Access to Anti-Biofilm Compounds from Endolichenic Fungi Using a Bioguided Networking Screening.

Endolichenic microorganisms represent a new source of bioactive natural compounds. Lichens, resulting from a symbiotic association between algae or cyanobacteria and fungi, constitute an original ecological niche for these microorganisms. Endolichenic fungi inhabiting inside the lichen thallus have been isolated and characterized. By cultivation on three different culture media, endolichenic fungi gave rise to a wide diversity of bioactive metabolites. A total of 38 extracts were screened for their anti-maturation effect on Candida albicans biofilms. The 10 most active ones, inducing at least 50% inhibition, were tested against 24 h preformed biofilms of C. albicans, using a reference strain and clinical isolates. The global molecular network was associated to bioactivity data in order to identify and priorize active natural product families. The MS-targeted isolation led to the identification of new oxygenated fatty acid in Preussia persica endowed with an interesting anti-biofilm activity against C. albicans yeasts.

Detection of ergosterol using liquid chromatography/electrospray ionization mass spectrometry: Investigation of unusual in-source reactions.

RATIONALE: In the field of natural products, de-replication of complex mixtures has become a usual practice to annotate known compounds and avoid their re-isolation. For this purpose, many groups rely on liquid chromatography coupled to high-resolution mass spectrometry (HPLC/MS) to deduce molecular formulae of compounds allowing comparison with public or in-house databases. Electrospray ionization (ESI) is usually considered as the method of choice for investigating a large panel of compounds but, in some cases, it may lead to unusual results as described in this article for ergosterol. METHODS: Ergosterol and other fungal sterols in methanolic solution were analysed using various chromatographic gradients with HPLC/MS using both ion trap time-of-flight MS and Orbitrap MS instruments fitted with an ESI source. Further flow injection analyses were performed to investigate the influence of the solvent composition. MS/MS fragmentation data were acquired to annotate the various ions observed. RESULTS: Contrary to other fungal sterols, ergosterol was found to be highly sensitive to oxidation during ESI. Putative structures were proposed based on MS/MS studies and known oxidation mechanisms of ergosterol by reactive oxygen species that could be formed in the ESI process. The proportion of acetonitrile in the eluent was found to influence this in-source oxidation, with an increased proportion of oxidized sodium adducts with higher proportions of acetonitrile. CONCLUSIONS: While ergosterol is a major sterol found in fungi, this study investigates its ionization by electrospray for the first time. The results reported here will help further detection and annotation of this compound in fungal extracts after HPLC/ESI-MS analyses.

4-cholesten-3-one decreases breast cancer cell viability and alters membrane raft-localized EGFR expression by reducing lipogenesis and enhancing LXR-dependent cholesterol transporters.

BACKGROUND: The alteration of lipid metabolism in cancer cells is recognized as one of the most important metabolic hallmarks of cancer. Membrane rafts defined as plasma membrane microdomains enriched in cholesterol and sphingolipids serve as platforms for signaling regulation in cancer. The main purpose of this study was to evaluate the effect of the cholesterol metabolite, 4-cholesten-3-one, on lipid metabolism and membrane raft integrity in two breast cancer cell lines, MCF-7 and MDA-MB-231. Its ability to reduce cell viability and migration has also been investigated. METHODS: RT-qPCR was performed to evaluate the expression of enzymes involved in lipogenesis and cholesterol synthesis, and ABCG1 and ABCA1 transporters involved in cholesterol efflux. Its effect on cell viability and migration was studied using the MTT assay, the wound healing assay and the Transwell migration assay, respectively. The effect of 4-cholesten-3-one on membrane rafts integrity was investigated by studying the protein expression of flotillin-2, a membrane raft marker, and raft-enriched EGFR by western blot. RESULTS: Interestingly, we found that 4-cholesten-3-one treatment decreased mRNA expression of different enzymes including ACC1, FASN, SCD1 and HMGCR. We further demonstrated that 4-cholesten-3-one increased the expression of ABCG1 and ABCA1. We also found that 4-cholesten-3-one decreased the viability of MCF-7 and MDA-MB-231 cells. This effect was neutralized after treatment with LXR inverse agonist or after LXRß knockdown by siRNA. As a result, we also demonstrated that 4-cholesten-3-one disrupts membrane rafts and cell migration capacity. CONCLUSION: Our results show that 4-cholesten-3-one exerts promising antitumor activity by altering LXR-dependent lipid metabolism in breast cancer cells without increasing lipogenesis.

