Dereplication Strategies for Targeted Isolation of New Antitrypanosomal Actinosporins A and B from a Marine Sponge Associated-Actinokineospora sp. EG49

High resolution Fourier transform mass spectrometry (HRFTMS) and nuclear magnetic resonance (NMR) spectroscopy were employed as complementary metabolomic tools to dereplicate the chemical profile of the new and antitrypanosomally active sponge-associated bacterium Actinokineospora sp. EG49 extract. Principal Component (PCA), hierarchical clustering (HCA), and orthogonal partial least square-discriminant analysis (OPLS-DA) were used to evaluate the HRFTMS and NMR data of crude extracts from four different fermentation approaches. Statistical analysis identified the best culture one-strain-many-compounds (OSMAC) condition and extraction procedure, which was used for the isolation of novel bioactive metabolites. As a result, two new O-glycosylated angucyclines, named actinosporins A (1) and B (2), were isolated from the broth culture of Actinokineospora sp. strain EG49, which was cultivated from the Red Sea sponge Spheciospongia vagabunda. The structures of actinosporins A and B were determined by 1D- and 2D-NMR techniques, as well as high resolution tandem mass spectrometry. Testing for antiparasitic properties showed that actinosporin A exhibited activity against Trypanosoma brucei brucei with an IC₅₀ value of 15 µM; however no activity was detected against Leishmania major and Plasmodium falciparum, therefore suggesting its selectivity against the parasite Trypanosoma brucei brucei; the causative agent of sleeping sickness.     

Production of Induced Secondary Metabolites by a Co-Culture of Sponge-Associated Actinomycetes,Actinokineospora sp. EG49 and Nocardiopsis sp. RV163

Two sponge-derived actinomycetes, Actinokineospora sp. EG49 and Nocardiopsis sp. RV163, were grown in co-culture and the presence of induced metabolites monitored by ¹H NMR. Ten known compounds, including angucycline, diketopiperazine and β-carboline derivatives 1–10, were isolated from the EtOAc extracts of Actinokineospora sp. EG49 and Nocardiopsis sp. RV163. Co-cultivation of Actinokineospora sp. EG49 and Nocardiopsis sp. RV163 induced the biosynthesis of three natural products that were not detected in the single culture of either microorganism, namely N-(2-hydroxyphenyl)-acetamide (11), 1,6-dihydroxyphenazine (12) and 5a,6,11a,12-tetrahydro-5a,11a-dimethyl[1,4]benzoxazino[3,2-b][1,4]benzoxazine (13a). When tested for biological activity against a range of bacteria and parasites, only the phenazine 12 was active against Bacillus sp. P25, Trypanosoma brucei and interestingly, against Actinokineospora sp. EG49. These findings highlight the co-cultivation approach as an effective strategy to access the bioactive secondary metabolites hidden in the genomes of marine actinomycetes.     

Bromopyrrole Alkaloids as Lead Compounds against Protozoan Parasites

In the present study, 13 bromopyrrole alkaloids, including the oroidin analogs hymenidin (2), dispacamide B (3) and dispacamide D (4), stevensine (5) and spongiacidin B (6), their derivatives lacking the imidazole ring bromoaldisin (7), longamide B (8) and longamide A (9), the dimeric oroidin derivatives sceptrin (10) and dibromopalau’amine (11), and the non-oroidin bromopyrrolohomoarginin (12), manzacidin A (13), and agelongine (14), obtained from marine sponges belonging to Axinella and Agelas genera have been screened in vitro against four parasitic protozoa, i.e., two Trypanosoma species (T. brucei rhodesiense and T. cruzi), Leishmania donovani and Plasmodium falciparum (K1 strain, a chloroquine resistant strain), responsible of human diseases with high morbidity and, in the case of malaria, high mortality. Our results indicate longamide B (8) and dibromopalau’amine (11) to be promising trypanocidal and antileishmanial agents, while dispacamide B (3) and spongiacidin B (6) emerge as antimalarial lead compounds. In addition, evaluation of the activity of the test alkaloids (2–14) against three different enzymes (PfFabI, PfFabG, PfFabZ) involved in the de novo fatty acid biosynthesis pathway of P. falciparum (PfFAS-II) identified bromopyrrolohomoarginin (12) as a potent inhibitor of PfFabZ. The structural similarity within the series of tested molecules allowed us to draw some preliminary structure-activity relationships. Tests against the mammalian L6 cells revealed important clues on therapeutic index of the metabolites. This is the first detailed study on the antiprotozoal potential of marine bromopyrrole alkaloids.     

