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This is the peer reviewed version of the following article: Wang S, Fang Q, Lu, Gao, Trembleau L, Ebel R, Andersen JH, Philips, Law, Deng H. Discovery and Biosynthetic Investigation of a New Antibacterial Dehydrated Non‐Ribosomal Tripeptide. Angewandte Chemie International Edition. 2020, which has been published in final form at https://doi.org/10.1002/anie.202012902. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions. Dehydroalanine (Dha) and dehydrobutyrine (Dhb) display considerable flexibility in a variety of chemical and biological reactions. Natural products containing Dha and/or Dhb residues are often found to display diverse biological activities. While the (Z) geometry is predominant in nature, only a handful of metabolites containing (E)‐Dhb have been found thus far. Here we report discovery of a new antimicrobial peptide, albopeptide, through NMR analysis and chemical synthesis, which contains two contiguous unsaturated residues, Dha‐(E)‐Dhb. It displays narrow‐spectrum activity against vancomycin‐resistant Enterococcus faecium. In‐vitro biochemical assays show that albopeptide originates from a noncanonical NRPS pathway featuring dehydration processes and catalysed by unusual condensation domains. Finally, we provide evidence of the occurrence of a previously untapped group of short unsaturated peptides in the bacterial kingdom, suggesting an important biological function in bacteria.

The Lacinutrix genus was discovered in 2005 and includes 12 Gram-negative bacterial species. To the best of our knowledge, the secondary metabolite production potential of this genus has not been explored before, and examination of Lacinutrix species may reveal novel chemistry. As part of a screening project of Arctic marine bacteria, the Lacinutrix sp. strain M09B143 was cultivated, extracted, fractionated and tested for antibacterial and cytotoxic activities. One fraction had antibacterial activity and was subjected to mass spectrometry analysis, which revealed two compounds with elemental composition that did not match any known compounds in databases. This resulted in the identification and isolation of two novel isobranched lyso-ornithine lipids, whose structures were elucidated by mass spectrometry and NMR spectroscopy. Lyso-ornithine lipids consist of a 3-hydroxy fatty acid linked to the alpha amino group of an ornithine amino acid through an amide bond. The fatty acid chains were determined to be iso-C15:0 (1) and iso-C16:0 (2). Compound 1 was active against the Gram-positive S. agalactiae, while 2 showed cytotoxic activity against A2058 human melanoma cells.

A series of novel quinoline-based tetracyclic ring-systems were synthesized and evaluated in vitro for their antiplasmodial, antiproliferative and antimicrobial activities. The novel hydroiodide salts 10 and 21 showed the most promising antiplasmodial inhibition, with compound 10 displaying higher selectivity than the employed standards. The antiproliferative assay revealed novel pyridophenanthridine 4b to be significantly more active against human prostate cancer (IC50 = 24 nM) than Puromycin (IC50 = 270 nM) and Doxorubicin (IC50 = 830 nM), which are used for clinical treatment. Pyridocarbazoles 9 was also moderately effective against all the employed cancer cell lines and moreover showed excellent biofilm inhibition (9a: MBIC = 100 µM, 9b: MBIC = 100 µM).

Siderophores are compounds with high affinity for ferric iron. Bacteria produce these compounds to acquire iron in iron-limiting conditions. Iron is one of the most abundant metals on earth, and its presence is necessary for many vital life processes. Bacteria from the genus Serratia contribute to the iron respiration in their environments, and previously several siderophores have been isolated from this genus. As part of our ongoing search for medicinally relevant compounds produced by marine microbes, a co-culture of a Shewanella sp. isolate and a Serratia sp. isolate, grown in iron-limited conditions, was investigated, and the rare siderophore serratiochelin A (1) was isolated with high yields. Compound 1 has previously been isolated exclusively from Serratia sp., and to our knowledge, there is no bioactivity data available for this siderophore to date. During the isolation process, we observed the degradation product serratiochelin C (2) after exposure to formic acid. Both 1 and 2 were verified by 1-D and 2-D NMR and high-resolution MS/MS. Here, we present the isolation of 1 from an iron-depleted co-culture of Shewanella sp. and Serratia sp., its proposed mechanism of degradation into 2, and the chemical and biological characterization of both compounds. The effects of 1 and 2 on eukaryotic and prokaryotic cells were evaluated, as well as their effect on biofilm formation by Staphylococcus epidermidis. While 2 did not show bioactivity in the given assays, 1 inhibited the growth of the eukaryotic cells and Staphylococcus aureus.

