Discovery of bioactive natural products of microbial origin as inhibitors of the PD-1/PD-L1 protein-protein interaction.

The PD-1/PD-L1 protein-protein interaction (PPI) controls an adaptive immune resistance mechanism exerted by tumor cells to evade immune responses. The large-molecule nature of current commercial monoclonal antibodies against this PPI hampers their effectiveness by limiting tumor penetration and inducing severe immune-related side effects. Synthetic small-molecule inhibitors may overcome such limitations and have demonstrated promising clinical translation, but their design is challenging. Microbial natural products (NPs) are a source of small molecules with vast chemical diversity that have proved anti-tumoral activities, but which immunotherapeutic properties as PD-1/PD-L1 inhibitors had remained uncharacterized so far. Here, we have developed the first cell-based PD-1/PD-L1 blockade reporter assay to screen NPs libraries. In this study, 6000 microbial extracts of maximum biosynthetic diversity were screened. A secondary metabolite called alpha-cyclopiazonic acid (α-CPA) of a bioactive fungal extract was confirmed as a new PD-1/PD-L1 inhibitor with low micromolar range in the cellular assay and in an additional cell-free competitive assay. Thermal denaturation experiments with PD-1 confirmed that the mechanism of inhibition is based on its stabilization upon binding to α-CPA. The identification of α-CPA as a novel PD-1 stabilizer proves the unprecedented resolution of this methodology at capturing specific PD-1/PD-L1 PPI inhibitors from chemically diverse NP libraries.

Naphthoquinone Derivatives from Angustimassarina populi CF-097565 Display Anti-Tumour Activity in 3D Cultures of Breast Cancer Cells.

Cancer is one of the leading causes of death worldwide, with breast cancer being the second cause of cancer-related mortality among women. Natural Products (NPs) are one of the main sources for drug discovery. During a screening campaign focused on the identification of extracts from Fundación MEDINA's library inhibiting the proliferation of cancer cell lines, a significant bioactivity was observed in extracts from cultures of the fungus Angustimassarina populi CF-097565. Bioassay-guided fractionation of this extract led to the identification and isolation of herbarin (1), 1-hydroxydehydroherbarin (4) plus other three naphthoquinone derivatives of which 3 and 5 are new natural products and 2 is herein described from a natural source for the first time. Four of these compounds (1, 3, 4 and 5) confirmed a specific cytotoxic effect against the human breast cancer cell line MCF-7. To evaluate the therapeutic potential of the compounds isolated, their efficacy was validated in 3D cultures, a cancer model of higher functionality. Additionally, an in-depth study was carried out to test the effect of the compounds in terms of cell mortality, sphere disaggregation, shrinkage, and morphology. The cell profile of the compounds was also compared to that of known cytotoxic compounds with the aim to distinguish the drug mode of action (MoA). The profiles of 1, 3 and 4 showed more biosimilarity between them, different to 5, and even more different to other known cytotoxic agents, suggesting an alternative MoA responsible for their cytotoxicity in 3D cultures.

Interactions of Different Streptomyces Species and Myxococcus xanthus Affect Myxococcus Development and Induce the Production of DK-Xanthenes.

The co-culturing of microorganisms is a well-known strategy to study microbial interactions in the laboratory. This approach facilitates the identification of new signals and molecules produced by one species that affects other species' behavior. In this work, we have studied the effects of the interaction of nine Streptomyces species (S. albidoflavus, S. ambofaciens, S. argillaceus, S. griseus, S. lividans, S. olivaceus, S. parvulus, S. peucetius, and S. rochei) with the predator bacteria Myxococcus xanthus, five of which (S. albidoflavus, S. griseus, S. lividans, S. olivaceus, and S. argillaceus) induce mound formation of M. xanthus on complex media (Casitone Yeast extract (CYE) and Casitone tris (CTT); media on which M. xanthus does not form these aggregates under normal culture conditions. An in-depth study on S. griseus-M. xanthus interactions (the Streptomyces strain producing the strongest effect) has allowed the identification of two siderophores produced by S. griseus, demethylenenocardamine and nocardamine, responsible for this grouping effect over M. xanthus. Experiments using pure commercial nocardamine and different concentrations of FeSO4 show that iron depletion is responsible for the behavior of M. xanthus. Additionally, it was found that molecules, smaller than 3 kDa, produced by S. peucetius can induce the production of DK-xanthenes by M. xanthus.