Activation of the Silent Secondary Metabolite Production by Introducing Neomycin-Resistance in a Marine-Derived Penicillium purpurogenum G59

Introduction of neomycin-resistance into a marine-derived, wild-type Penicillium purpurogenum G59 resulted in activation of silent biosynthetic pathways for the secondary metabolite production. Upon treatment of G59 spores with neomycin and dimethyl sulfoxide (DMSO), a total of 56 mutants were obtained by single colony isolation. The acquired resistance of mutants to neomycin was testified by the resistance test. In contrast to the G59 strain, the EtOAc extracts of 28 mutants inhibited the human cancer K562 cells, indicating that the 28 mutants have acquired the capability to produce bioactive metabolites. HPLC-photodiode array detector (PDAD)-UV and HPLC-electron spray ionization (ESI)-MS analyses further indicated that diverse secondary metabolites have been newly produced in the bioactive mutant extracts. Followed isolation and characterization demonstrated that five bioactive secondary metabolites, curvularin (1), citrinin (2), penicitrinone A (3), erythro-23-O-methylneocyclocitrinol (4) and 22E-7α-methoxy-5α,6α-epoxyergosta-8(14),22-dien-3β-ol (5), were newly produced by a mutant, 4-30, compared to the G59 strain. All 1–5 were also not yet found in the secondary metabolites of other wild type P. purpurogenum strains. Compounds 1–5 inhibited human cancer K562, HL-60, HeLa and BGC-823 cells to varying extents. Both present bioassays and chemical investigations demonstrated that the introduction of neomycin-resistance into the marine-derived fungal G59 strain could activate silent secondary metabolite production. The present work not only extended the previous DMSO-mediated method for introducing drug-resistance in fungi both in DMSO concentrations and antibiotics, but also additionally exemplified effectiveness of this method for activating silent fungal secondary metabolites. This method could be applied to other fungal isolates to elicit their metabolic potentials to investigate secondary metabolites from silent biosynthetic pathways.     

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.     

Different Culture Metabolites of the Red Sea Fungus Fusarium equiseti Optimize the Inhibition of Hepatitis C Virus NS3/4A Protease (HCV PR)

The endophytic fungus Fusarium equiseti was isolated from the brown alga Padina pavonica, collected from the Red Sea. The fungus was identified by its morphology and 18S rDNA. Cultivation of this fungal strain in biomalt-peptone medium led to isolation of 12 known metabolites of diketopeprazines and anthraquinones. The organic extract and isolated compounds were screened for their inhibition of hepatitis C virus NS3/4A protease (HCV PR). As a result, the fungal metabolites showed inhibition of HCV protease (IC₅₀ from 19 to 77 μM), and the fungus was subjected to culture on Czapek’s (Cz) media, with a yield of nine metabolites with potent HCV protease inhibition ranging from IC₅₀ 10 to 37 μM. The Cz culture extract exhibited high-level inhibition of HCV protease (IC₅₀ 27.6 μg/mL) compared to the biomalt culture extract (IC₅₀ 56 μg/mL), and the most potent HCV PR isolated compound (Griseoxanthone C, IC₅₀ 19.8 μM) from the bio-malt culture extract showed less of an inhibitory effect compared to isolated ω-hydroxyemodin (IC₅₀ 10.7 μM) from the optimized Cz culture extract. Both HCV PR active inhibitors ω-hydroxyemodin and griseoxanthone C were considered as the lowest selective safe constituents against Trypsin inhibitory effect with IC₅₀ 48.5 and 51.3 μM, respectively.     

