Bioassay-Guided Fractionation Leads to the Detection of Cholic Acid Generated by the Rare Thalassomonas sp.

Bacterial symbionts of marine invertebrates are rich sources of novel, pharmaceutically relevant natural products that could become leads in combatting multidrug-resistant pathogens and treating disease. In this study, the bioactive potential of the marine invertebrate symbiont Thalassomonas actiniarum was investigated. Bioactivity screening of the strain revealed Gram-positive specific antibacterial activity as well as cytotoxic activity against a human melanoma cell line (A2058). The dereplication of the active fraction using HPLC-MS led to the isolation and structural elucidation of cholic acid and 3-oxo cholic acid. T. actiniarum is one of three type species belonging to the genus Thalassomonas. The ability to generate cholic acid was assessed for all three species using thin-layer chromatography and was confirmed by LC-MS. The re-sequencing of all three Thalassomonas type species using long-read Oxford Nanopore Technology (ONT) and Illumina data produced complete genomes, enabling the bioinformatic assessment of the ability of the strains to produce cholic acid. Although a complete biosynthetic pathway for cholic acid synthesis in this genus could not be determined based on sequence-based homology searches, the identification of putative penicillin or homoserine lactone acylases in all three species suggests a mechanism for the hydrolysis of conjugated bile acids present in the growth medium, resulting in the generation of cholic acid and 3-oxo cholic acid. With little known currently about the bioactivities of this genus, this study serves as the foundation for future investigations into their bioactive potential as well as the potential ecological role of bile acid transformation, sterol modification and quorum quenching by Thalassomonas sp. in the marine environment.

Adding Zooplankton to the OSMAC Toolkit: Effect of Grazing Stress on the Metabolic Profile and Bioactivity of a Diatom.

"One strain many compounds" (OSMAC) based approaches have been widely used in the search for bioactive compounds. Introducing stress factors like nutrient limitation, UV-light or cocultivation with competing organisms has successfully been used in prokaryote cultivation. It is known that diatom physiology is affected by changed cultivation conditions such as temperature, nutrient concentration and light conditions. Cocultivation, though, is less explored. Hence, we wanted to investigate whether grazing pressure can affect the metabolome of the marine diatom Porosira glacialis, and if the stress reaction could be detected as changes in bioactivity. P. glacialis cultures were mass cultivated in large volume bioreactor (6000 L), first as a monoculture and then as a coculture with live zooplankton. Extracts of the diatom biomass were screened in a selection of bioactivity assays: inhibition of biofilm formation, antibacterial and cell viability assay on human cells. Bioactivity was found in all bioassays performed. The viability assay towards normal lung fibroblasts revealed that P. glacialis had higher bioactivity when cocultivated with zooplankton than in monoculture. Cocultivation with diatoms had no noticeable effect on the activity against biofilm formation or bacterial growth. The metabolic profiles were analyzed showing the differences in diatom metabolomes between the two culture conditions. The experiment demonstrates that grazing stress affects the biochemistry of P. glacialis and thus represents a potential tool in the OSMAC toolkit.

Two Novel Lyso-Ornithine Lipids Isolated from an Arctic Marine Lacinutrix sp. Bacterium.

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.

Characterization of Rhamnolipids Produced by an Arctic Marine Bacterium from the Pseudomonas fluorescence Group.

The marine environment is a rich source of biodiversity, including microorganisms that have proven to be prolific producers of bioactive secondary metabolites. Arctic seas are less explored than warmer, more accessible areas, providing a promising starting point to search for novel bioactive compounds. In the present work, an Arctic marine Pseudomonas sp. belonging to the Pseudomonas (P.) fluorescence group was cultivated in four different media in an attempt to activate biosynthetic pathways leading to the production of antibacterial and anticancer compounds. Culture extracts were pre-fractionated and screened for antibacterial and anticancer activities. One fraction from three of the four growth conditions showed inhibitory activity towards bacteria and cancer cells. The active fractions were dereplicated using molecular networking based on MS/MS fragmentation data, indicating the presence of a cluster of related rhamnolipids. Six compounds were isolated using HPLC and mass-guided fractionation, and by interpreting data from NMR and high-resolution MS/MS analysis; the structures of the compounds were determined to be five mono-rhamnolipids and the lipid moiety of one of the rhamnolipids. Molecular networking proved to be a valuable tool for dereplication of these related compounds, and for the first time, five mono-rhamnolipids from a bacterium within the P. fluorescence group were characterized, including one new mono-rhamnolipid.

Ponasterone A and F, Ecdysteroids from the Arctic Bryozoan Alcyonidium gelatinosum.

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 Edgeøya, 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 α and ß. 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.

Targeted Dereplication of Microbial Natural Products by High-Resolution MS and Predicted LC Retention Time.

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.

The immunomodulatory effects of barettin and involvement of the kinases CAMK1α and RIPK2.

BACKGROUND: Barettin is a marine natural compound with reported anti-inflammatory and antioxidant properties. The combination of these effects led us to explore barettin further as an inhibitor of atherosclerosis development. METHODS: The effect of barettin on MCP-1 and IL-10 secretion from activated immune cells was detected by ELISA. Determination of cell viability of oxidized low-density lipoprotein (oxLDL) and barettin exposed HUVEC cells were investigated by using CellTiter 96® AQ(ueous) One Solution. The kinase inhibition assays were performed using a radioactive ((33)P-ATP) filter binding assay at the University of Dundee, UK. RESULTS: Barettin reduces the secretion of monocyte chemotactic protein-1 (MCP-1) from LPS-stimulated monocytes, but was not able to prevent oxLDL-induced cell death in HUVEC. Barettin has inhibitory activity against two protein kinases related to inflammation, namely the receptor-interacting serine/threonine kinase 2 (RIPK2) and calcium/calmodulin-dependent protein kinase 1α (CAMK1α). We also demonstrate that barettin reduce the production of the anti-inflammatory cytokine interleukin-10 (IL-10) in a dose and time-dependent manner, possibly by inhibiting CAMK1α. CONCLUSIONS: The anti-inflammatory activity of barettin is exerted through the regulation of inflammatory mediators such as MCP-1 and IL-10, possibly via inhibition of kinases.