Acoustic droplet ejection facilitates cell-based high-throughput screenings using natural products.

Natural Products (NPs) are one of the main sources for drug discovery. Many clinical drugs are NPs or NP-inspired compounds, and recently discovered New Chemical Entities (NCEs) of NPs are emerging as promising new drugs. High-Throughput Screening (HTS) of large sample sets or libraries has grown to be vital for the drug discovery field. Industrial-scale HTS of NP libraries can be limited due to the difficulties entailed in working with tiny extract volumes and the variability in viscosity of NP extracts. For these reasons, the implementation of new technologies to miniaturize different reagent volumes grows to be fundamental. Since Acoustic Droplet Ejection (ADE) emerged as a helpful tool in HTS campaigns for the transference of compound libraries. The aim of this work was to test the effectiveness of ADE for the dispensation of NP extract libraries in cell-based HTS assays.

Development and Validation of a HTS Platform for the Discovery of New Antifungal Agents against Four Relevant Fungal Phytopathogens.

Fungal phytopathogens are the major agents responsible for causing severe damage to and losses in agricultural crops worldwide. Botrytis cinerea, Colletotrichum acutatum, Fusarium proliferatum, and Magnaporthe grisea are included in the top ten fungal phytopathogens that impose important plant diseases on a broad range of crops. Microbial natural products can be an attractive alternative for the biological control of phytopathogens. The objective of this work was to develop and validate a High-throughput Screening (HTS) platform to evaluate the antifungal potential of chemicals and natural products against these four important plant pathogens. Several experiments were performed to establish the optimal assay conditions that provide the best reproducibility and robustness. For this purpose, we have evaluated two media formulations (SDB and RPMI-1640), several inoculum concentrations (1 × 106, 5 × 105 and 5 × 106 conidia/mL), the germination curves for each strain, each strain's tolerance to dimethyl sulfoxide (DMSO), and the Dose Response Curves (DRC) of the antifungal control (Amphotericin B). The assays were performed in 96-well plate format, where absorbance at 620 nm was measured before and after incubation to evaluate growth inhibition, and fluorescence intensity at 570 nm excitation and 615 nm emission was monitored after resazurin addition for cell viability evaluation. Quality control parameters (RZ' Factors and Signal to Background (S/B) ratios) were determined for each assay batch. The assay conditions were finally validated by titrating 40 known relevant antifungal agents and testing 2400 microbial natural product extracts from the MEDINA Library through both HTS agar-based and HTS microdilution-based set-ups on the four phytopathogens.

Delftia tsuruhatensis TC1 symbiont suppresses malaria transmission by anopheline mosquitoes.

Malaria control demands the development of a wide range of complementary strategies. We describe the properties of a naturally occurring, non-genetically modified symbiotic bacterium, Delftia tsuruhatensis TC1, which was isolated from mosquitoes incapable of sustaining the development of Plasmodium falciparum parasites. D. tsuruhatensis TC1 inhibits early stages of Plasmodium development and subsequent transmission by the Anopheles mosquito through secretion of a small-molecule inhibitor. We have identified this inhibitor to be the hydrophobic molecule harmane. We also found that, on mosquito contact, harmane penetrates the cuticle, inhibiting Plasmodium development. D. tsuruhatensis TC1 stably populates the mosquito gut, does not impose a fitness cost on the mosquito, and inhibits Plasmodium development for the mosquito's life. Contained field studies in Burkina Faso and modeling showed that D. tsuruhatensis TC1 has the potential to complement mosquito-targeted malaria transmission control.