Untargeted Metabolomics Approach for the Discovery of Environment-Related Pyran-2-Ones Chemodiversity in a Marine-Sourced Penicillium restrictum

Very little is known about chemical interactions between fungi and their mollusc host within marine environments. Here, we investigated the metabolome of a Penicillium restrictum MMS417 strain isolated from the blue mussel Mytilus edulis collected on the Loire estuary, France. Following the OSMAC approach with the use of 14 culture media, the effect of salinity and of a mussel-derived medium on the metabolic expression were analysed using HPLC-UV/DAD-HRMS/MS. An untargeted metabolomics study was performed using principal component analysis (PCA), orthogonal projection to latent structure discriminant analysis (O-PLSDA) and molecular networking (MN). It highlighted some compounds belonging to sterols, macrolides and pyran-2-ones, which were specifically induced in marine conditions. In particular, a high chemical diversity of pyran-2-ones was found to be related to the presence of mussel extract in the culture medium. Mass spectrometry (MS)- and UV-guided purification resulted in the isolation of five new natural fungal pyran-2-one derivatives—5,6-dihydro-6S-hydroxymethyl-4-methoxy-2H-pyran-2-one (1), (6S, 1’R, 2’S)-LL-P880β (3), 5,6-dihydro-4-methoxy-6S-(1’S, 2’S-dihydroxy pent-3’(E)-enyl)-2H-pyran-2-one (4), 4-methoxy-6-(1’R, 2’S-dihydroxy pent-3’(E)-enyl)-2H-pyran-2-one (6) and 4-methoxy-2H-pyran-2-one (7)—together with the known (6S, 1’S, 2’S)-LL-P880β (2), (1’R, 2’S)-LL-P880γ (5), 5,6-dihydro-4-methoxy-2H-pyran-2-one (8), (6S, 1’S, 2’R)-LL-P880β (9), (6S, 1’S)-pestalotin (10), 1’R-dehydropestalotin (11) and 6-pentyl-4-methoxy-2H-pyran-2-one (12) from the mussel-derived culture medium extract. The structures of 1-12 were determined by 1D- and 2D-MMR experiments as well as high-resolution tandem MS, ECD and DP4 calculations. Some of these compounds were evaluated for their cytotoxic, antibacterial, antileishmanial and in-silico PTP1B inhibitory activities. These results illustrate the utility in using host-derived media for the discovery of new natural products.     

Activation of Dormant Secondary Metabolite Production by Introducing Neomycin Resistance into the Deep-Sea Fungus,Aspergillus versicolor ZBY-3

A new ultrasound-mediated approach has been developed to introduce neomycin-resistance to activate silent pathways for secondary metabolite production in a bio-inactive, deep-sea fungus, Aspergillus versicolor ZBY-3. Upon treatment of the ZBY-3 spores with a high concentration of neomycin by proper ultrasound irradiation, a total of 30 mutants were obtained by single colony isolation. The acquired resistance of the mutants to neomycin was confirmed by a resistance test. In contrast to the ZBY-3 strain, the EtOAc extracts of 22 of the 30 mutants inhibited the human cancer K562 cells, indicating that these mutants acquired a capability to produce antitumor metabolites. HPLC-photodiode array detector (PDAD)-UV and HPLC-electron spray ionization (ESI)-MS analyses of the EtOAc extracts of seven bioactive mutants and the ZBY-3 strain indicated that diverse secondary metabolites have been newly produced in the mutant extracts in contrast to the ZBY-3 extract. The followed isolation and characterization demonstrated that six metabolites, cyclo(d-Pro-d-Phe) (1), cyclo(d-Tyr-d-Pro) (2), phenethyl 5-oxo-l-prolinate (3), cyclo(l-Ile-l-Pro) (4), cyclo(l-Leu-l-Pro) (5) and 3β,5α,9α-trihydroxy-(22E,24R)-ergosta-7,22-dien-6-one (6), were newly produced by the mutant u2n2h3-3 compared to the parent ZBY-3 strain. Compound 3 was a new compound; 2 was isolated from a natural source for the first time, and all of these compounds were also not yet found in the metabolites of other A. versicolor strains. Compounds 1–6 inhibited the K562 cells, with inhibition rates of 54.6% (1), 72.9% (2), 23.5% (3), 29.6% (4), 30.9% (5) and 51.1% (6) at 100 μg/mL, and inhibited also other human cancer HL-60, BGC-823 and HeLa cells, to some extent. The present study demonstrated the effectiveness of the ultrasound-mediated approach to activate silent metabolite production in fungi by introducing acquired resistance to aminoglycosides and its potential for discovering new compounds from silent fungal metabolic pathways. This approach could be applied to elicit the metabolic potentials of other fungal isolates to discover new compounds from cryptic secondary metabolites.     

