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The suomilide and the banyasides are highly modified and functionalized non-ribosomal peptides produced by cyanobacteria of the order Nostocales. These compound classes share several substructures, including a complex azabicyclononane core, which was previously assumed to be derived from the amino acid tyrosine. In our study we were able to isolate and determine the structures of four suomilides, named suomilide B – E (1–4). The compounds differ from the previously isolated suomilide A by the functionalization of the glycosyl group. Compounds 1–4 were assayed for anti-proliferative, anti-biofilm and anti-bacterial activities, but no significant activity was detected. The sequenced genome of the producer organism Nostoc sp. KVJ20 enabled us to propose a biosynthetic gene cluster for suomilides. Our findings indicated that the azabicyclononane core of the suomilides is derived from prephenate and is most likely incorporated by a proline specific non-ribosomal peptide synthetase-unit.

Source at: http://doi.org/10.3389/fphar.2018.01006 Despite the discovery and development of novel therapies, cancer is still a leading cause of death worldwide. In order to grow, tumor cells require large quantities of nutrients involved in metabolic processes, and an increase in iron levels is known to contribute to cancer proliferation. Iron plays an important role in the active site of a number of proteins involved in energy metabolism, DNA synthesis and repair, such as ribonucleotide reductase, which induce G0/S phase arrest and exert a marked antineoplastic effect, particularly in leukemia and neuroblastoma. Iron-depletion strategies using iron chelators have been shown to result in cell cycle arrest and apoptosis. Deferoxamine (DFO) was the first FDA-approved drug for the treatment of iron overload pathologies, and has also been recognized as having anticancer properties. The high cost, low permeability and short plasma half-life of DFO led to the development of other iron-chelating drugs. Pyridoxal isonicotinoyl hydrazone (PIH) and its analogs chelate cellular iron by tridentate binding, and inhibit DNA synthesis more robustly than DFO, demonstrating an effective antiproliferative activity. Here, we investigated the biological effects of a PIH derivative, 3-chloro-N′-(2-hydroxybenzylidene)benzohydrazide (CHBH), known to be a lysine-specific histone demethylase 1A inhibitor. We showed that CHBH is able to induce cell proliferation arrest in several human cancer cell lines, including lung, colon, pancreas and breast cancer, at micromolar levels. Our findings indicate that CHBH exerts a dual anticancer action by strongly impairing iron metabolism and modulating chromatin structure and function.

Infectious diseases have been a problem for humans since the beginning of human existence. The “golden age” of antibiotics started at the end of the 1920s, with the discovery of penicillin by Sir Alexander Fleming. This was followed by the discovery of several life-saving antibiotics. The number of new marketed antibiotics has declined, and most pharmaceutical companies are no longer working in antibiotic development. This in itself would be unproblematic, had it not been for the rapid ability of bacteria to become resistant towards previously debilitating agents. The need for new antibiotics is therefore eminent. Natural products have been important contributors for antibiotic drug discovery and development. Microorganisms have been a particularly proliferative source of antibiotics, providing us with among others the penicillins, aminoglycosides, tetracyclines and polymyxins. Most naturally derived pharmaceuticals, including antibiotics, originate from terrestrial organisms. This is mainly because the terrestrial environment historically has been easier to access compared to the marine environment below the intertidal zone. The marine environment is highly diverse, and there is still a huge biodiversity that is yet to be explored. In this project, Arctic and sub-Arctic marine bacteria and fungi were cultivated and studied for their production of natural products. The cultures were extracted and fractionated, and the fractions were tested for bioactivity, mainly focusing on antibacterial activity. Using bioactivity-guided isolation, compounds were isolated and structurally characterized. Finally, the bioactivity of the isolated compounds was broadly evaluated. In paper I, a known siderophore, serratiochelin A, was isolated from a co-culture of two bacteria, Serratia sp. and Shewanella sp. The compound was not detected in axenic cultures, indicating that cocultivation triggered production. The acid-catalyzed degradation of serratiochelin A into serratiochelin C was also observed. Serratiochelin A had weak activity against Staphylococcus aureus, melanoma cells and non-malignant lung fibroblasts. No activity was observed for the degradation product serratiochelin C, indicating that the oxazoline moiety in the original compound is essential for the bioactivity. In paper II, a marine bacterium Lacinutrix sp. was cultivated and studied for its ability to produce bioactive natural products. Through bioactivity-guided isolation, two new lyso-ornithine lipids were isolated, and their structures elucidated, showing that they only differed by the length of the hydrocarbon tail. Analysis by UHPLC-HR-MS indicate that the purified solutions are mixtures of isomers, but these were not possible to separate by preparative HPLC-MS. The compounds were evaluated for antibacterial activity and antiproliferative activity against human cells. Compound 1 displayed weak activity against Streptococcus agalactiae, while compound 2 had weak activity against melanoma cells. In paper III, a new dimeric naphthopyrone substituted with a sulphate group was isolated in high yields from cultures of an obligate marine fungus in the family Lulworthiaceae. The compound was tested against an extended panel of clinical bacterial isolates and showed potent antibacterial activity against several clinical methicillin-resistant Staphylococcus aureus isolates, with MICs down to 1.56 μg/mL. Acid-catalyzed degradation was also observed. The compound also displayed moderate activities against three human cell lines: melanoma, hepatocellular carcinoma, and non-malignant lung fibroblast. In paper IV, a new chlovalicin variant, chlovalicin B, was isolated from cultures of the obligate marine fungus Digitatispora marina. The fungus has previously been studied for its distribution in the marine environment but has not been extensively studied for its biosynthetic potential. The compound was isolated in low yields, and the structure was elucidated by NMR and HRMS experiments. The compound was assessed for a range of bioactivities and had weak antiproliferative activity against human melanoma cells.

