Isolation and comparative genotoxicity screening of trichokonins VI and VIII on CHO-K1 cells.

Peptaibols are fungal peptides that exhibit efficacy against pathogen microorganisms. Trichokonin VI (TK-VI) and trichokonin VIII (TK-VIII) are known peptaibols isolated from the endolichenic fungi Hypocrea sp. Previous investigations reported that trichokonin VI presents antiproliferative effects on tumor cells. This study is pioneering in elucidating the genotoxic effects of TK-VI and TK-VIII, contributing to the thorough assessment of their safety as potential therapeutic agents. The present investigation aimed to evaluate the genotoxicity of TK-VI and TK-VIII on CHO-K1 cells. Cytotoxicity was evaluated using the XTT assay and clonogenic survival assays, followed by evaluation of DNA damage using the comet assay and micronucleus test conducted in vitro. The XTT assay results indicated IC50 values of 10.30 µM and 9.89 µM for TK-VI and TK-VIII, respectively. The clonogenic survival assay indicated that concentrations of 10 µM or higher completely inhibited the cell colony formation. In the comet assay, both TK-VI and TK-VIII increased the DNA damage score and the frequency of comet nuclei in all tested concentrations. In the micronucleus assay, TK-VI and TK-VIII at 10 µM increased the frequency of MN in CHO-K1 cells. Both TK-VI and TK-VIII exhibited genotoxic effects. Our findings underscore the importance of considering the genotoxicological safety of peptaibols, particularly when assessing their potential for other biological activities.

Structure and Biosynthesis of Perochalasins A-C, Open-Chain Merocytochalasans Produced by the Marine-Derived Fungus Peroneutypa sp. M16.

Novel open-chain merocytochalasans, perochalasins A-C (1-3), containing an unusual N-O six-membered heterocyclic moiety, were isolated from cultures of the marine-derived Peroneutypa sp. M16 fungus, along with cytochalasin Z27 (4), cytochalasin Z28 (5), [12]-cytochalasin (6), and phenochalasin B (7). The structures of compounds 1-3 were established by analysis of the spectroscopic data. Full genome sequencing of Peroneutypa sp. M16 enabled the identification of a cytochalasan biosynthetic gene cluster and a proposal for the biosynthetic assembly of perochalasins. The proposal is supported by the nonenzymatic conversion of phenochalasin B (7) into 1-3, based on isotope-labeled hydroxylamine (15NH2OH and ND2OD) feeding studies in vivo and in vitro. In contrast to other merocytochalasans, these are the first cytochalasans confirmed to arise via nucleophilic addition and at a distinct location from the reactive macrocycle olefin, potentially expanding further the range of merocytochalasans to be discovered or engineered. Cytochalasin Z27 (4) exhibited antiplasmodial activities in the low micromolar range against the chloroquine-sensitive Plasmodium falciparum 3D7 strain as well as against resistant strains of the parasite (Dd2, TM90C6B, and 3D7r_MMV848).

NP3 MS Workflow: An Open-Source Software System to Empower Natural Product-Based Drug Discovery Using Untargeted Metabolomics.

Natural products (or specialized metabolites) are historically the main source of new drugs. However, the current drug discovery pipelines require miniaturization and speeds that are incompatible with traditional natural product research methods, especially in the early stages of the research. This article introduces the NP3 MS Workflow, a robust open-source software system for liquid chromatography-tandem mass spectrometry (LC-MS/MS) untargeted metabolomic data processing and analysis, designed to rank bioactive natural products directly from complex mixtures of compounds, such as bioactive biota samples. NP3 MS Workflow allows minimal user intervention as well as customization of each step of LC-MS/MS data processing, with diagnostic statistics to allow interpretation and optimization of LC-MS/MS data processing by the user. NP3 MS Workflow adds improved computing of the MS2 spectra in an LC-MS/MS data set and provides tools for automatic [M + H]+ ion deconvolution using fragmentation rules; chemical structural annotation against MS2 databases; and relative quantification of the precursor ions for bioactivity correlation scoring. The software will be presented with case studies and comparisons with equivalent tools currently available. NP3 MS Workflow shows a robust and useful approach to select bioactive natural products from complex mixtures, improving the set of tools available for untargeted metabolomics. It can be easily integrated into natural product-based drug-discovery pipelines and to other fields of research at the interface of chemistry and biology.

