Pyrenosetins A–C, New Decalinoylspirotetramic Acid Derivatives Isolated by Bioactivity-Based Molecular Networking from the Seaweed-Derived Fungus Pyrenochaetopsis sp. FVE-001

Marine algae represent a prolific source of filamentous fungi for bioprospecting. In continuation of our search for new anticancer leads from fungi derived from the brown alga Fucus vesiculosus, an endophytic Pyrenochaetopsis sp. FVE-001 was selected for an in-depth chemical analysis. The crude fungal extract inhibited several cancer cell lines in vitro, and the highest anticancer activity was tracked to its CHCl3–soluble portion. A bioactivity-based molecular networking approach was applied to C18-SPE fractions of the CHCl3 subextract to predict the bioactivity scores of metabolites in the fractions and to aid targeted purification of anticancer metabolites. This approach led to a rapid isolation of three new decalinoylspirotetramic acid derivatives, pyrenosetins A–C (1–3) and the known decalin tetramic acid phomasetin (4). The structures of the compounds were elucidated by extensive NMR, HR-ESIMS, FT-IR spectroscopy, [α]D and Mosher’s ester method. Compounds 1 and 2 showed high anticancer activity against malignant melanoma cell line A-375 (IC50 values 2.8 and 6.3 μM, respectively), in line with the bioactivity predictions. This is the first study focusing on secondary metabolites of a marine-derived Pyrenochaetopsis sp. and the second investigation performed on the member of the genus Pyrenochaetopsis

Pyrenosetin D, a New Pentacyclic Decalinoyltetramic Acid Derivative from the Algicolous Fungus Pyrenochaetopsis sp. FVE-087

The fungal genus Pyrenochaetopsis is commonly found in soil, terrestrial, and marine environments, however, has received little attention as a source of bioactive secondary metabolites so far. In a recent work, we reported the isolation and characterization of three new anticancer decalinoyltetramic acid derivatives, pyrenosetins A-C, from the Baltic Fucus vesiculosus-derived endophytic fungus Pyrenochaetopsis sp. FVE-001. Herein we report a new pentacyclic decalinoylspirotetramic acid derivative, pyrenosetin D (1), along with two known decalin derivatives wakodecalines A (2) and B (3) from another endophytic strain Pyrenochaetopsis FVE-087 isolated from the same seaweed and showed anticancer activity in initial screenings. The chemical structures of the purified compounds were elucidated by comprehensive analysis of HR-ESIMS, FT-IR, [a]D, 1D and 2D NMR data coupled with DFT calculations of NMR parameters and optical rotation. Compounds 1-3 were evaluated for their anticancer and toxic potentials against the human malignant melanoma cell line (A-375) and the non-cancerous keratinocyte cell line (HaCaT). Pyrenosetin D (1) showed toxicity towards both A-375 and HaCaT cells with IC50 values of 77.5 and 39.3 μM, respectively, while 2 and 3 were inactive. This is the third chemical study performed on the fungal genus Pyrenochaetopsis and the first report of a pentacyclic decalin ring system from the fungal genus Pyrenochaetopsis

Application of Feature-Based Molecular Networking for Comparative Metabolomics and Targeted Isolation of Stereoisomers from Algicolous Fungi

Seaweed endophytic (algicolous) fungi are talented producers of bioactive natural products. We have previously isolated two strains of the endophytic fungus, Pyrenochaetopsis sp. FVE-001 and FVE-087, from the thalli of the brown alga Fucus vesiculosus. Initial chemical studies yielded four new decalinoylspirotetramic acid derivatives with antimelanoma activity, namely pyrenosetins A–C (1–3) from Pyrenochaetopsis sp. strain FVE-001, and pyrenosetin D (4) from strain FVE-087. In this study, we applied a comparative metabolomics study employing HRMS/MS based feature-based molecular networking (FB MN) on both Pyrenochaetopsis strains. A higher chemical capacity in production of decalin derivatives was observed in Pyrenochaetopsis sp. FVE-087. Notably, several decalins showed different retention times despite the same MS data and MS/MS fragmentation pattern with the previously isolated pyrenosetins, indicating they may be their stereoisomers. FB MN-based targeted isolation studies coupled with antimelanoma activity testing on the strain FVE-087 afforded two new stereoisomers, pyrenosetins E (5) and F (6). Extensive NMR spectroscopy including DFT computational studies, HR-ESIMS, and Mosher’s ester method were used in the structure elucidation of compounds 5 and 6. The 3′R,5′R stereochemistry determined for compound 6 was identical to that previously reported for pyrenosetin C (3), whose stereochemistry was revised as 3′S,5′R in this study. Pyrenosetin E (5) inhibited the growth of human malignant melanoma cells (A-375) with an IC50 value of 40.9 μM, while 6 was inactive. This study points out significant variations in the chemical repertoire of two closely related fungal strains and the versatility of FB MN in identification and targeted isolation of stereoisomers. It also confirms that the little-known fungal genus Pyrenochaetopsis is a prolific source of complex decalinoylspirotetramic acid derivatives