Micrococcin P1 and P2 from Epibiotic Bacteria Associated with Isolates of Moorea producens from Kenya

Epibiotic bacteria associated with the filamentous marine cyanobacterium Moorea producens were explored as a novel source of antibiotics and to establish whether they can produce cyclodepsipeptides on their own. Here, we report the isolation of micrococcin P1 (1) (C₄₈H₄₉N₁₃O₉S₆; obs. m/z 1144.21930/572.60381) and micrococcin P2 (2) (C₄₈H₄₇N₁₃O₉S₆; obs. m/z 1142.20446/571.60370) from a strain of Bacillus marisflavi isolated from M. producens’ filaments. Interestingly, most bacteria isolated from M. producens’ filaments were found to be human pathogens. Stalked diatoms on the filaments suggested a possible terrestrial origin of some epibionts. CuSO₄·5H₂O assisted differential genomic DNA isolation and phylogenetic analysis showed that a Kenyan strain of M. producens differed from L. majuscula strain CCAP 1446/4 and L. majuscula clones. Organic extracts of the epibiotic bacteria Pseudoalteromonas carrageenovora and Ochrobactrum anthropi did not produce cyclodepsipeptides. Further characterization of 24 Firmicutes strains from M. producens identified extracts of B. marisflavi as most active. Our results showed that the genetic basis for synthesizing micrococcin P1 (1), discovered in Bacillus cereus ATCC 14579, is species/strain-dependent and this reinforces the need for molecular identification of M. producens species worldwide and their epibionts. These findings indicate that M. producens-associated bacteria are an overlooked source of antimicrobial compounds.     

Bioactive Phenylalanine Derivatives and Cytochalasins from the Soft Coral-Derived Fungus,Aspergillus elegans

One new phenylalanine derivative 4′-OMe-asperphenamate (1), along with one known phenylalanine derivative (2) and two new cytochalasins, aspochalasin A1 (3) and cytochalasin Z24 (4), as well as eight known cytochalasin analogues (5–12) were isolated from the fermentation broth of Aspergillus elegans ZJ-2008010, a fungus obtained from a soft coral Sarcophyton sp. collected from the South China Sea. Their structures and the relative configurations were elucidated using comprehensive spectroscopic methods. The absolute configuration of 1 was determined by chemical synthesis and Marfey’s method. All isolated metabolites (1–12) were evaluated for their antifouling and antibacterial activities. Cytochalasins 5, 6, 8 and 9 showed strong antifouling activity against the larval settlement of the barnacle Balanus amphitrite, with the EC₅₀ values ranging from 6.2 to 37 μM. This is the first report of antifouling activity for this class of metabolites. Additionally, 8 exhibited a broad spectrum of antibacterial activity, especially against four pathogenic bacteria Staphylococcus albus, S. aureus, Escherichia coli and Bacillus cereus.     

Isolation of Araguspongine M,a New Stereoisomer of an Araguspongine/Xestospongin alkaloid,and Dopamine from the Marine Sponge Neopetrosia exigua Collected in Palau

A new stereoisomer of an araguspongine/xestospongin alkaloid, named araguspongine M (1), has been isolated together with 12 known compounds, araguspongines B (2) and D (3), dopamine, three galactosyl diacylglycerols, 24-methyl cholesterol, 5,6-dihydrocholesterol, β-sitosterol, and three 5α,8α-epidioxy sterols (11–13), from the marine sponge Neopetrosia exigua (formerly Xestospongia exigua) collected in Palau. The structure of 1 was assigned on the basis of its spectral data analysis. This is the first report on the isolation of dopamine from a marine sponge. This compound may be produced by an endosymbiotic Synechococcus-like cyanobacterium. Compounds 1–3 and 11–13 showed cytotoxicity against HL-60 at IC₅₀’s of 5.5, 5.5, 5.9, 22.4, 9.5, and 9.6 μM, respectively. The possible biosynthesis origin of the isolated metabolites is discussed.     