Microalgae have been shown to be excellent producers of lipids, pigments, carbohydrates, and a plethora of secondary metabolites with possible applications in the pharmacological, nutraceutical, and cosmeceutical sectors. Recently, various microalgal raw extracts have been found to have anti-inflammatory properties. In this study, we performed the fractionation of raw extracts of the diatom Cylindrotheca closterium, previously shown to have anti-inflammatory properties, obtaining five fractions. Fractions C and D were found to significantly inhibit tumor necrosis factor alpha (TNF-⍺) release in LPS-stimulated human monocyte THP-1 cells. A dereplication analysis of these two fractions allowed the identification of their main components. Our data suggest that lysophosphatidylcholines and a breakdown product of chlorophyll, pheophorbide a, were probably responsible for the observed anti-inflammatory activity. Pheophorbide a is known to have anti-inflammatory properties. We tested and confirmed the anti-inflammatory activity of 1-palmitoyl-sn-glycero-3-phosphocholine, the most abundant lysophosphatidylcholine found in fraction C. This study demonstrated the importance of proper dereplication of bioactive extracts and fractions before isolation of compounds is commenced.

Streptomyces remains one of the prolific sources of structural diversity, and a reservoir to mine for novel natural products. Continued screening for new Streptomyces strains in our laboratory led to the isolation of Streptomyces sp. RK44 from the underexplored areas of Kintampo waterfalls, Ghana, Africa. Preliminary screening of the metabolites from this strain resulted in the characterization of a new 2-alkyl-4-hydroxymethylfuran carboxamide (AHFA) 1 together with five known compounds, cyclo-(L-Pro-Gly) 2, cyclo-(L-Pro-L-Phe) 3, cyclo-(L-Pro-L-Val) 4, cyclo-(L-Leu-Hyp) 5, and deferoxamine E 6. AHFA 1, a methylenomycin (MMF) homolog, exhibited anti-proliferative activity (EC50 = 89.6 &micro, M) against melanoma A2058 cell lines. This activity, albeit weak is the first report amongst MMFs. Furthermore, the putative biosynthetic gene cluster (ahfa) was identified for the biosynthesis of AHFA 1. DFO-E 6 displayed potent anti-plasmodial activity (IC50 = 1.08&micro, M) against P. falciparum 3D7. High-resolution electrospray ionization mass spectrometry (HR ESIMS) and molecular network assisted the targeted-isolation process, and tentatively identified six AHFA analogues, 7&ndash, 12 and six siderophores 13&ndash, 18.

A key step in the process of isolating microbial natural products is the preparation of an extract from a culture. This step determines which molecules will be available for detection in the subsequent chemical and biological analysis of a biodiscovery pipeline. In the present study we wanted to document potential differences in performance between liquid–liquid extraction using ethyl acetate and liquid–solid extraction using a poly-benzyl-resin. For the comparison of the two extraction protocols, we spiked a culture of Flavobacterium sp. with a diverse selection of natural products of microbial and plant origin to investigate whether the methods were comparable with respect to selectivity. We also investigated the efficiency of the two extraction methods quantitatively, using water spiked with a selection of natural products, and studied the quantitative effect of different pH levels of the aqueous solutions on the extraction yields of the two methods. The same compounds were extracted by the two methods, but the solid-phase extract contained more media components compared with the liquid-phase extract. Quantitatively, the two extraction methods varied in their recovery rates. We conclude that practical aspects could be more important when selecting one of the extraction protocols, as their efficiencies in extracting specific compounds were quite similar.