Global trends show that interest in fish products and fish consumption are increasing, while marine fishery resources are decreasing. Fish processing industry produces a high amount of residual raw materials that have nutritious proteins and other valuable compounds. An optimization of residual raw materials’ utilization can help meet the growing demand for fish products and help reduce environmental problems. A promising valorization method is enzymatic protein hydrolysis. In this project, enzymatic protein hydrolysis was performed to produce protein hydrolysates from complex material of Atlantic cod heads. Three types of material from cod heads (muscle, skin and bone) were hydrolyzed by 23 different proteases. The produced hydrolysates were analyzed and evaluated based on yield, molecular weight (determined by SEC and SDS-PAGE), selectivity of proteases towards collagen and myofibrillar proteins (selectivity ratio), and bioactivity properties (anti-proliferative, antioxidant and anti-inflammatory). It was determined that the highest yield from muscle was produced by Tail 191, from skin by Tail 194, and from bone by Tail 190. Different proteases produced hydrolysates with different average MW. Notably, Tail 189 produced hydrolysates with the lowest average MW from all three types of raw material. The SDS-PAGE patterns of the hydrolysates from skin indicated that Tail 193 and Endocut 01 might have selectivity towards peptide bonds they cleave. The selectivity ratio identified that Endocut 01 had the highest selectivity towards myofibrillar proteins and Flavourzyme was the only enzyme selective to collagen. The results of bioactivity assays showed no antiproliferative or anti-inflammatory activity of the hydrolysates, however, all hydrolysates demonstrated antioxidant activity. The hydrolysates made from muscle showed higher antioxidant activity than the hydrolysates prepared from skin and bone. Based on the results, conditions for a scale-up experiment (from 5 g of raw material to 250 g) were suggested, which included recommendation of several enzymes per material, adjustment of temperature to optimal for each enzyme, monitoring of hydrolysis process and determination of ash content in the product.

Fungi are among the most prolific producers of secondary metabolites, many of which have huge utility for society as various anti-infective and anticancer drugs. The genes encoding these secondary metabolites are arranged in continuous biosynthetic gene clusters (BGCs). Each fungus can have tens of different BGCs in its genome, possessing the ability to produce a multitude of secondary metabolites with potential for medical utility. However, most of the BGCs are silent under standard laboratory conditions, packaged in heterochromatin, inaccessible for transcription. To activate these clusters the heterochromatin must be converted to euchromatin, the transcription accessible form. The addition of small molecule epigenetic modifiers, inhibiting the activity of transcription repressive epigenetic enzymes, have been proven to effectively induce secondary metabolism in many fungi. As this approach does not require genetic manipulation, it is potentially applicable to a broad range of fungi, recalcitrant to genetic manipulation. This method could therefore have the potential for implementation. the high-throughput screening of microorganisms at Marbio. In this thesis, the method of manipulating the epigenetic machinery of marine filamentous fungi by the application of epigenetic modifiers 5-azacytidine, SBHA, and nicotinamide were employed as a means for inducing activation of biosynthetic gene clusters. This is the first time epigenetic modifiers have been applied to any microbial cultures at Marbio. The fungi investigated were cultivated in solid and liquid media with added epigenetic inhibitors in different combinations to study the effects of the modifiers on the fungi. The fungal cultures were subsequently extracted and subjected to bioactivity testing. The bioactivity screening revealed that epigenetic modifiers did not induce the production of bioactive secondary metabolites in detectable amounts. However, SBHA was found to alter colony morphology and pigmentation in some cultures, indicating that these fungi are accessible for epigenetic manipulation by HDAC inhibitors. Increased concentrations or applications of more potent HDAC inhibitors such as SAHA, might be necessary to observe more evident changes in the fungal metabolomes.