Novel α-Amylase Inhibitor Hemi-Pyocyanin Produced by Microbial Conversion of Chitinous Discards

α-Amylase inhibitors (aAIs) have been applied for the efficient management of type 2 diabetes. The aim of this study was to search for potential aAIs produced by microbial fermentation. Among various bacterial strains, Pseudomonas aeruginosa TUN03 was found to be a potential aAI-producing strain, and shrimp heads powder (SHP) was screened as the most suitable C/N source for fermentation. P. aeruginosa TUN03 exhibited the highest aAIs productivity (3100 U/mL) in the medium containing 1.5% SHP with an initial pH of 7–7.5, and fermentation was performed at 27.5 °C for two days. Further, aAI compounds were investigated for scaled-up production in a 14 L-bioreactor system. The results revealed a high yield (4200 U/mL) in a much shorter fermentation time (12 h) compared to fermentation in flasks. Bioactivity-guided purification resulted in the isolation of one major target compound, identified as hemi-pyocyanin (HPC) via gas chromatography-mass spectrometry and nuclear magnetic resonance. Its purity was analyzed by high-performance liquid chromatography. HPC demonstrated potent α-amylase inhibitory activity comparable to that of acarbose, a commercial antidiabetic drug. Notably, HPC was determined as a new aAI. The docking study indicated that HPC inhibits α-amylase by binding to amino acid Arg421 at the biding site on enzyme α-amylase with good binding energy (−9.3 kcal/mol) and creating two linkages of H-acceptors.     

Characterization of Bioactivities and Biosynthesis of Angucycline/Angucyclinone Derivatives Derived from Gephyromycinifex aptenodytis gen. nov., sp. nov.

Strain NJES-13^T is the type strain and currently the only species of the newly established actinobacteria genera Aptenodytes in the family Dermatophilaceae isolated from the gut microbiota of the Antarctic emperor penguin. This strain demonstrated excellent bioflocculation activity with bacteria-derived exopolysaccharides (EPSs). Moreover, it produced bioactive angucycline/angucyclinone derivatives (ADs) and contained one type III polyketide synthase (T3PKS), thus demonstrating great potential to produce novel bioactive compounds. However, the low productivity of the potential new AD metabolite was the main obstacle for its chemical structure elucidation. In this study, to increase the concentration of targeted metabolites, the influence of cellular morphology on AD metabolism in strain NJES-13^T was determined using glass bead-enhanced fermentation. Based on the cellular ultra-structural observation driven by bacterial EPSs, and quantitative analysis of the targeted metabolites, the successful increasing of the productivity of three AD metabolites was achieved. Afterward, a new frigocyclinone analogue was isolated and then identified as 2-hydroxy-frigocyclinone, as well as two other known ADs named 2-hydroxy-tetrangomycin (2-HT) and gephyromycin (GPM). Three AD metabolites were found to demonstrate different bioactivities. Both C-2 hydroxyl substitutes, 2-hydroxy-tetrangomycin and 2-hydroxy-frigocyclinone, exhibited variable inhibitory activities against Staphylococcus aureus, Bacillus subtilis and Candida albicans. Moreover, the newly identified 2-hydroxy-frigocyclinone also showed significant cytotoxicity against three tested human-derived cancerous cell lines (HL-60, Bel-7402 and A549), with all obtained IC₅₀ values less than 10 µM. Based on the genetic analysis after genomic mining, the plausible biogenetic pathway of the three bioactive ADs in strain NJES-13^T was also proposed.     

Investigation of Indolglyoxamide and Indolacetamide Analogues of Polyamines as Antimalarial and Antitrypanosomal Agents