Testacosides A-D, glycoglycerolipids produced by Microbacterium testaceum isolated from Tedania brasiliensis.

Marine bacteria living in association with marine sponges have proven to be a reliable source of biologically active secondary metabolites. However, no studies have yet reported natural products from Microbacterium testaceum spp. We herein report the isolation of a M. testaceum strain from the sponge Tedania brasiliensis. Molecular networking analysis of bioactive pre-fractionated extracts from culture media of M. testaceum enabled the discovery of testacosides A-D. Analysis of spectroscopic data and chemical derivatizations allowed the identification of testacosides A-D as glycoglycerolipids bearing a 1-[α-glucopyranosyl-(1 → 3)-(α-mannopyranosyl)]-glycerol moiety connected to 12-methyltetradecanoic acid for testacoside A (1), 14-methylpentadecanoic acid for testacoside B (2), and 14-methylhexadecanoic acid for testacosides C (3) and D (4). The absolute configuration of the monosaccharide residues was determined by 1H-NMR analysis of the respective diastereomeric thiazolidine derivatives. This is the first report of natural products isolated from cultures of M. testaceum. KEY POINTS: • The first report of metabolites produced by Microbacterium testaceum. • 1-[α-Glucopyranosyl-(1 → 3)-(α-mannopyranosyl)]-glycerol lipids isolated and identified. • Microbacterium testaceum strain isolated from the sponge Tedania brasiliensis.

Metabolomics approach to understand molecular mechanisms involved in fungal pathogen-citrus pathosystems.

Citrus is a crucial crop with a significant economic impact globally. However, postharvest decay caused by fungal pathogens poses a considerable threat, leading to substantial financial losses. Penicillium digitatum, Penicillium italicum, Geotrichum citri-aurantii and Phyllosticta citricarpa are the main fungal pathogens, causing green mold, blue mold, sour rot and citrus black spot diseases, respectively. The use of chemical fungicides as a control strategy in citrus raises concerns about food and environmental safety. Therefore, understanding the molecular basis of host-pathogen interactions is essential to find safer alternatives. This review highlights the potential of the metabolomics approach in the search for bioactive compounds involved in the pathogen-citrus interaction, and how the integration of metabolomics and genomics contributes to the understanding of secondary metabolites associated with fungal virulence and the fungal infection mechanisms. Our goal is to provide a pipeline combining metabolomics and genomics that can effectively guide researchers to perform studies aiming to contribute to the understanding of the fundamental chemical and biochemical aspects of pathogen-host interactions, in order to effectively develop new alternatives for fungal diseases in citrus cultivation. We intend to inspire the scientific community to question unexplored biological systems, and to employ diverse analytical approaches and metabolomics techniques to address outstanding questions about the non-studied pathosystems from a chemical biology perspective.

Stilbenes, phenanthrenes and antiproliferative activity of Cattleya intermedia.

The phytochemical study of Cattleya intermedia (Orchidaceae) led to the isolation of two new stilbenoids and one new 9,10-dihydrophenanthrene, 4',5-dihydroxy-2',3-dimethoxy-dihydrostilbene (1), 3,6'-dihydroxy-4'-methoxy-dihydrostilbene (2) and 1,2,6-trihydroxy-3,8-dimethoxy-9,10-dihydrophenanthrene (3), named cattleymediol, cattleyol and phenanmediol, respectively, in addition to other five known compounds (4-8). The structural elucidations of the isolated compounds were carried out through the analyses of the one-dimensional 1H,1³C and NOE NMR spectra, and the 2D HSQC, HMBC, COSY and NOESY spectra, besides high-resolution mass spectrometry. In addition to this, the crude extract and its main fractions were analysed by ultra-high performance liquid chromatography coupled to high resolution mass spectrometry (UHPLC-QTOF-MS/MS), leading to the putative identification of several other compounds, including flavonoids and organic acids derivatives. Finally, the main fractions of the crude extract, and the pure compounds cattleymediol (1) and lusiantridine (7), had their antiproliferative activities evaluated against human cancerous HeLa and non-cancerous VERO cells.

Improvement of Targeted Fungi Secondary Metabolite Production Using a Systematic Experimental Design and Chemometrics Analysis.

Fungi are well-known producers of chemically diverse and biologically active secondary metabolites. However, their production yields through fermentation may hamper structural analysis and biological activity downstream investigations. Herein, a systematic experimental design that varies multiple cultivation parameters, followed by chemometrics analysis on HPLC-UV-MS or UHPLC-HRMS/MS data, is presented to enhance the production yield of fungal natural products. The overall procedure typically requires 3-4 months of work when first developed, and up to 3 months as a routine procedure.