Microalgae have proven to contain a vast amount of beneficial, high value compounds like proteins, lipids and powerful antioxidants as well as some interesting bioactive compounds. Nevertheless, microalgae are severely underrepresented in conjunction with marine bioactive natural product discovery. This thesis aims to unlock bioactivity potential of a microalgae species from the most abundant and diverse group of microalgae, namely the diatoms, by bioassay guided isolation. This diatom species is isolated from northern Arctic waters, where research on bioactivity potential in diatoms are poorly investigated. Five samples of raw biomass from a diatom species cultivated at five different conditions were extracted, fractionated through FLASH chromatography and screened in five different bioassays; an antibacterial assay against five bacteria strains (Staphylococcus aureus, Escherichia coli, Enterococcus faecalis, Pseudomonas aeruginosa and Streptococcus agalactiae), an anti-biofilm assay (against S. epidermidis biofilm-formation), a MTS cell viability assay with three different cell lines (human melanoma A2058, human colon carcinoma HT29, and human pulmonary fibroblast MRC-5), a cellular antioxidant activity assay (with a THP-1 cell line) and an anti-inflammatory assay (with a HepG2 cell line). Some selected samples were fractionated further by HPLC chromatography and screened again for anti-biofilm and anti-inflammatory properties. Bioactivity was detected in all assays, and interestingly, some variation was observed within the assays for the different cultivation conditions. This indicated that the metabolite bioactivity profile of the diatom might have changed due to the varying pre-experiment cultivation conditions. The results demonstrate the huge bioactivity potential of diatoms, and that modification of cultivation conditions might be used to our advantage to obtain bioactive fractions with a different range of activities.

The marine environment is an untapped source for biodiversity and has a great potential to provide the drugs of the future. Antibiotic resistance is an increasing threat worldwide and the need for discovering new antibacterial compounds is urgent. Marine microorganisms produce a wide range of bioactive compounds, and marine fungi have only been exploited to a small extent. This creates a great potential for finding novel antibacterial compounds in marine fungi. In this study, the antibacterial and anticancer potential for five marine fungi Acremonium sp. TS7, Typhula sp., Amylocarpus encephaloides, Pseudogymnoascus sp. TS12 and Digitatispora marina have been investigated for antibacterial and anticancer activity. The One-strain-many-compounds (OSMAC)-approach was to try to induce the production of secondary metabolites by applying stress to the marine fungi. These five marine fungi were fermented on four different media and at two different temperatures. Half of the fermentations were co-cultivated with the marine bacteria Leeuwenhoekiella sp. The active fractions were dereplicated with UPLC-QToF-MS. The antibacterial compounds were identified as rhamnolipids and were found in all active samples. Rhamnolipids were also identified in the bacteria controls with Leeuwenhoekiella sp., suggesting that Leeuwenhoekiella sp. is the producer of rhamnolipids. The bioactivity effects of our OSMAC-approach were not as expected, this is probably due to that the culture conditions selected for this study did not trigger the production of secondary metabolites. The full bioactivity potential for Acremonium sp. TS7, Typhula sp., Amylocarpus encephaloides, Pseudogymnoascus sp. TS12 and Digitatispora marina has not been fully investigated in this study, but should be further explored.