Madurastatins with Imidazolidinone Rings: Natural Products or Side-Reaction Products from Extraction Solvents?

Madurastatins are a group of pentapeptides containing an oxazoline moiety, and, in a few cases, an imidazolidinone ring as an additional structural feature. In our search for new potential antiparasitic metabolites from natural sources, we studied the acetone extracts from a culture of Actinomadura sp. CA-135719. The LC/HRMS analysis of this extract identified the presence of the known madurastatins C1 (1), D1 (4), and D2 (5) together with additional members of the family that were identified as the new madurastatins H2 (2) and 33-epi-D1 (3) after isolation and spectroscopic analysis. The planar structures of the new compounds were established by HRMS, ESI-qTOF-MS/MS, and 1D and 2D NMR data, and their absolute configuration was proposed using Marfey's and bioinformatic analyses of the biosynthetic gene cluster (BGC). A revision of the absolute configuration of madurastatins D1 and D2 is proposed. Additionally, madurastatins containing imidazolidinone rings are proved to be artifacts originating during acetone extraction of the bacterial cultures.

Kribbellichelins A and B, Two New Antibiotics from Kribbella sp. CA-293567 with Activity against Several Human Pathogens.

Current needs in finding new antibiotics against emerging multidrug-resistant superbugs are pushing the scientific community into coming back to Nature for the discovery of novel active structures. Recently, a survey of halophilic actinomyectes from saline substrates of El Saladar del Margen, in the Cúllar-Baza depression (Granada, Spain), led us to the isolation and identification of 108 strains from the rhizosphere of the endemic plant Limonium majus. Evaluation of the potential of these strains to produce new anti-infective agents against superbug pathogens was performed through fermentation in 10 different culture media using an OSMAC approach and assessment of the antibacterial and antifungal properties of their acetone extracts. The study allowed the isolation of two novel antibiotic compounds, kribbellichelin A (1) and B (2), along with the known metabolites sandramycin (3), coproporphyrin III (4), and kribelloside C (5) from a bioassay-guided fractionation of scaled-up active extracts of the Kribbella sp. CA-293567 strain. The structures of the new molecules were elucidated by ESI-qTOF-MS/MS, 1D and 2D NMR, and Marfey's analysis for the determination of the absolute configuration of their amino acid residues. Compounds 1-3 and 5 were assayed against a panel of relevant antibiotic-resistant pathogenic strains and evaluated for cytotoxicity versus the human hepatoma cell line HepG2 (ATCC HB-8065). Kribbellichelins A (1) and B (2) showed antimicrobial activity versus Candida albicans ATCC-64124, weak potency against Acinetobacter baumannii MB-5973 and Pseudomonas aeruginosa MB-5919, and an atypical dose-dependent concentration profile against Aspergillus fumigatus ATCC-46645. Sandramycin (3) confirmed previously reported excellent growth inhibition activity against MRSA MB-5393 but also presented clear antifungal activity against C. albicans ATCC-64124 and A. fumigatus ATCC-46645 associated with lower cytotoxicity observed in HepG2, whereas Kribelloside C (5) displayed high antifungal activity only against A. fumigatus ATCC-46645. Herein, we describe the processes followed for the isolation, structure elucidation, and potency evaluation of these two new active compounds against a panel of human pathogens as well as, for the first time, the characterization of the antifungal activities of sandramycin (3).

Grapevine Xylem Sap Is a Potent Elicitor of Antibiotic Production in Streptomyces spp.