Polyketide- and Terpenoid-Derived Metabolites Produced by a Marine-Derived Fungus, Peroneutypa sp.

Bioassay-guided investigation of the EtOAc-soluble extract of a culture of the marine-derived fungus Peroneutypa sp. M16 led to the isolation of seven new polyketide- and terpenoid-derived metabolites (1, 2, 4-8), along with known polyketides (3, 9-13). Structures of compounds 1, 2, and 4-8 were established by analysis of spectroscopic data. Absolute configurations of compounds 1, 2, 4, 6, 7, and 8 were determined by the comparison of experimental ECD spectra with calculated CD data. Compound 5 exhibited moderate antiplasmodial activity against both chloroquine-sensitive and -resistant strains of Plasmodium falciparum.

Metabolomics Reveals a 26-Membered Macrolactone Produced by Endophytic Colletotrichum spp. from Alcatrazes Island, Brazil.

Comparative metabolomics analysis of nonphytotoxic endophytic Colletotrichum spp. isolated from Anthurium alcatrazense endemic to Alcatrazes island (Brazil) and phytopathogenic Colletotrichum spp. isolated from the mainland of Brazil revealed significant differences in chemical composition. Examination of endophytic Colletotrichum spp. from Alcatrazes island led to the discovery of a 26-member macrolactone, colletotrichumolide (1), containing a phosphatidyl choline side chain. Further examination of the phytopathogenic strains from the mainland identified a family of phytopathogenic metabolites not present in the nonpathogenic island-derived strains, suggesting that geographical isolation could influence the secondary metabolism of fungal strains.

Comparative study of composition of methanolic extracts of the paratoid gland secretions (PGS) of Rhinella jimi (cururu toad) from northeastern Brazil: Gender, seasonality and geographic occurrence.

Toads belonging to the Bufonidae family have a pair of paratoid glands that store highly toxic a biological secretion with varied chemical composition, that act as a chemical defense against microbial infections and predators. The paratoid gland secretion (PGS) of bufonids is rich in bioactive steroids, alkaloids, proteins, bufadienolides and bufotoxins. In the present investigation we performed a systematic analysis of the chemical profile of PGS obtained from the Bufonidae toad Rhinella jimi ("Cururu" toad) collected at three different regions of Piauí state, Northeastern Brazil. Our aim was to investigate the PGS variation related to the season of animals collection, geographic distribution and gender of the animals. The methanolic extracts of PGS were analyzed by UPLC-QToF-MS/MS. Principal component analysis (PCA) were applied to the data set obtained by the UPLC-QToF-MS/MS analyses. Among 23 compounds identified, dehydrobufotenine, suberoyl arginine, 3-(N-suberoyl-argininyl) telocinobufagin, 3-(N-suberoyl-argininyl) marinobufagin, telocinobufagin, marinobufagin and 3-(N-suberoyl-argininyl) bufalin were detected in all PGS. Minimal variations in the composition of paratoid secretions of R. jimi were observed related to distinct geographical and seasonal parameters. R. jimi female animals presented the most diverse chemical composition in its PGS. With this comparative study, unprecedented for the species, it was possible to observe that the secretions of the paratoid glands produced by R. jimi from different regions of the state of Piauí, at different times of the year, presented consistent chemical composition, with discrete particularities in the number and nature chemistry of its constituents.

Isolation of Water-Soluble Metabolites from Marine Invertebrates and Microorganisms.

The isolation of pure organic compounds from biological sources, reaction media, or other complex molecular matrixes is the first step to overcome before further biological and chemical investigations. While the isolation of chemicals soluble in organic solvents is commonly accomplished, the isolation of water-soluble organic compounds is less often addressed. We present here a simple method for the isolation of water-soluble organic compounds, using adsorptive macroporous resins and reversed-phase chromatography.

Feature-Based Molecular Networking Discovery of Bromopyrrole Alkaloids from the Marine Sponge Agelas dispar.