A new strategy for the identification of known compounds in Streptomyces extracts that can be applied in the discovery of natural products is presented. The strategy incorporates screening a database of 5555 natural products including 5098 structures from Streptomyces sp., using a high-throughput LCMS data processing algorithm that utilizes HRMS data and predicted LC retention times (tR) as filters for rapid identification of known compounds in the natural product extract. The database, named StrepDB, contains for each compound the structure, molecular formula, molecular mass, and predicted LC retention time. All identified compounds are annotated and color coded for easier visualization. It is an indirect approach to quickly assess masses (which are not annotated) that may potentially lead to the discovery of new or novel structures. In addition, a spectral database named MbcDB was generated using the ACD/Spectrus DB Platform. MbcDB contains 665 natural products, each with structure, experimental HRESIMS, MS/MS, UV, and NMR spectra. StrepDB was used to screen a mutant Streptomyces albus extract, which led to the identification and isolation of two new compounds, legonmaleimides A and B, the structures of which were elucidated with the aid of MbcDB and spectroscopic techniques. The structures were confirmed by computer-assisted structure elucidation (CASE) methods using ACD/Structure Elucidator Suite. The developed methodology suggests a pipeline approach to the dereplication of extracts and discovery of novel natural products.

This is an Accepted Manuscript of an article published by Taylor & Francis in Natural Product Research on 20 February 2019, available online: https://www.tandfonline.com/doi/full/10.1080/14786419.2019.1574788. The new guanidine alkaloid Dendrobeaniamine A (1) was isolated from the organic extract of the Arctic marine bryozoan Dendrobeania murrayana. The chemical structure of 1 was elucidated by spectroscopic experiments, including 1D and 2D NMR and HRESIMS analysis. Compound 1 is a lipoamino acid, consisting of a C12 fatty acid anchored to the amino acid arginine. The bioactivity of 1 was evaluated using cellular and biochemical assays, but the compound did not show cytotoxic, antimicrobial, anti-inflammatory or antioxidant activities

Source at https://doi.org/10.1016/j.ejmech.2019.111671. The rapid emergence and spread of multi-resistant bacteria have created an urgent need for new antimicrobial agents. We report here a series of amphipathic α,α-disubstituted β-amino amide derivatives with activity against 30 multi-resistant clinical isolates of Gram-positive and Gram-negative bacteria, including isolates with extended spectrum β-lactamase – carbapenemase (ESBL-CARBA) production. A variety of halogenated aromatic side-chains were investigated to improve antimicrobial potency and minimize formation of Phase I metabolites. Net positive charge and cationic character of the derivatives had an important effect on toxicity against human cell lines. The most potent and selective derivative was the diguanidine derivative 4e with 3,5-di-brominated benzylic side-chains. Derivative 4e displayed minimum inhibitory concentrations (MIC) of 0.25–8 μg/mL against Gram-positive and Gram-negative reference strains, and 2–32 μg/mL against multi-resistant clinical isolates. Derivative 4e showed also low toxicity against human red blood cells (EC50 > 200 μg/mL), human hepatocyte carcinoma cells (HepG2: EC50 > 64 μg/mL), and human lung fibroblast cells (MRC-5: EC50 > 64 μg/mL). The broad-spectrum antimicrobial activity and low toxicity of diguanylated derivatives such as 4e make them attractive as lead compounds for development of novel antimicrobial drugs.

Accepted manuscript version. Published version available at https://doi.org/10.1016/j.bioorg.2018.11.024. Licensed CC BY-NC-ND 4.0. The marine environment remains a rich source for the discovery and development of novel bioactive compounds. The present paper describes the design, synthesis and biological evaluation of a library of small molecule heterocyclic mimetics of the marine 2,5-diketopiperazine barettin which is a powerful natural antioxidant. By mainly focusing on the influence from the brominated indole and heterocyclic core of barettin, a library of 19 compounds was prepared. The compounds comprised a heterocyclic core, either a 2,5 diketopiperazine, an imidazolidinedione or a thioxothiazolidinone, which were mainly monosubstituted with ranging bulky substituents. The prepared compounds were screened for activity in a cellular lipid peroxidation assay using HepG2 cells. Several of the synthetic compounds showed antioxidant properties superior to the positive control barettin. Two of the prepared compounds displayed inhibitory activity similar to commercial antioxidants with significant inhibition at low µg/mL concentrations. The toxicity of the compounds was also investigated against MRC-5 lung fibroblasts and none of the included compounds displayed any toxicity at 50 µg/mL.