A new siderophore containing a 4,5-dihydroimidazole moiety was isolated from Pseudoalteromonas piscicida S2040 together with myxochelins A and B, alteramide A and its cycloaddition product, and bromo- and dibromoalterochromides. The structure of pseudochelin A was established by spectroscopic techniques including 2D NMR and MS/MS fragmentation data. In bioassays selected fractions of the crude extract of S2040 inhibited the opportunistic pathogen Pseudomonas aeruginosa. Pseudochelin A displayed siderophore activity in the chrome azurol S assay at concentrations higher than 50 mu M, and showed weak activity against the fungus Aspergillus fumigatus, but did not display antibacterial, anti-inflammatory or anticonvulsant activity. (C) 2017 Elsevier Ltd. All rights reserved.

Bioprospecting is the systematic search for and discovery of products in nature, with the purpose of developing commercial products. The marine environment displays a rich biological diversity, as well as a diversity within environmental factors. This environment has necessitated the production of potent secondary metabolites by marine organisms in their arms race against predators and pathogens, in the battle for space and to increase chances of reproduction. The resulting compounds are generally known to have unique chemical features, often unknown from terrestrial sources, as well as interesting biological activities. Due to these factors, they are believed to hold an immense potential as lead compounds in development of commercial products. The aim of this thesis was to isolate and characterise secondary metabolites from extracts of eight Arctic, marine invertebrates. Prefractionated extracts were screened for anticancer activity, and active fractions were dereplicated to investigate if the bioactive compound(s) was novel or had been previously reported. Three compounds believed to be novel were isolated, structure elucidated and biologically characterised. A novel compound, named BI-L-665.6 in this thesis, was isolated from the organic extract of Bryozoa indet. In addition, Ponasterone A (Pon A) and dehydroxy-Pon A were isolated from the organic extract of Alcyonidium gelatinosum. Pon A was first isolated from Podocarpus nakaii in 1966, but this is the first time that this compound has been isolated from A.gelatinosum. Biological characterisation of the isolated compounds detected no anticancer or antibacterial activity at the test concentrations employed in the assays. The results from this thesis show that bioprospecting of collected marine invertebrates enables discovery of secondary metabolites with novel chemistry, as well as previously reported compounds in new species.

The number of compounds being isolated from the marine environment is increasing, and there is a great potential for discovering new marine derived drug candidates. Improved collection techniques has strengthened bioprospecting on a wider diversity of marine microorganisms. The focus on microorganisms has led to the realisation that many of the natural products originally isolated from macroorganisms, are metabolic products produced by their associated microorganisms. This, and the fact that most marketed antimicrobial drugs originate from microorganisms, motivated the work conducted as part of this thesis. In this study, two Arctic marine bacteria of the genus Leifsonia and Polaribacter were studied. The “One Strain-Many Compounds” (OSMAC) approach was utilised when cultivating the bacteria, in an attempt to trigger the bacteria into activating different metabolic pathways and producing compounds with interesting chemistry and bioactivity. Seven different cultivation treatments were used, varying different parameters e.g. media composition and temperature. The secondary metabolites secreted by the cultivated bacteria were harvested, extracted and prefractionated. The fractions were screened for antibacterial activity, inhibition of biofilm formation and anticancer activity. The bioactivity screening resulted in eight active fractions. Dereplication of the active fractions gave several candidates that could be responsible for the observed bioactivity. The results from this thesis give a valuable starting point for further research on cultivation of Arctic marine bacteria, with the purpose of producing bioactive secondary metabolites.