Investigation of the marine sponge Agelas dispar MeOH fractions using feature-based molecular networking, dereplication, and isolation led to the discovery of new bromopyrrole-derived metabolites. An in-house library of bromopyrrole alkaloids previously isolated from A. dispar and Dictyonella sp. was utilized, along with the investigation of an MS/MS fragmentation of these compounds. Our strategy led to the isolation and identification of the disparamides A-C (1-3), with a novel carbon skeleton. Additionally, new dispyrins B-F (4-8) and nagelamides H2 and H3 (9 and 10) and known nagelamide H (11), citrinamine B (12), ageliferin (13), bromoageliferin (14), and dibromoageliferin (15) were also isolated and identified by analysis of spectroscopic data. Analysis of MS/MS fragmentation data and molecular networking analysis indicated the presence of hymenidin (16), oroidin (17), dispacamide (18), monobromodispacamide (19), keramadine (20), longamide B (21), methyl ester of longamide B (22), hanishin (23), methyl ester of 3-debromolongamide B (24), and 3-debromohanishin (25). Antibacterial activity of ageliferin (13), bromoageliferin (14), and dibromoageliferin (15) was evaluated against susceptible and multi-drug-resistant ESKAPE pathogenic bacteria Klabsiella pneumoniae, Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii, and Enterococcus faecalis. Dibromoageliferin (15) displayed the most potent antimicrobial activity against all tested susceptible and MDR strains. Compounds 13-15 presented no significant hemolytic activity up to 100 µM.

Absolute configuration of cytotoxic anthraquinones from a Brazilian cave soil-derived fungus, Aspergillus sp. SDC28.

Microbial strains isolated from extreme and understudied environments, such as caves, are still poorly investigated for the production of bioactive secondary metabolites. Investigation of the ethyl acetate extract from the growth medium produced by the soil-derived fungus Aspergillus sp. SDC28, isolated from a Brazilian cave, yielded two anthraquinones: versicolorin C (1) and versiconol (2). The complete assignment of nuclear magnetic resonance and mass spectroscopic data of 1 and 2 was performed for the first time. Moreover, the yet unreported absolute configuration of both compounds was unambiguously established by analysis of experimental and theoretical electronic circular dichroism data. Vibrational circular dichroism was also applied to confirm the absolute stereochemistry of 2. Compounds 1 and 2 showed cytotoxic activity against human ovarian cancer cells (OVCAR3).

The isolation of water-soluble natural products - challenges, strategies and perspectives.

Covering period: up to 2019Water-soluble natural products constitute a relevant group of secondary metabolites notably known for presenting potent biological activities. Examples are aminoglycosides, ß-lactam antibiotics, saponins of both terrestrial and marine origin, and marine toxins. Although extensively investigated in the past, particularly during the golden age of antibiotics, hydrophilic fractions have been less scrutinized during the last few decades. This review addresses the possible reasons on why water-soluble metabolites are now under investigated and describes approaches and strategies for the isolation of these natural compounds. It presents examples of several classes of hydrosoluble natural products and how they have been isolated. Novel stationary phases and chromatography techniques are also reviewed, providing a perspective towards a renaissance in the investigation of water-soluble natural products.

Post-synthetic functionalization of tryptophan protected peptide sequences through indole (C-2) photocatalytic alkylation.

We report a selective, mild, and efficient C-H functionalization of tryptophan and tryptophan-containing peptides with activated α-bromo-carbonyl compounds under visible-light irradiation. The protocol efficiency is outlined by the wide substrate scope and excellent tolerance of sensitive functional groups present in the amino acid side chains. The method can be successfully extended to access pharmaco-peptide conjugate scaffolds.

Phytotoxic Tryptoquialanines Produced In Vivo by Penicillium digitatum Are Exported in Extracellular Vesicles.