Pure compound screening has previously identified the indolglyoxylamidospermidine ascidian metabolites didemnidine A and B (2 and 3) to be weak growth inhibitors of Trypanosoma brucei rhodesiense (IC₅₀ 59 and 44 μM, respectively) and Plasmodium falciparum (K1 dual drug resistant strain) (IC₅₀ 41 and 15 μM, respectively), but lacking in selectivity (L6 rat myoblast, IC₅₀ 24 μM and 25 μM, respectively). To expand the structure–activity relationship of this compound class towards both parasites, we have prepared and biologically tested a library of analogues that includes indoleglyoxyl and indoleacetic “capping acids”, and polyamines including spermine (PA3-4-3) and extended analogues PA3-8-3 and PA3-12-3. 7-Methoxy substituted indoleglyoxylamides were typically found to exhibit the most potent antimalarial activity (IC₅₀ 10–92 nM) but with varying degrees of selectivity versus the L6 rat myoblast cell line. A 6-methoxyindolglyoxylamide analogue was the most potent growth inhibitor of T. brucei (IC₅₀ 0.18 μM) identified in the study: it, however, also exhibited poor selectivity (L6 IC₅₀ 6.0 μM). There was no apparent correlation between antimalarial and anti-T. brucei activity in the series. In vivo evaluation of one analogue against Plasmodium berghei was undertaken, demonstrating a modest 20.9% reduction in parasitaemia.     

Complementary Analytical Platforms of NMR Spectroscopy and LCMS Analysis in the Metabolite Profiling of Isochrysis galbana

This study was designed to profile the metabolites of Isochrysis galbana, an indigenous and less explored microalgae species. ¹H Nuclear Magnetic Resonance (NMR) spectroscopy and Liquid Chromatography-Mass Spectrometry (LCMS) were used to establish the metabolite profiles of five different extracts of this microalga, which are hexane (Hex), ethyl acetate (EtOAc), absolute ethanol (EtOH), EtOH:water 1:1 (AqE), and 100% water (Aq). Partial least square discriminant analysis (PLS–DA) of the generated profiles revealed that EtOAc and Aq extracts contain a diverse range of metabolites as compared to the other extracts with a total of twenty-one metabolites, comprising carotenoids, polyunsaturated fatty acids, and amino acids, that were putatively identified from the NMR spectra. Meanwhile, thirty-two metabolites were successfully annotated from the LCMS/MS data, ten of which (palmitic acid, oleic acid, α-linolenic acid, arachidic acid, cholesterol, DHA, DPA, fucoxanthin, astaxanthin, and pheophytin) were similar to those present in the NMR profile. Another eleven glycerophospholipids were discovered using MS/MS-based molecular network (MN) platform. The results of this study, besides providing a better understanding of I. galbana’s chemical make-up, will be of importance in exploring this species potential as a feed ingredient in the aquaculture industry.     

Antiplasmodial Activities of Homogentisic Acid Derivative Protein Kinase Inhibitors Isolated from a Vanuatu Marine Sponge Pseudoceratina sp.

As part of our search for new antimalarial drugs in South Pacific marine sponges, we have looked for inhibitors of Pfnek-1, a specific protein kinase of Plasmodium falciparum. On the basis of promising activity in a preliminary screening, the ethanolic crude extract of a new species of Pseudoceratina collected in Vanuatu was selected for further investigation. A bioassay-guided fractionation led to the isolation of a derivative of homogentisic acid [methyl (2,4-dibromo-3,6-dihydroxyphenyl)acetate, 4a] which inhibited Pfnek-1 with an IC₅₀ around 1.8 μM. This product was moderately active in vitro against a FcB1 P. falciparum strain (IC₅₀ = 12 μM). From the same sponge, we isolated three known compounds [11,19-dideoxyfistularin-3 (1), 11-deoxyfistularin-3 (2) and dibromo-verongiaquinol (3)] which were inactive against Pfnek-1. Synthesis and biological evaluation of some derivatives of 4a are reported.     

Influence of Temperature on Growth and Production of Pectenotoxin-2 by a Monoclonal Culture of Dinophysis caudata

The effects of temperature on growth and production of Lipophilic Toxins (LT) by a monoclonal culture of Dinophysis caudata was studied. The cell density of D. caudata increased significantly with increasing temperature, and was the highest under 27, 30, and 32.5 °C. Temperature affected the average specific growth rate (µ) during the exponential growth phase (EG), which increased from 15 °C to 30 °C, and then decreased at 32.5 °C. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) revealed that this strain of D. caudata produced only pectenotoxin-2 (PTX-2) whose concentration increased significantly with incubation period, except at 32.5 °C. It was significantly different between temperatures ≤18 °C, ≥21 °C, and 32.5 °C. The cellular toxin production (CTP, pg·cell⁻¹·day⁻¹) showed variation with growth phase and temperature, except at 32.5 °C. The average net toxin production (R_tox) was not affected by temperature. During EG, the average specific toxin production rate (µ_tox) increased significantly with increase in temperature, reaching a peak of 0.66 ± 0.01 day⁻¹ at 30 °C, and then decreased. Over the entire growth span, µ_tox was significantly correlated to µ, and this correlation was most significant at 27 and 30 °C. During EG, µ_tox was affected by both temperature and growth. This study shows that temperature affects growth and toxin production of this strain of D. caudata during EG. In addition, a positive correlation was found between toxin production and growth.     