The diversity of the marine species inhabiting the oceans makes them highly interesting as potential sources of bioactive molecules for various applications. The extreme environment in the Barents Sea, in terms of temperature and light availability, have forced its organisms to adapt to their surroundings by developing unique biomolecules, and Arctic marine organisms are therefore an excellent starting point when searching for compounds with novel bioactivities. In this thesis, the focus has been on revealing potential antioxidant and anti-inflammatory effects of barettin, a compound originally isolated and described from the marine sponge Geodia barretti in 1986. Several papers have previously highlighted the strong anti-fouling properties of the molecule. In paper I, we presented novel antioxidant and anti-inflammatory properties of barettin. We found that barettin had strong dose-dependent antioxidant effect in both biochemical and cellular assays and follow-up experiments in an inflammatory cell model showed that barettin inhibited LPS-induced production of IL-1β and TNFα in a dose-dependent manner. The combination of the antioxidant and anti-inflammatory effects led us to explore barettin as a potent inhibitor of atherosclerosis development. In paper II, barettin was found to also inhibit LPS-induced production of MCP-1 in THP-1 cells, a chemokine strongly linked to monocyte recruitment and atherosclerosis. When THP-1 cells were co-stimulated with LPS and IL-4, IL-4 being a cytokine with pleiotropic effects, barettin increased the anti-inflammatory effect of IL-4. In paper II, we also presented a biochemical kinase inhibitory profile of barettin, showing inhibitory effect of three kinases, namely salt-inducible kinase 2 (SIK2), calcium/calmodulin-dependent kinase 1α (CAMK1α) and receptor-interacting protein kinase 2 (RIPK2), the two latter being involved in inflammatory processes. The inhibition observed on IL-1β production could be explained by the ability of barettin to inhibit RIPK2, as this kinase have a role in NF-κB activation and possibly also IL-1β maturation. The inhibitory effect of barettin on CAMK1α, which is involved in IL-10 regulation, probably result in an observed reduction of the anti-inflammatory cytokine IL-10 in THP-1 cells. In an ex vivo whole blood model stimulated with LPS, the effect of barettin on tissue factor (TF), TNFα, MCP-1, IL-10, TxB2, LTB4 and HMGB1 was evaluated (paper III). Barettin reduced TF, TNFα, TxB2, LTB4, MCP-1 and IL-10, whereas the effect on HMGB1 was modest. In summary, the overall results show that barettin affects several cellular processes and the effect of barettin should be further evaluated both in vitro and in vivo to find the true potential of the molecule.

Marine bioprospecting is a promising field of research that has gained interest worldwide for the past few decades, and can be characterised as a systematic and purpose oriented search for bioactive compounds or genetic material from marine sources. Several compounds have been isolated and some have found their way into the commercial market. The main aim of this master thesis was to investigate the antibacterial activity of bioactive compounds in the extracts of the marine ascidian Halocynthia pyriformis. Two other marine organisms Metridium senile and Parastichopus tremulus were also screened for antibacterial activity, but they were purified in two other parallel projects. Aqueous and organic extracts from H. pyriformis were purified using preparative HPLC and screened for antibacterial activity. The antibacterial activity was mostly found in the organic fractions against the Gram-positive bacteria E.faecalis, S. aureus and MRSA. The active fractions from the primary screening were subjected to a secondary screening where they did not display any clear activity. Three of the fractions showed some activity and was pooled and refractionated and later subjected to a third screening. The active fractions were analysed by ESI-TOF and ESI-Q without obtaining any results. A dose-response test was carried out to investigate the potency of the raw-extract. Due to weak results it was decided to fractionate the organic extract of H. pyriformis once again before it was subjected to a new primary screening. This time the fractions were concentrated and three of them displayed antibacterial activity and were subjected to MS-analyses in positive and negative mode. The results from the MS-analyses indicated that one of the fractions contained two active compounds with the m/z values 269.2094 and 505.4248. The calculated elemental composition proposed the empirical formula C16H28O3 for the molecular weight 268.2021. A search in databases containing known bioactive compounds proposed four different compounds with the same empirical formula. The elemental composition of the m/z value 505.4248, proposed two empirical formulas C32H56O4 and C31H54N4. None of these two formulas gave any hits in the database over known bioactive compounds. This could indicate that this is a novel compound. It was impossible to know the identity of these compounds without analysing them in NMR. NMR analyses require large amounts of pure compounds which was difficult to obtain in this thesis.

Metridium senile, Halocynthia pyriformis and ParaParastichopus tremulus were subjected to organic and aqueous extraction. The aqueous extract was made with water, while the organic was made with dichloromethane and methanol in 1:1 ratio. The aqueous and organic extracts were fractionated on reversed-phase HPLC and tested for antiviral activity against Herpes simplex virus-1. All the three organisms displayed anti-viral activity in the primary screening, but when retested their activity were lost. During the work with the thesis Dendrodoa aggregata was included.Screening of D.aggregata had shown that deoxytubastrine had antiviral activity in pilot studies. Tubasterine has shown antiviral activity in earlier studies so it was decided to purify and test deoxytubastrine against HSV-1. Analysis on LC-MS ESI-TOF in positive mode indicated the active compound with m/z 178.0906 amu which gave us the elemental composition C9H11N30. The identity of deoxytubastrine was confirmed by LC-MS ESI-TOF in positive mode and 1H-NMR. However, pure deoxytubastrine did not show any pronounced anti-viral activity.