Streptomyces bacteria produce a wide number of antibiotics and antitumor compounds that have attracted the attention of pharmaceutical and biotech companies. In this study, we provide evidence showing that the xylem sap from grapevines has a positive effect on the production of different antibiotics by several Streptomyces species, including S. ambofaciens ATCC 23877 and S. argillaceus ATCC 12596 among others. The production of several already known compounds was induced: actinomycin D, chromomycin A3, fungichromin B, mithramycin A, etc., and four compounds with molecular formulas not included in the Dictionary of Natural Products (DNP v28.2) were also produced. The molecules present in the xylem sap that acts as elicitors were smaller than 3 kDa and soluble in water and insoluble in ether, ethyl acetate, or methanol. A combination of potassium citrate and di-D-fructose dianhydrides (related to levanbiose or inulobiose) seemed to be the main effectors identified from the active fraction. However, the level of induction obtained in the presence of these compounds mix was weaker and delayed with respect to the one got when using the whole xylem sap or the 3 kDa sap fraction, suggesting that another, not identified, elicitor must be also implied in this induction.

Comoclathrin, a novel potent skin-whitening agent produced by endophytic Comoclathris strains associated with Andalusia desert plants.

As part of our screening program for the discovery of molecules of microbial origin with skin-whitening activity, 142 diverse fungal endophytes from a wide variety of Andalusia arid plants were screened, applying the OSMAC approach. The fungal strains CF-090361 and CF-090766, isolated from xerophytic plants, were selected as the most promising, while phylogenetic analysis revealed that both strains could represent a new species within the genus Comoclathris. The effect of different fermentation conditions on the production of tyrosinase inhibitory activity was examined, in order to identify the optimum cultivation conditions. LCMS based metabolomics was applied to determine significant differences between the strains and fermentation conditions, and to identify potential bioactive secondary metabolites. Bioassay-guided purification of the main active components led to the isolation of three new compounds (1-3), along with the known compounds graphostrin B (4) and brevianamide M (5). Compound 1 (Comoclathrin) demonstrated the strongest anti-tyrosinase activity (IC50 0.16 µΜ), which was 90-times higher than kojic acid (IC50 14.07 µΜ) used as positive control. Additionally, comoclathrin showed no significant cytotoxicity against a panel of cancer cell lines (HepG2, A2058, A549, MCF-7 and MIA PaCa-2) and normal BJ fibroblasts. These properties render comoclathrin an excellent development candidate as whitening agent.

Activation and Identification of a Griseusin Cluster in Streptomyces sp. CA-256286 by Employing Transcriptional Regulators and Multi-Omics Methods.

Streptomyces are well-known producers of a range of different secondary metabolites, including antibiotics and other bioactive compounds. Recently, it has been demonstrated that "silent" biosynthetic gene clusters (BGCs) can be activated by heterologously expressing transcriptional regulators from other BGCs. Here, we have activated a silent BGC in Streptomyces sp. CA-256286 by overexpression of a set of SARP family transcriptional regulators. The structure of the produced compound was elucidated by NMR and found to be an N-acetyl cysteine adduct of the pyranonaphtoquinone polyketide 3'-O-α-d-forosaminyl-(+)-griseusin A. Employing a combination of multi-omics and metabolic engineering techniques, we identified the responsible BGC. These methods include genome mining, proteomics and transcriptomics analyses, in combination with CRISPR induced gene inactivations and expression of the BGC in a heterologous host strain. This work demonstrates an easy-to-implement workflow of how silent BGCs can be activated, followed by the identification and characterization of the produced compound, the responsible BGC, and hints of its biosynthetic pathway.

Metabolomic Analysis of The Chemical Diversity of South Africa Leaf Litter Fungal Species Using an Epigenetic Culture-Based Approach.