Barettin, 8,9-dihydrobarettin, bromoconicamin and a novel brominated marine indole were isolated from the boreal sponge Geodia barretti collected off the Norwegian coast. The compounds were evaluated as inhibitors of electric eel acetylcholinesterase. Barettin and 8,9-dihydrobarettin displayed significant inhibition of the enzyme, with inhibition constants (Ki) of 29 and 19 μM respectively towards acetylcholinesterase via a reversible noncompetitive mechanism. These activities are comparable to those of several other natural acetylcholinesterase inhibitors of marine origin. Bromoconicamin was less potent against acetylcholinesterase, and the novel compound was inactive. Based on the inhibitory activity, a library of 22 simplified synthetic analogs was designed and prepared to probe the role of the brominated indole, common to all the isolated compounds. From the structure-activity investigation it was shown that the brominated indole motif is not sufficient to generate a high acetylcholinesterase inhibitory activity, even when combined with natural cationic ligands for the acetylcholinesterase active site. The four natural compounds were also analysed for their butyrylcholinesterase inhibitory activity in addition and shown to display comparable activities. The study illustrates how both barettin and 8,9-dihydrobarettin display additional bioactivities which may help to explain their biological role in the producing organism. The findings also provide new insights into the structure-activity relationship of both natural and synthetic acetylcholinesterase inhibitors.

The marine secondary metabolite stryphnusin (1) was isolated from the boreal sponge Stryphnus fortis, collected off the Norwegian coast. Given its resemblance to other natural acetylcholinesterase antagonists, it was evaluated against electric eel acetylcholinesterase and displayed inhibitory activity. A library of twelve synthetic phenethylamine analogs, 2a–7a and 2b–7b, containing tertiary and quaternary amines respectively were synthesized to investigate the individual structural contributions to the activity. Compound 7b was the strongest competitive inhibitor of both acetylcholinesterase and butyrylcholinesterase with IC50 values of 57 and 20 μM, respectively. This inhibitory activity is one order of magnitude higher than the positive control physostigmine, and is comparable with several other marine acetylcholinesterase inhibitors. The physiological effect of compound 7b on muscle function and neuromuscular transmission was studied and revealed a selective mode of action at the investigated concentration. This data is of importance as the interference of therapeutic acetylcholinesterase inhibitors with neuromuscular transmission can be problematic and lead to unwanted side effects. The current findings also provide additional insights into the structure–activity relationship of both natural and synthetic acetylcholinesterase inhibitors.

A new ecdysteroid, ponasterone F (1) and the previously reported compound ponasterone A (2) were isolated from specimens of the Arctic marine bryozoan Alcyonidium gelatinosum collected at Hopenbanken, off the coast of Edge&oslash, ya, Svalbard. The structure of 1 was elucidated, and the structure of 2 confirmed by spectroscopic methods including 1D and 2D NMR and analysis of HR-MS data. The compounds were evaluated for their ability to affect bacterial survival and cell viability, as well as their agonistic activities towards the estrogen receptors &alpha, and &beta, The compounds were not active in these assays. Compound 2 is an arthropod hormone controlling molting and are known to act as an allelochemical when produced by plants. Even though its structure has been previously reported, this is the first time a ponasterone has been isolated from a bryozoan. A. gelatinosum produced 1 and 2 in concentrations surpassing those expected of hormonal molecules, indicating their function as defence molecules against molting predators. This work adds to the chemical diversity reported from marine bryozoans and expanded our knowledge of the chemical modifications of the ponasterones.

Nature is an important source for anti-cancer therapeutics, and nearly half of the currently marketed cancer drugs are derived from natural products. Most of the therapeutic natural products are derived from terrestrial sources, such as paclitaxel, vincristine, epothilones, doxorubicin, etoposide and camptothecin. However, the oceans have received growing interest as a source for new useful bioactive compounds, and there are currently several drugs derived from marine natural products for the treatment of cancer on the market. The current recommended chemotherapy of acute myeloid leukemia (AML) is founded on cytarabine, a molecule derived from a natural product isolated from a marine sponge. However, in order to increase the efficiency of the chemotherapy used in the treatment of AML, it is necessary to develop more targeted drugs with less pronounced side effects. In this review, we argue that marine natural products have many of the desired properties of such a drug, and that prefractionated extract libraries of marine plants, animals and microorganisms should be a part of the screening efforts for new AML chemotherapeutics.