Steroids from Marine-Derived Fungi: Evaluation of Antiproliferative and Antimicrobial Activities of Eburicol.

The most common sterol in fungi is ergosterol, which has frequently been investigated in human pathogenic fungal strains. This sterol, and others isolated from fungal strains, has also demonstrated cytotoxicity against cancer cell lines and antimicrobial activities. Marine fungi can produce high amounts of bioactive compounds. So, a screening was performed to study sterol composition using GC/MS in 19 marine fungal strains and ergosterol was always the major one. One strain, Clonostachys rosea MMS1090, was selected due to its high amount of eburicol and a one strain many compounds approach was performed on seven culture media to optimize its production. After purification and structural identification by NMR, eburicol was assessed against four cancer cell lines, MCF-7, MDA-MB-231, NSCLC-N6-L16 and A549, and seven human pathogenic bacteria Staphylococcus aureus, Bacillus sp., Bacillus cereus, Listeria ivanovii, Escherichia coli, Citrobacter freundii and Salmonella spp. The most significant activity was cytotoxicity against MCF-7 cells (2 µM). This is the first report of such an accumulation of eburicol in the marine fungal strain C. rosea confirming its potential in the production of bioactive lipids.

Targeting bioactive compounds in natural extracts - Development of a comprehensive workflow combining chemical and biological data.

In natural product drug discovery, several strategies have emerged to highlight specifically bioactive compound(s) within complex mixtures (fractions or crude extracts) using metabolomics tools. In this area, a great deal of interest has raised among the scientific community on strategies to link chemical profiles and associated biological data, leading to the new field called "biochemometrics". This article falls into this emerging research by proposing a complete workflow, which was divided into three major steps. The first one consists in the fractionation of the same extract using four different chromatographic stationary phases and appropriated elution conditions to obtain five fractions for each column. The second step corresponds to the acquisition of chemical profiles using HPLC-HRMS analysis, and the biological evaluation of each fraction. The last step evaluates the links between the relative abundances of molecules present in fractions (peak area) and the global bioactivity level observed for each fraction. To this purpose, an original bioinformatics script (encoded with R Studio software) using the combination of four statistical models (Spearman, F-PCA, PLS, PLS-DA) was here developed leading to the generation of a "Super list" of potential bioactive compounds together with a predictive score. This strategy was validated by its application on a marine-derived Penicillium chrysogenum extract exhibiting antiproliferative activity on breast cancer cells (MCF-7 cells). After the three steps of the workflow, one main compound was highlighted as responsible for the bioactivity and identified as ergosterol. Its antiproliferative activity was confirmed with an IC50 of 0.10 µM on MCF-7 cells. The script efficiency was further demonstrated by comparing the results obtained with a different recently described approach based on NMR profiling and by virtually modifying the data to evaluate the computational tool behaviour. This approach represents a new and efficient tool to tackle some of the bottlenecks in natural product drug discovery programs.

UHPLC-MS-based HDAC Assay Applied to Bio-guided Microfractionation of Fungal Extracts.

INTRODUCTION: Histone deacetylases (HDAC) are considered as promising targets for cancer treatment. Today, four HDAC inhibitors, vorinostat, romidepsin, belinostat, and panobinostat, have been approved by the Food and Drug Administration (FDA) for cancer treatment, while others are in clinical trials. Among them, several are naturally occurring fungal metabolites. OBJECTIVE: To develop and optimise an enzyme assay for bio-guided identification of HDAC inhibitors in fungal strains. METHODS: Fluorescence and MS-based HDAC enzymatic assays were compared during the bio-guided fractionation of Penicillium griseofulvum. The MS-based approach was then optimised to evaluate HDAC selectivity using the human recombinant class I isoform HDAC1 and the class II isoform HDAC6. RESULTS: Fluorescence-based assays have several drawbacks when used for bio-guided fractionation because of the native fluorescence and the trypsin inhibitory ability of compounds present in many extracts. The MS-based method led to the isolation of gliocladride C, which is selective for HDAC1 and salirepol, which showed an HDAC6 selectivity. Their activity and presence in P. griseofulvum is described here for the first time. CONCLUSION: The UHPLC-ESI-MS/MS-based method using specific HDAC isoforms is suitable to isolate selective HDAC inhibitors by bio-guided fractionation of fungal strains. Also, it decreases potential interferences with natural products compared to the fluorescence-based assay.