Microbial natural products are an invaluable resource for the biotechnological industry. Genome mining studies have highlighted the huge biosynthetic potential of fungi, which is underexploited by standard fermentation conditions. Epigenetic effectors and/or cultivation-based approaches have successfully been applied to activate cryptic biosynthetic pathways in order to produce the chemical diversity suggested in available fungal genomes. The addition of Suberoylanilide Hydroxamic Acid to fermentation processes was evaluated to assess its effect on the metabolomic diversity of a taxonomically diverse fungal population. Here, metabolomic methodologies were implemented to identify changes in secondary metabolite profiles to determine the best fermentation conditions. The results confirmed previously described effects of the epigenetic modifier on the metabolism of a population of 232 wide diverse South Africa fungal strains cultured in different fermentation media where the induction of differential metabolites was observed. Furthermore, one solid-state fermentation (BRFT medium), two classic successful liquid fermentation media (LSFM and YES) and two new liquid media formulations (MCKX and SMK-II) were compared to identify the most productive conditions for the different populations of taxonomic subgroups.

Biological Evaluation and In Silico Study of Benzoic Acid Derivatives from Bjerkandera adusta Targeting Proteostasis Network Modules.

Abstract: A main cellular functional module that becomes dysfunctional during aging is the proteostasis network. In the present study, we show that benzoic acid derivatives isolated from Bjerkandera adusta promote the activity of the two main protein degradation systems, namely the ubiquitin-proteasome (UPP) and especially the autophagy-lysosome pathway (ALP) in human foreskin fibroblasts. Our findings were further supported by in silico studies, where all compounds were found to be putative binders of both cathepsins B and L. Among them, compound 3 (3-chloro-4-methoxybenzoic acid) showed the most potent interaction with both enzymes, which justifies the strong activation of cathepsins B and L (467.3 ± 3.9%) on cell-based assays. Considering that the activity of both the UPP and ALP pathways decreases with aging, our results suggest that the hydroxybenzoic acid scaffold could be considered as a promising candidate for the development of novel modulators of the proteostasis network, and likely of anti-aging agents.

Design of High-Throughput Screening of Natural Extracts to Identify Molecules Bypassing Primary Coenzyme Q Deficiency in Saccharomyces cerevisiae.

Coenzyme Q10 (CoQ10) deficiency syndrome is a rare disease included in the family of mitochondrial diseases, which is a heterogeneous group of genetic disorders characterized by defective energy production. CoQ10 biosynthesis in humans requires at least 11 gene products acting in a multiprotein complex within mitochondria. The high-throughput screening (HTS) method based on the stabilization of the CoQ biosynthesis complex (Q-synthome) produced by the COQ8 gene overexpression is proven here to be a successful method for identifying new molecules from natural extracts that are able to bypass the CoQ6 deficiency in yeast mutant cells. The main features of the new approach are the combination of two yeast targets defective in genes with different functions on CoQ6 biosynthesis to secure the versatility of the molecule identified, the use of glycerol as a nonfermentable carbon source providing a wide growth window, and the stringent conditions required to mark an extract as positive. The application of this pilot approach to a representative subset of 1200 samples of the Library of Natural Products of Fundación MEDINA resulted in the finding of nine positive extracts. The fractionation of three of the nine extracts allowed the identification of five molecules; two of them are present in molecule databases of natural extracts and three are nondescribed molecules. The use of this screening method opens the possibility of discovering molecules with CoQ10-bypassing action useful as therapeutic agents to fight against mitochondrial diseases in human patients.

Extending the Metabolite Diversity of the Endophyte Dimorphosporicola tragani.