Source at https://doi.org/10.1038/s41598-017-15980-8 Microalgae and plankton can be a rich source of bioactivity. However, induction of secondary metabolite production in lab conditions can be difficult. One simple way of bypassing this issue is to collect biomass in the field and screen for bioactivity. Therefore, bulk net samples from three areas along the coast of northern Norway and Spitsbergen were collected, extracted and fractionated. Biomass samples from a strain of a mass-cultivated diatom Porosira glacialis were used as a reference for comparison to field samples. Screening for bioactivity was performed with 13 assays within four therapeutic areas: antibacterial, anticancer, antidiabetes and antioxidation. We analysed the metabolic profiles of the samples using high resolution - mass spectroscopy (HR-MS). Principal component analysis showed a marked difference in metabolite profiles between the field samples and the photobioreactor culture; furthermore, the number of active fractions and extent of bioactivity was different in the field compared to the photobioreactor samples. We found varying levels of bioactivity in all samples, indicating that complex marine field samples could be used to investigate bioactivities from otherwise inaccessible sources. Furthermore, we hypothesize that metabolic pathways that would otherwise been silent under controlled growth in monocultures, might have been activated in the field samples.

A library of 29 small 1,4-substituted 1,2,3-triazoles was prepared for studies of antimicrobial activity. The pharmacophore model investigated with these substrates was based on small peptidomimetics of antimicrobial peptides and antimicrobials isolated from marine organisms from sub-arctic regions. Using methyl 1,2,3-triazole-carboxylates and 1,2,3-triazole methyl ketones prepared through “click” chemistry we were able to synthesize the different cationic amphiphiles through three steps or less. Several structural modifications to the lipopohilic side and hydrophilic sides of the amphiphiles were investigated and compared with regards to antimicrobial activity and cytotoxicity in particular. The most promising amphiphile 10f displayed minimum inhibitory concentrations (MICs) between 4 - 16 µg/mL against Gram-positive Enterococcus faecalis, Staphylococcus aureus, Streptococcus agalacticae, and Gram-negative Escherichia coli and Pseudomonas aeruginosa. The decent level of antimicrobial activity and biofilm inhibition, short synthesis, and accessible reagents, makes this type of amphiphilic mimics interesting leads for further development. © 2017 Elsevier Ltd. All rights reserved. This is the authors' accepted and refereed manuscript to the article. Locked until 29 July 2019 due to copyright restrictions

This document is the Accepted Manuscript version of a Published Work that appeared in final form in Journal of natural products, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see https://doi.org/10.1021/np401002s. Pulmonarins A and B are two new dibrominated marine acetylcholinesterase inhibitors that were isolated and characterized from the sub-Arctic ascidian Synoicum pulmonaria collected off the Norwegian coast. The structures of natural pulmonarins A and B were tentatively elucidated by spectroscopic methods and later verified by comparison with synthetically prepared material. Both pulmonarins A and B displayed reversible, noncompetitive acetylcholinesterase inhibition comparable to several known natural acetylcholinesterase inhibitiors. Pulmonarin B was the strongest inhibitor, with an inhibition constant (Ki) of 20 μM. In addition to reversible, noncompetitive acetylcholinesterase inhibition, the compounds displayed weak antibacterial activity but no cytotoxicity or other investigated bioactivities.

A metabolomic approach was used to identify known and new natural products from the marine sponges Geodia baretti and G. macandrewii. G. baretti is known to produce bioactive natural products such as barettin (1), 8,9-dihydrobarettin (2), and bromobenzisoxazolone barettin (3), while secondary metabolites from G. macandrewii are not reported in the literature. Specimens of the two sponges were collected from different sites along the coast of Norway, and their extracts were analyzed using UHPLC-HR-MS. Metabolomic analyses revealed that extracts from both species contained barettin (1) and 8,9-dihydrobarettin (2), and all samples of G. baretti contained higher amounts of both compounds compared to G. macandrewii. The analysis of the MS data also revealed that samples of G. macandrewii contained a compound that was not present in any of the G. baretti samples. This new compound was isolated and identified as the N-acyl-taurine geodiataurine (4), and it was tested for antioxidant, anticancer, and antibacterial properties.