Atrasentan and renal events in patients with type 2 diabetes and chronic kidney disease (SONAR): a double-blind, randomised, placebo-controlled trial

Background: Short-term treatment for people with type 2 diabetes using a low dose of the selective endothelin A receptor antagonist atrasentan reduces albuminuria without causing significant sodium retention. We report the long-term effects of treatment with atrasentan on major renal outcomes. Methods: We did this double-blind, randomised, placebo-controlled trial at 689 sites in 41 countries. We enrolled adults aged 18–85 years with type 2 diabetes, estimated glomerular filtration rate (eGFR)25–75 mL/min per 1·73 m 2 of body surface area, and a urine albumin-to-creatinine ratio (UACR)of 300–5000 mg/g who had received maximum labelled or tolerated renin–angiotensin system inhibition for at least 4 weeks. Participants were given atrasentan 0·75 mg orally daily during an enrichment period before random group assignment. Those with a UACR decrease of at least 30% with no substantial fluid retention during the enrichment period (responders)were included in the double-blind treatment period. Responders were randomly assigned to receive either atrasentan 0·75 mg orally daily or placebo. All patients and investigators were masked to treatment assignment. The primary endpoint was a composite of doubling of serum creatinine (sustained for ≥30 days)or end-stage kidney disease (eGFR <15 mL/min per 1·73 m 2 sustained for ≥90 days, chronic dialysis for ≥90 days, kidney transplantation, or death from kidney failure)in the intention-to-treat population of all responders. Safety was assessed in all patients who received at least one dose of their assigned study treatment. The study is registered with ClinicalTrials.gov, number NCT01858532. Findings: Between May 17, 2013, and July 13, 2017, 11 087 patients were screened; 5117 entered the enrichment period, and 4711 completed the enrichment period. Of these, 2648 patients were responders and were randomly assigned to the atrasentan group (n=1325)or placebo group (n=1323). Median follow-up was 2·2 years (IQR 1·4–2·9). 79 (6·0%)of 1325 patients in the atrasentan group and 105 (7·9%)of 1323 in the placebo group had a primary composite renal endpoint event (hazard ratio [HR]0·65 [95% CI 0·49–0·88]; p=0·0047). Fluid retention and anaemia adverse events, which have been previously attributed to endothelin receptor antagonists, were more frequent in the atrasentan group than in the placebo group. Hospital admission for heart failure occurred in 47 (3·5%)of 1325 patients in the atrasentan group and 34 (2·6%)of 1323 patients in the placebo group (HR 1·33 [95% CI 0·85–2·07]; p=0·208). 58 (4·4%)patients in the atrasentan group and 52 (3·9%)in the placebo group died (HR 1·09 [95% CI 0·75–1·59]; p=0·65). Interpretation: Atrasentan reduced the risk of renal events in patients with diabetes and chronic kidney disease who were selected to optimise efficacy and safety. These data support a potential role for selective endothelin receptor antagonists in protecting renal function in patients with type 2 diabetes at high risk of developing end-stage kidney disease. Funding: AbbVie

Inducing the Chemodiversity of Fucus vesiculosus-Derived Fungi by OSMAC Strategy for Discovery of New Anticancer Leads