Fungi are one of the most prolific sources of microbial secondary metabolites. The production of new metabolites can be achieved using multiple fermentation conditions and by adding small-molecule effectors, including epigenetic modifiers. In the framework of our Natural Product screening programme targeting the discovery of new antimicrobial compounds, we applied multiple fermentation conditions and adsorptive polymeric resins on a large collection of fungal endophytes, to increase and stimulate their fungal secondary metabolite production. During this work the endophytic fungus Dimorphosporicola tragani CF-090383 showed antimicrobial activity only when grown in presence of adsorptive polymeric resins. In addition, seven epigenetic modifiers were added to fermentations of this endophytic fungus, in an attempt to activate its cryptic pathways as well as to analyse the metabolites produced under these conditions. D. tragani was seen to produce three different mycotoxin dendrodolides when the epigenetic modifiers 5-azacytidine and valproic acid were added to the fermentations, and these compounds were further characterized. However, the fungus produced the fatty acid synthesis inhibitor cerulenin, a molecule not previously described to be produced by this fungal species, only when cultivated in presence of the XAD-16 resin. We have found that the addition of XAD-16 resin resulted in four-fold higher titers in the production of cerulenin when compared to the best production conditions described in literature for the original fungal producer strain, Cephalosporium caerulens KF-140 (=Sarocladium oryzae), in a zeolite-based fermentation, used as an ammonium ion-trapping agent. The production of cerulenin by this strain of D. tragani, represents an alternative source for the improved production of cerulenin with better yields.

Bioactivities and Extract Dereplication of Actinomycetales Isolated From Marine Sponges.

In the beginning of the twenty-first century, humanity faces great challenges regarding diseases and health-related quality of life. A drastic rise in bacterial antibiotic resistance, in the number of cancer patients, in the obesity epidemics and in chronic diseases due to life expectation extension are some of these challenges. The discovery of novel therapeutics is fundamental and it may come from underexplored environments, like marine habitats, and microbial origin. Actinobacteria are well-known as treasure chests for the discovery of novel natural compounds. In this study, eighteen Actinomycetales isolated from marine sponges of three Erylus genera collected in Portuguese waters were tested for bioactivities with the main goal of isolating and characterizing the responsible bioactive metabolites. The screening comprehended antimicrobial, anti-fungal, anti-parasitic, anti-cancer and anti-obesity properties. Fermentations of the selected strains were prepared using ten different culturing media. Several bioactivities against the fungus Aspergillus fumigatus, the bacteria Staphylococcus aureus methicillin-resistant (MRSA) and the human liver cancer cell line HepG2 were obtained in small volume cultures. Screening in higher volumes showed consistent anti-fungal activity by strain Dermacoccus sp. #91-17 and Micrococcus luteus Berg02-26. Gordonia sp. Berg02-22.2 showed anti-parasitic (Trypanosoma cruzi) and anti-cancer activity against several cell lines (melanoma A2058, liver HepG2, colon HT29, breast MCF7 and pancreatic MiaPaca). For the anti-obesity assay, Microbacterium foliorum #91-29 and #91-40 induced lipid reduction on the larvae of zebrafish (Danio rerio). Dereplication of the extracts from several bacteria showed the existence of a variety of secondary metabolites, with some undiscovered molecules. This work showed that Actinomycetales are indeed good candidates for drug discovery.

Ultraviolet (IUV) and mass spectrometry (IMS) imaging for the deconvolution of microbial interactions.

BACKGROUND: Spatial localization of natural products or proteins during microbial interactions can help to identify new antimicrobials both as offensive or defensive agents. Visible spatial interactions have been used for decades to enhance Drug Discovery processes both in industry and academia. RESULTS: Herein we describe an automated micro-extraction methodology, that coupled with the previously described HPLC-Studio 2.0 software and the new development, the MASS-Studio 1.0 software, can combine multiple chemical analyses to generate ultraviolet (UV) and mass spectrometry (MS) images from traditional affordable analytical equipment. As a proof of concept, we applied this methodology on two microbial antagonisms observed among co-habitant endophytes isolated from endemic plants of arid areas of the south of Europe. CONCLUSIONS: The use of UV and MS images highlighted interacting naturals products and allowed clear identification of induced molecules of interest not produced by the strains when cultured individually.

Fungal endophytes from arid areas of Andalusia: high potential sources for antifungal and antitumoral agents.