The milk protein lactoferrin (Lf) has multiple functions, including immune stimulation and antiviral activity towards herpes simplex virus 1 and 2 (HSV-1 and HSV-2); antiviral activity has also been reported for the N-terminal pepsin-derived fragment lactoferricin (Lfcin). The anti-HSV mode of action of Lf and Lfcin is assumed to involve, in part, their interaction with the cell surface glycosaminoglycan heparan sulfate, thereby blocking of viral entry. In this study we investigated the ability of human and bovine Lf and Lfcin to inhibit viral cell-to-cell spread as well as the involvement of cell surface glycosaminoglycans during viral cell-to-cell spread. Lf and Lfcin from both human and bovine origin, inhibited cell-to-cell spread of both HSV-1 and HSV-2. Inhibition of cell-to-cell spread by bovine Lfcin involved cell surface chondroitin sulfate. Based on transmission electron microscopy studies, human Lfcin, like bovine Lfcin, was randomly distributed intracellularly, thus differences in their antiviral activity could not be explained by differences in their distribution. In contrast, the cellular localization of iron-saturated (holo)-Lf appeared to differ from that of apo-Lf, indicating that holo- and apo-Lf may exhibit different antiviral mechanisms.

Ten highly cationic, α-helical peptides were synthesized and tested for antiviral activity against herpes simplex virus 1 and 2 (HSV-1 and HSV-2). Several of the peptides were found to exhibit antiviral activity. The peptides affinity for heparan sulfate (HS) increased with the number of cationic residues. Net charge could be decisive for the anti-HSV-1 activity, while secondary structure of the peptides seems more important for the anti-HSV-2 activity. The peptides were able to inhibit the entry of HSV-1 into the host cell, probably by blocking HS at the cell surface. HSV plaque formation was inhibited in a dose-dependent manner when cells were exposed to the peptides prior to the addition of virus. Lower inhibition activity was observed when the virus was allowed to attach to the cell surface before the addition of peptide. However, the plaque size was smaller compared to the untreated control, indicating that the peptides may also interfere with cell-to-cell spread of the virus. The two most potent antiviral peptides exhibited synergy with acyclovir against HSV.

The enzyme gamma-glutamyltransferase (GGT) is frequently overexpressed in cancer cells and tissues and has significant utility as a cancer marker. Significant heterogeneity of the enzyme has been described due to both transcriptional and post-translational variations. For possible use in diagnosis and follow-up of patients with colorectal cancer, a search was performed for specific mRNA subtypes and glycan structures of the enzyme in liver metastases. We found no differences in the distribution of three GGT mRNA subtypes (fetal liver, HepG2, placenta) in metastatic tissue and normal liver tissue. Furthermore, the three subtypes were present in leukocytes isolated from both normal individuals and cancer patients. Two colon carcinoma cell lines (Colo 205 and HCC 2998) also displayed the three forms and no consistent changes in mRNA composition were noted after butyrate-induced differentiation of the cells. Thus, neither of the GGT mRNA subforms appear to be tumor-specific, although some qualitative and quantitative variations were noted. Two distinct glycosylation features were detected for GGT in metastatic tissue in contrast to normal liver GGT; an extreme sialic acid heterogeneity and a significant increase in beta1,6GlcNAc branching. The GGT glycans from the two colon carcinoma cell lines also possessed these features. As butyrate treatment of the cells resulted in an increased sialic acid content and a reduced beta1,6GlcNAc branching, the described carbohydrate structures appear to be part of a tumor-related pattern. We were, however, unable to identify such GGT isoforms in serum from patients with advanced colorectal cancer. This indicates that their usefulness in diagnostic use is doubtful.

Lactoferrin (LF) is a multifunctional glycoprotein, which plays an important role in immune regulation and defense mechanisms against bacteria, fungi, and viruses. Upon peptic digestion of LF, a peptide called lactoferricin (Lfcin) is generated. Lfcin corresponds to the N-terminal part of the protein. In this study we investigated the antiviral activity of bovine and human Lfcin against Herpes simplex virus (HSV)-1 and HSV-2. The 50% effective concentrations (EC(50)) for LF and Lfcin against several clinical isolates of HSV-1 and HSV-2, including acyclovir (ACV)-resistant strains, were determined. We further evaluated the effect of the combination of either LF or Lfcin with ACV against HSV-1 and HSV-2. Synergy was observed between both LF or Lfcin in combination with ACV against the HSV laboratory strains. The 50% effective concentration (EC(50)) for ACV and LF or Lfcin, when combined with ACV, could be reduced by two- to sevenfold compared to the EC(50) when the drugs were used alone.

