habitats or associated with other organisms has allowed to uncover new BGCs for unknown compounds. The in-house "Carlos Sialer (CS)" strain collection consists of seventy-one Streptomyces strains isolated from the cuticle of leaf-cutting ants of the tribe Attini. Genomes from twelve of these strains have been sequenced and mined using bioinformatics tools, highlighting their potential to encode secondary metabolites. In this work, we have screened in silico those genomes, using KtzT as a hook to identify BGCs encoding piperazic acid-containing compounds. This resulted in uncovering the new BGC dpn in Streptomyces sp. CS113, which encodes the biosynthesis of the hybrid polyketide-depsipeptide diperamycin. Analysis of the diperamycin polyketide synthase (PKS) and NRPS reveals their functional similarity to those from the aurantimycin A biosynthetic pathway. Experimental proof linking the dpn BGC to its encoded compound was achieved by determining the growth conditions for the expression of the cluster and by inactivating the NRPS encoding gene dpnS2 and the piperazate synthase gene dpnZ. The identity of diperamycin was confirmed by High-Resolution Mass Spectrometry (HRMS) and Nuclear Magnetic Resonance (NMR) and by analysis of the domain composition of modules from the DpnP PKS and DpnS NRPS. The identification of the dpn BGC expands the number of BGCs that have been confirmed to encode the relatively scarcely represented BGCs for depsipeptides of the azinothricin family of compounds and will facilitate the generation of new-to-nature analogues by combinatorial biosynthesis.

Destruxin A inhibits the hemocytin-mediated hemolymph immunity of host insects to facilitate Metarhizium infection.

Insects have an effective innate immune system to protect themselves against fungal invasion. Metarhizium employs a toxin-based strategy using a nonribosomal peptide called destruxin A (DA) to counteract the host immune response. However, the mechanism by which DA inhibits insect immunity is still unclear. Here, we identified 48 DA-binding proteins in silkworm hemolymph, with the binding affinity (KD) ranging from 2 to 420 µM. Among these proteins, hemocytin, an important immune factor, was determined to be the strongest DA-binding protein. DA binds to hemocytin and regulates its conformation in a multisite manner. Furthermore, DA exerts a significant inhibitory effect on hemocytin-mediated hemocyte aggregation. By disrupting the interaction between hemocytin, actin A3, and gelsolin, DA prevents the transformation of granules into vesicles in hemocytes. These vesicles are responsible for storing, maturing, and exocytosing hemocytin. Therefore, hemocytin secretion is reduced, and the formation of structures that promote aggregation in outer hemocytes is inhibited.

A Semisynthesis Platform for the Efficient Production and Exploration of Didemnin-Based Drugs.

Plitidepsin (or dehydrodidemnin B), an approved anticancer drug, belongs to the didemnin family of cyclic depsipeptides, which are found in limited quantities in marine tunicate extracts. Herein, we introduce a new approach that integrates microbial and chemical synthesis to generate plitidepsin and its analogues. We screened a Tistrella strain library to identify a potent didemnin B producer, and then introduced a second copy of the didemnin biosynthetic gene cluster into its genome, resulting in a didemnin B titer of approximately 75 mg/L. Next, we developed two straightforward chemical strategies to convert didemnin B into plitidepsin, one of which involved a one-step synthetic route giving over 90 % overall yield. Furthermore, we synthesized 13 new didemnin derivatives and three didemnin probes, enabling research into structure-activity relationships and interactions between didemnin and proteins. Our study highlights the synergistic potential of biosynthesis and chemical synthesis in overcoming the challenge of producing complex natural products sustainably and at scale.

A GSH-resistant FK228 analogue containing a stable disulfide bond.

FK228 is a potent natural pan HDAC inhibitor approved by the FDA for the treatment of cutaneous T-cell lymphoma as well as peripheral T-cell lymphoma. It is generally believed that the mechanism of FK228 acting on HDACs is by reducing its disulfide bond after entering the cell, and the dithiol group may chelate with Zn2+ and form a weak reversible covalent bond with cysteine in the catalytic pocket of HDACs, therefore inhibiting the activity of HDACs. However, due to the weak stability of the disulfide bond in FK228, it has been difficult to obtain direct evidence for the above conjecture. Thus, improving the stability of the FK228 disulfide bond will help to explore the exact mechanism of FK228. In this study, based on the stability and target-induced covalent properties of the Cysteine-Penicillamine (Cys-Pen) disulfide bond reported previously, the Pen was introduced into the modification of FK228. Specifically, the d-Cys in FK228 was replaced by d-Pen, the total synthetic pathway was optimized, and the novel synthetic FK228 analogue (FK-P) stability was verified. FK-P can also be used as a new drug molecule in the future to participate in the research of related biological mechanisms or the treatment of diseases.

Mebamamide C, a deoxy analogue of mebamamides in Bryopsis marine green algae and Elysia sacoglossan mollusks.

Kahalalides, originally isolated from the sacoglossan mollusk Elysia rufescens, have been found in various Elysia and Bryopsis species, with over 20 variants identified to date. These compounds are biosynthesized by Candidatus Endobryopsis kahalalidefaciens within Bryopsis species. In this study, we report the isolation and structural determination of a new cyclic depsipeptide, mebamamide C (1), from Bryopsis sp. The planar structure was determined by spectroscopic data analyses, and the absolute configurations were determined using Marfey's method and modified Mosher's method. Additionally, our study explores the chemical relationship between Bryopsis algae and Elysia mollusks. The individual chemical profiles of these marine organisms highlight a fascinating aspect of marine chemical ecology. The distinct, species-specific chemical profiles observed in Elysia species imply the possibility of a symbiotic relationship with the kahalalide-producing bacteria.