Bioactive Bromotyrosine Derivatives from the Pacific Marine Sponge Suberea clavata (Pulitzer-Finali, 1982)

Chemical investigation of the South-Pacific marine sponge Suberea clavata led to the isolation of eight new bromotyrosine metabolites named subereins 1–8 (2–9) along with twelve known co-isolated congeners. The detailed configuration determination of the first representative major compound of this family 11-epi-fistularin-3 (11R,17S) (1) is described. Their chemical characterization was achieved by HRMS and integrated 1D and 2D NMR (nuclear magnetic resonance) spectroscopic studies and extensive comparison with literature data. For the first time, a complete assignment of the absolute configurations for stereogenic centers C-11/17 of the known members (11R,17S) 11-epi-fistularin-3 (1) and 17-deoxyfistularin-3 (10) was determined by a combination of chemical modifications, Mosher’s technology, and ECD spectroscopy. Consequently, the absolute configurations of all our new isolated compounds 2–9 were determined by the combination of NMR, Mosher’s method, ECD comparison, and chemical modifications. Interestingly, compounds 2–7 were obtained by chemical transformation of the major compound 11-epi-fistularin-3 (1). Evaluation for acetylcholinesterase inhibition (AChE), DNA methyltransferase 1 (DNMT1) modulating activity and antifouling activities using marine bacterial strains are also presented.     

Secondary Metabolite Production Potential of Mangrove-Derived Streptomyces olivaceus

Mangroves are intertidal extreme environments with rich microbial communities. Actinobacteria are well known for producing antibiotics. The search for biosynthetic potential of Actinobacteria from mangrove environments could provide more possibilities for useful secondary metabolites. In this study, whole genome sequencing and MS/MS analysis were used to explore the secondary metabolite production potential of one actinobacterial strain of Streptomyces olivaceus sp., isolated from a mangrove in Macau, China. The results showed that a total of 105 gene clusters were found in the genome of S. olivaceus sp., and 53 known secondary metabolites, including bioactive compounds, peptides, and other products, were predicted by genome mining. There were 28 secondary metabolites classified as antibiotics, which were not previously known from S. olivaceus. ISP medium 2 was then used to ferment the S. olivaceus sp. to determine which predicted secondary metabolite could be truly produced. The chemical analysis revealed that ectoine, melanin, and the antibiotic of validamycin A could be observed in the fermentation broth. This was the first observation that these three compounds can be produced by a strain of S. olivaceus. Therefore, it can be concluded that Actinobacteria isolated from the mangrove environment have unknown potential to produce bioactive secondary metabolites.     

Antiviral Activity and Mechanisms of Seaweeds Bioactive Compounds on Enveloped Viruses—A Review

In the last decades, the interest in seaweed has significantly increased. Bioactive compounds from seaweed’s currently receive major attention from pharmaceutical companies as they express several interesting biological activities which are beneficial for humans. The structural diversity of seaweed metabolites provides diverse biological activities which are expressed through diverse mechanisms of actions. This review mainly focuses on the antiviral activity of seaweed’s extracts, highlighting the mechanisms of actions of some seaweed molecules against infection caused by different types of enveloped viruses: influenza, Lentivirus (HIV-1), Herpes viruses, and coronaviruses. Seaweed metabolites with antiviral properties can act trough different pathways by increasing the host’s defense system or through targeting and blocking virus replication before it enters host cells. Several studies have already established the large antiviral spectrum of seaweed’s bioactive compounds. Throughout this review, antiviral mechanisms and medical applications of seaweed’s bioactive compounds are analyzed, suggesting seaweed’s potential source of antiviral compounds for the formulation of novel and natural antiviral drugs.