Synoxazolidinone C is a new member of the synoxazolidinone family of compounds which are dibrominated guanidinium 4-oxazolidinones isolated from the sub-Arctic ascidian Synoicum pulmonaria. The compound was isolated along with synoxazolidinones A and B from the same ascidian extract. Spectroscopic methods revealed that the synoxazolidinone C differed from the other synoxazolidinones by an unprecedented bicyclic partial structure, containing an additional pyrrolidine ring. Synoxazolidinone C exhibited both antibacterial and anticancer activities.

Lactoferrin is mainly produced by polymorphonuclear leukocytes and has been demonstrated in mammalian milk and external secretions. Lactoferrin is an iron-binding, multifunctional protein and may play an important role in immune regulation and in defense mechanisms against bacteria, fungi and viruses. Lactoferricin is a potent antimicrobial peptide generated from the N-terminal part of lactoferrin by pepsin cleavage. We demonstrate that lactoferrins from different species and its N-terminal peptide lactoferricin (particularly the cyclic form) inhibit expression of early and late antigens, as well as production of infectious viral progeny during human cytomegalovirus (HCMV) infection in vitro. Iron-saturated lactoferrin did not affect HCMV antigen expression. Heparin had the same effects as iron-depleted lactoferrin. Yet, mixtures of lactoferrin and heparin did not inhibit HCMV multiplication i.e. lactoferrin and heparin seemed to mutually block each other's antiviral activities. HCMV-infected cells exposed to lactoferrin and cyclic lactoferricin contained less intracellular virus than unexposed cells. The antiviral activity of cyclic lactoferricin was more than seven-fold weaker than that of the maternal molecule. Lactoferrin and cyclic lactoferricin prevented HCMV entrance into the host cell.

The marine polyunsaturated lipid-derived natural product all-(Z)-5,7-dihydroxy-2-(4Z,7Z,10Z,13Z,16Z-nonadecapentaenyl) (1) and four analogs 5-8 have been synthesized and evaluated as antioxidants in two cell-based assays. The natural product 1 and the analog 5 exhibited interesting antioxidant effects with IC50-values of 14±9 and 29±3μM, respectively, in a cellular lipid peroxidation antioxidant activity assay using HepG2 cells. Moreover, in the HepG2 cellular antioxidant activity assay, the natural product 1 exhibited strong protective effects against reactive oxygen species with an IC50=160±25μM.

Three known bromophenols, 2,3-dibromo-4,5-dihydroxybenzylaldehyde ( 1 ), 2,2 ,3-tribromo-3 ,4,4 ,5-tetrahydroxy-6 -hydroxymethyldiphenylmethane ( 2 ) and bis(2, 3-dibromo-4,5-dihydroxylbenzyl) ether ( 3 ), and one new one, 5,5 Ž-oxybis(methylene)bis (3-bromo-4-(2 ,3 -dibromo-4 ,5 -dihydroxylbenzyl)benzene-1,2-diol) ( 4 ), were isolated from an extract of the red alga, Vertebrata lanosa . The antioxidant activity of these four bromophenols was examined using one biochemical and two cellular assays: Oxygen Radical Absorbance Capacity (ORAC), Cellular Antioxidant Activity (CAA) and Cellular Lipid Peroxidation Antioxidant Activity (CLPAA) assays. Compound 2 distinguished itself by showing potent activity, having a better antioxidant effect than luteolin in both the CAA and CLPAA assays and of quercetin in the CLPAA assay. Although several bromophenols are known to be potent antioxidants in biochemical assays, this is the first time their cellular antioxidant activity has been demonstrated.