Penicillium digitatum is the most aggressive pathogen of citrus fruits. Tryptoquialanines are major indole alkaloids produced by P. digitatum It is unknown if tryptoquialanines are involved in the damage of citrus fruits caused by P. digitatum. To investigate the pathogenic roles of tryptoquialanines, we initially asked if tryptoquialanines could affect the germination of Citrus sinensis seeds. Exposure of the citrus seeds to tryptoquialanine A resulted in a complete inhibition of germination and an altered metabolic response. Since this phytotoxic effect requires the extracellular export of tryptoquialanine A, we investigated the mechanisms of extracellular delivery of this alkaloid in P. digitatum We detected extracellular vesicles (EVs) released by P. digitatum both in culture and during infection of citrus fruits. Compositional analysis of EVs produced during infection revealed the presence of a complex cargo, which included tryptoquialanines and the mycotoxin fungisporin. The EVs also presented phytotoxicity activity in vitro and caused damage to the tissues of citrus seeds. Through molecular networking, it was observed that the metabolites present in the P. digitatum EVs are produced in all of its possible hosts. Our results reveal a novel phytopathogenic role of P. digitatum EVs and tryptoquialanine A, implying that this alkaloid is exported in EVs during plant infection.IMPORTANCE During the postharvest period, citrus fruits can be affected by phytopathogens such as Penicillium digitatum, which causes green mold disease and is responsible for up to 90% of total citrus losses. Chemical fungicides are widely used to prevent green mold disease, leading to concerns about environmental and health risks. To develop safer alternatives to control phytopathogens, it is necessary to understand the molecular basis of infection during the host-pathogen interaction. In the P. digitatum model, the virulence strategies are poorly known. Here, we describe the production of phytotoxic extracellular vesicles (EVs) by P. digitatum during the infection of citrus fruits. We also characterized the secondary metabolites in the cargo of EVs and found in this set of molecules an inhibitor of seed germination. Since EVs and secondary metabolites have been related to virulence mechanisms in other host-pathogen interactions, our data are important for the comprehension of how P. digitatum causes damage to its primary hosts.

The bacterial and fungal communities associated with Anthurium ssp. leaves: Insights into plant endemism and microbe association.

It is known that different plant species select specific microbes to live inside their tissues in a process determined by the host genotype, phenotype and geographic location, which can introduce discussion on plant endemism and the assembly of specific microbial communities. Herein, we report the results of an investigation relating the geographic distribution of plant species and the composition of microbial communities associated with plant hosts. The bacterial and fungal community associated with Anthurium plant leaves was mapped to assess the diversity and ecology of the endophytic community associated with Anthurium spp. collected on islands and on the Brazilian mainland. Twenty-six Anthurium specimens were surveyed, distributed throughout the São Paulo state coastline, including Alcatrazes Island, some coastal islands and distinct mainland environments. Bacterial and fungal endophytes were obtained from the leaves of A. alcatrazense, A. loefgrenii, A. penthaphyllum, A. urvellianum and A. intermedium and subjected to massive bacterial 16S rRNA and fungal ITS sequencing. The results indicated that A. alcatrazense, endemic to Alcatrazes Island, hosted a specific bacterial community structure, while its fungal community was similar to that of Anthurium species from other locations. Betaproteobacteria showed a high differential occurrence in A. alcatrazense. Some groups of fungi were found mainly inhabiting A. loefgrenii plants. While Alphaproteobacteria, Gammaproteobacteria, Actinobacteria and Sordariomycetes, Dothiodeomycetes and Tremellomycetes composed the core microbial community among Anthurium plants. The results suggest crucial role for the bacterial communities to endemic plants, while endophytic fungal diversity is less specifically distributed among endemic and nonendemic plant species.

Metabolomics Reveals Minor Tambjamines in a Marine Invertebrate Food Chain.

Metabolomics analysis detected tambjamine alkaloids in aqueous and EtOAc extracts of the marine invertebrates Virididentula dentata, Tambja stegosauriformis, Tambja brasiliensis, and Roboastra ernsti. Among several tambjamines, the new amino acid derivatives tambjamines M-O (17-19) were identified by Marfey's advanced analysis, UPLC-MS/MS analyses, and total synthesis. The tambjamine diversity increased from the bryozoan V. dentata to its nudibranch predators T. stegosauriformis and T. brasiliensis and attained a higher diversity in R. ernsti, the nudibranch that preys upon T. stegosauriformis and T. brasiliensis. The total tambjamine content also increases among the trophic levels, probably due to biomagnification. Tambjamines A (1), C (3), and D (4) are the major metabolites in the tissues of V. dentata, T. stegosauriformis, T. brasiliensis, and R. ernsti and are likely the main chemical defenses of these marine invertebrates.

The chemistry and biology of guanidine secondary metabolites.

Covering: 2017-2019Guanidine natural products isolated from microorganisms, marine invertebrates and terrestrial plants, amphibians and spiders, represented by non-ribosomal peptides, guanidine-bearing polyketides, alkaloids, terpenoids and shikimic acid derived, are the subject of this review. The topics include the discovery of new metabolites, total synthesis of natural guanidine compounds, biological activity and mechanism-of-action, biosynthesis and ecological functions.