Native plant communities from arid areas present distinctive characteristics to survive in extreme conditions. The large number of poorly studied endemic plants represents a unique potential source for the discovery of novel fungal symbionts as well as host-specific endophytes not yet described. The addition of adsorptive polymeric resins in fungal fermentations has been seen to promote the production of new secondary metabolites and is a tool used consistently to generate new compounds with potential biological activities. A total of 349 fungal strains isolated from 63 selected plant species from arid ecosystems located in the southeast of the Iberian Peninsula, were characterized morphologically as well as based on their ITS/28S ribosomal gene sequences. The fungal community isolated was distributed among 19 orders including Basidiomycetes and Ascomycetes, being Pleosporales the most abundant order. In total, 107 different genera were identified being Neocamarosporium the genus most frequently isolated from these plants, followed by Preussia and Alternaria. Strains were grown in four different media in presence and absence of selected resins to promote chemical diversity generation of new secondary metabolites. Fermentation extracts were evaluated, looking for new antifungal activities against plant and human fungal pathogens, as well as, cytotoxic activities against the human liver cancer cell line HepG2. From the 349 isolates tested, 126 (36%) exhibited significant bioactivities including 58 strains with exclusive antifungal properties and 33 strains with exclusive activity against the HepG2 hepatocellular carcinoma cell line. After LCMS analysis, 68 known bioactive secondary metabolites could be identified as produced by 96 strains, and 12 likely unknown compounds were found in a subset of 14 fungal endophytes. The chemical profiles of the differential expression of induced activities were compared. As proof of concept, ten active secondary metabolites only produced in the presence of resins were purified and identified. The structures of three of these compounds were new and herein are elucidated.

Co-culturing of Fungal Strains Against Botrytis cinerea as a Model for the Induction of Chemical Diversity and Therapeutic Agents.

New fungal SMs (SMs) have been successfully described to be produced by means of in vitro-simulated microbial community interactions. Co-culturing of fungi has proved to be an efficient way to induce cell-cell interactions that can promote the activation of cryptic pathways, frequently silent when the strains are grown in laboratory conditions. Filamentous fungi represent one of the most diverse microbial groups known to produce bioactive natural products. Triggering the production of novel antifungal compounds in fungi could respond to the current needs to fight health compromising pathogens and provide new therapeutic solutions. In this study, we have selected the fungus Botrytis cinerea as a model to establish microbial interactions with a large set of fungal strains related to ecosystems where they can coexist with this phytopathogen, and to generate a collection of extracts, obtained from their antagonic microbial interactions and potentially containing new bioactive compounds. The antifungal specificity of the extracts containing compounds induced after B. cinerea interaction was determined against two human fungal pathogens (Candida albicans and Aspergillus fumigatus) and three phytopathogens (Colletotrichum acutatum, Fusarium proliferatum, and Magnaporthe grisea). In addition, their cytotoxicity was also evaluated against the human hepatocellular carcinoma cell line (HepG2). We have identified by LC-MS the production of a wide variety of known compounds induced from these fungal interactions, as well as novel molecules that support the potential of this approach to generate new chemical diversity and possible new therapeutic agents.

Hormonemate Derivatives from Dothiora sp., an Endophytic Fungus.

A search for cytotoxic agents from cultures of the endophytic fungus Dothiora sp., isolated from the endemic plant Launaea arborescens, led to the isolation of six new compounds structurally related to hormonemate, with moderate cytotoxic activity against different cancer cell lines. By using a bioassay-guided fractionation approach, hormonemates A-D (1-4), hormonemate (5), and hormonemates E (6) and F (7) were obtained from the acetone extract of this fungus. Their structures were determined using a combination of HRMS, ESI-qTOF-MS/MS, 1D and 2D NMR experiments, and chemical degradation. The cytotoxic activities of these compounds were evaluated by microdilution colorimetric assays against human breast adenocarcinoma (MCF-7), human liver cancer cells (HepG2), and pancreatic cancer cells (MiaPaca_2). Most of the compounds displayed cytotoxic activity against this panel.