The marine opisthobranch Scaphander lignarius has been analyzed in the systematic search for novel bioactive compounds in Arctic marine organisms using bioassay guided fractionation. A number of highly cytotoxic fractions were shown to contain mainly polyunsaturated fatty acids (PUFAs). Selected PUFAs were isolated and identified using both liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR). It was shown that the opisthobranch contained unusual PUFAs such as several ω3 fatty acids and the ω7 heneicosa-5,8,11,14-tetraenoic acid (21:4 n-7) not isolated before. The organism was shown to be a very rich source of PUFAs and the activity of the isolated compounds against a range of human cancer cell lines (melanoma, colon carcinoma and breast carcinoma) is further reported. The ω7 PUFA was significantly more cytotoxic in comparison with reference ω6 arachidonic and ω3 eicosapentaenoic acid. A noteworthy non-selective cytotoxicity against normal lung fibroblasts was also established. The paper contains isolation protocols in addition to cytotoxicity data of the isolated compounds. The potential of marine mollusks as a source for rare PUFAs is also discussed.

Low-molecular weight antioxidants receive increased interest both as protectors from disease and therapeutics, but are also used in other applications such as sun screens and food additives. Antioxidants are present in all organisms and are well characterized in terrestrial organisms whereas little knowledge exists for marine organisms. Only in Norwegian Arctic and sub-Arctic waters, thousands of species (invertebrates, algae, bacteria) are yet to be chemically characterized. Here we describe cell-based antioxidant assays (CAA and CLPAA in microplate format, and the Comet assay) measuring both hydrophilic and lipophilic antioxidant activities of single compounds and extract mixtures. Contrary to our chemical methods (ORAC and FRAP), cell-based assays assess cell membrane permeability and operate under more physiologically relevant conditions. A brief overview of our group’s work on finding novel antioxidants in marine organisms including testing activities of semipurified natural product extracts, identification of active compounds, purification of compounds in greater amounts, as well as how antioxidants can be tested in animals and humans is also described.

Bioassay-guided fractionation of the sub-Arctic ascidian Synoicum pulmonaria collected off the Norwegian coast led to the isolation of a novel family of brominated guanidinium oxazolidinones named synoxazolidinones A and B (1 and 2). The backbone of the compounds contains a 4-oxazolidinone ring rarely seen in natural products. The structure of the compounds was determined by spectroscopic methods. The synoxazolidinones exhibited antibacterial and antifungal activities.

Lactoferrin (LF) is a multifunctional glycoprotein, which plays an important role in immune regulation and defense mechanisms against bacteria, fungi, and viruses. Lactoferricin (Lfcin) is a potent antimicrobial peptide generated from the N-terminal part of LF by pepsin cleavage. In this study, we investigated the mechanisms of the anti-herpes simplex virus (anti-HSV) activity of LF and Lfcin. The results demonstrated that LF and Lfcin inhibited the entry of HSV into Vero cells. LF had no effect against HSV after the virus had entered the cells, while Lfcin exerted antiviral activity also after the initial binding of the virus to the host cell. The distribution of LF and Lfcin in the cells was investigated by immunogold-labeling and transmission electron microscope (TEM). LF was found mainly at the cell surface in cells expressing heparan sulphate. Lfcin was randomly distributed intracellularly. LF must be present at the cell surface to exert antiviral activity, while Lfcin exert its antiviral activity also when found mainly intracellularly. Both LF and Lfcin were dependent on the presence of heparan sulphate at the cell surface to exert their antiviral activity.

Two bacterial isolates from the Barents Sea, both belonging to the genus Algibacter, were found to yield extracts with anti-bacterial bioactivity. Mass spectrometry guided dereplication and purification of the active extracts lead to the isolation of the same active principle in both extracts. The structure of the bioactive compound was identified via mass spectrometry and nuclear resonance spectroscopy and it turned out to be the known lipopeptide Lipid 430. We discovered and determined its previously unknown anti-bacterial activity against Streptococcus agalactiae and revealed a cytotoxic effect against the A2058 human melanoma cell line at significantly lower concentrations compared to its anti-bacterial concentration. Flow cytometry and microscopy investigations of the cytotoxicity against the melanoma cell line indicated that Lipid 430 did not cause immediate cell lysis. The experiments with melanoma cells suggest that the compound functions trough more complex pathways than acting as a simple detergent.