Algae-derived (algicolous) fungi has been regarded as an important source for discovery of novel bioactive natural products. As one of the most-widespread brown algae that occur in the shallow coastal regions of Baltic Sea, Fucus vesiculosus provides habitat for many invertebrates and vertebrates. However, the fungal community associated with F. vesiculosus has not been investigated for their chemical constituents or bioactivity potential. This Ph.D. project aimed to pursue a culture-based approach to isolate fungi from F. vesiculosus for discovery of new anticancer lead compounds. In order to induce the chemical space of fungi associated with Fucus vesiculosus, an OSMAC approach that included variations in media composition and culture regimes (liquid/solid) was applied. The crude extracts of ten fungal isolates showed anticancer bioactivities against at least one cancer cell line under one culture condition. MS/MS-based molecular networking (MN) combined with bioactivity mapping was applied to those crude extracts, allowing the identification and prioritization of two endophytic fungal strain, Pyrenochaetopsis sp. FVE-001 and FVE-087 with anticancer activity. Both strains were selected for large-scale fermentation followed by massive metabolomics analysis and chemical work-up. For Pyrenochaetopsis sp. FVE-001, a modified Kupchan partition method was applied to crude extract to separate the crude extract into three subextracts. Bioactivity was tracked to the chloroform subextract, which was fractionated on a C18 solid-phase extraction (SPE) cartridge. The anticancer activity was mapped onto molecular networks (the so-called bioactivity-based MN), and with the aid of additional bioinformatics application, the anticancer activity of the compounds in the network was predicted. The compound isolation was carried out in a targeted manner from the bioactive fractions to yield three new decalinoylspirotetramic acid derivatives, pyrenosetins A-C, and a known decalin derivative, phomasetin. As expected, pyrenosetins A and B showed strong inhibitory potential against malignant melanoma cell A-375 with IC50 values of 2.8 and 6.3 μM, respectively. The bioactivity-guided isolation of the second Pyrenochaetopsis sp. FVE-087 strain yielded one new decalinoyltetramic acid derivative, pyrenosetin D, and two known compounds wakodecalines A and B. Pyrenosetin D possesses an unusual pentacyclic ring system, which is rare in nature. Bioassay results showed that pyrenosetins D exhibited moderate anticancer bioactivity against A-375 with IC50 value of 77.5 μM, while wakodecalines A and B were inactive. The current study represents a successful application of OSMAC in inducing new compounds with anticancer activity in algicolous fungi. Further combination of MN and additional information layers such as bioactivity data has successfully led to rapid purification of new compounds pyrenosetins A-D from two Fucus vesiculosus-derived Pyrenochaetopsis sp. strains. This is the first study focusing on secondary metabolites of algal-derived Pyrenochaetopsis sp

Activation of Hepatic Stellate Cells is Inhibited by microRNA-378a-3p via Wnt10a

Background/Aims: Wnt/β-catenin pathway is involved in liver fibrosis and microRNAs (miRNAs) are considered as key regulators of the activation of hepatic stellate cells (HSCs). A recent study showed the protective role of miR-378a-3p against cardiac fibrosis. However, whether miR-378a-3p suppresses Wnt/β-catenin pathway in liver fibrosis is largely unknown. Methods: miR-378a-3p expression was detected in carbon tetrachloride-induced liver fibrosis and activated HSCs. Effects of miR-378a-3p overexpression on HSC activation and Wnt/β-catenin pathway were analyzed. Bioinformatic analysis was employed to identify the potential targets of miR-378a-3p. Serum miR-378a-3p expression was analyzed in patients with cirrhosis. Results: Reduced miR-378a-3p expression was observed in the fibrotic liver tissues and activated HSCs. Up-regulation of miR-378a-3p inhibited HSC activation including cell proliferation, α-smooth muscle actin (α-SMA) and collagen expression. Moreover, miR-378a-3p overexpression resulted in Wnt/β-catenin pathway inactivation. Luciferase reporter assays demonstrated that Wnt10a, a member of Wnt/β-catenin pathway, was confirmed to be a target of miR-378a-3p. By contrast, miR-378a-3p inhibitor contributed to HSC activation, with an increase in cell proliferation, α-SMA and collagen expression. But all these effects were blocked down by silencing of Wnt10a. Notably, sera from patients with cirrhosis contained lower levels of miR-378a-3p than sera from healthy controls. Receiver operating characteristic curve analysis suggested that serum miR-378a-3p differentiated liver cirrhosis patients from healthy controls, with an area under the curve of ROC curve of 0.916. Conclusion: miR-378a-3p suppresses HSC activation, at least in part, via targeting of Wnt10a, supporting its potential utility as a novel therapeutic target for liver fibrosis

Influence of OSMAC-Based Cultivation in Metabolome and Anticancer Activity of Fungi Associated with the Brown Alga Fucus vesiculosus

The fungi associated with marine algae are prolific sources of metabolites with high chemical diversity and bioactivity. In this study, we investigated culture-dependent fungal communities associated with the Baltic seaweed Fucus vesiculosus. Altogether, 55 epiphytic and endophytic fungi were isolated and identified. Twenty-six strains were selected for a small-scale One-Strain-Many-Compounds (OSMAC)-based fermentation in four media under solid and liquid culture regimes. In total, 208 fungal EtOAc extracts were tested for anticancer activity and general cytotoxicity. Ten most active strains (i.e., 80 extracts) were analyzed for their metabolome by molecular networking (MN), in-silico MS/MS fragmentation analysis (ISDB&ndash;UNPD), and manual dereplication. Thirty-six metabolites belonging to 25 chemical families were putatively annotated. The MN clearly distinguished the impact of culture conditions in chemical inventory and anticancer activity of the fungal extracts that was often associated with general toxicity. The bioactivity data were further mapped into MN to seek metabolites, exclusively expressed in the active extracts. This is the first report of cultivable fungi associated with the Baltic F. vesiculosus that combined an OSMAC and an integrated MN-based untargeted metabolomics approaches for efficient assessment and visualization of the impact of the culture conditions on chemical space and anticancer potential of the fungi