Short cationic antimicrobial peptides bind to human alpha-1 acid glycoprotein with no implications for the in vitro bioactivity.

Several drugs interact with the major plasma proteins serum albumin and alpha-1 acid glycoprotein. Such binding may be either beneficial or disadvantageous from a pharmacokinetic perspective. In the present paper, we investigate the thermodynamics involved in the binding of a series of promising cationic antimicrobial peptides to the alpha-1 acid glycoprotein using isothermal titration calorimetry. The drug-like peptides are able to effectively destroy multiresistant bacterial strains, and members of this peptide class are currently in clinical phase II trials. Similar peptides, in a previous study, have been shown to bind to serum albumin resulting in a 10-fold reduction in the peptides ability to kill bacteria in vitro. Here, it is shown that the peptides also are ligands for alpha-1 glycoprotein with moderate binding affinities. The binding mode is investigated in detail using molecular docking, which maps the interaction to sub-pockets I, II and III of the binding site. Despite this interaction, protein binding is shown to have little or no effect on the ability of the peptides to kill bacteria in vitro, either at normal physiological or acute phase concentrations. The results show that although the peptides interact with the binding pocket of alpha-1 acid glycoprotein, the low stoichiometric binding ratio ensures that the interaction is not an obstacle for further development of these promising peptides as antimicrobial therapies.

A High-Affinity Peptide Ligand Targeting Syntenin Inhibits Glioblastoma.

Despite the recent advances in cancer therapeutics, highly aggressive cancer forms, such as glioblastoma (GBM), still have very low survival rates. The intracellular scaffold protein syntenin, comprising two postsynaptic density protein-95/discs-large/zona occludens-1 (PDZ) domains, has emerged as a novel therapeutic target in highly malignant phenotypes including GBM. Here, we report the development of a novel, highly potent, and metabolically stable peptide inhibitor of syntenin, KSL-128114, which binds the PDZ1 domain of syntenin with nanomolar affinity. KSL-128114 is resistant toward degradation in human plasma and mouse hepatic microsomes and displays a global PDZ domain selectivity for syntenin. An X-ray crystal structure reveals that KSL-128114 interacts with syntenin PDZ1 in an extended noncanonical binding mode. Treatment with KSL-128114 shows an inhibitory effect on primary GBM cell viability and significantly extends survival time in a patient-derived xenograft mouse model. Thus, KSL-128114 is a novel promising candidate with therapeutic potential for highly aggressive tumors, such as GBM.

Bioactivity of Serratiochelin A, a Siderophore Isolated from a Co-Culture of Serratia sp. and Shewanella sp.

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.

Field sampling marine plankton for biodiscovery.

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.

Light and temperature effects on bioactivity in diatoms.

Isolates of five pelagic North Atlantic marine diatoms (Bacillariophyceae): Attheya longicornis, Chaetoceros socialis, Chaetoceros furcellatus, Skeletonema marinoi and Porosira glacialis were cultivated in large photobioreactors at two light and two temperature regimes to test if this affected bioactivity. We screened for bioactivity in assays representing five different therapeutic areas: diabetes II (PTP1b), cancer (melanoma cells, A2058), anti-oxidants (FRAP), immunomodulation (TNFa) and anti-infection (MRSA, Enterococcus faecalis, Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa). All the diatom strains showed activity in two or more assays. We detected differences in bioactivity both between species and within species cultivated with different light and temperature regimes. Our results demonstrate the potential for a more exhaustive exploitation of diatom metabolites that can be obtained by manipulation of the cultivation conditions.

Heterogeneity in gamma-glutamyltransferase mRNA expression and glycan structures. Search for tumor-specific variants in human liver metastases and colon carcinoma cells.

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 and lactoferricin inhibit Herpes simplex 1 and 2 infection and exhibit synergy when combined with acyclovir.

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.

Anti-HSV activity of lactoferricin analogues is only partly related to their affinity for heparan sulfate.

Earlier studies have shown that the heparan sulfate (HS) on the cell surface acts as a receptor for herpes simplex virus (HSV). We have recently shown that bovine lactoferricin (LfcinB), a small part of the milk protein lactoferrin, inhibits HSV-1 and HSV-2 infection, probably by blocking the entry of the virus. The human homologue (18-42), which shares 36% sequence similarity with LfcinB (17-41), displayed much lower antiviral activity. In the present study, a set of cyclic and linear human and bovine Lfcin derivatives were constructed to investigate the relation between their affinity to HS and chondroitin sulfate (CS) and their antiviral activity against HSV-1 and HSV-2. The lactoferrin (LF) proteins and several of the Lfcin derivatives exhibited similar affinity for HS, but the LF proteins possess a much higher antiviral activity than the smaller peptides. Our structure-activity relationship studies on the Lfcin derivates confirmed that affinity for HS, that was correlated to the net positive charge, is an important factor, but does not well predict the antiviral activity. Structural parameters such as hydrophobicity, molecular size, spatial distribution of charged and lipophilic amino acids, and the cyclic structure of Lfcin also seem to be important factors to govern antiviral activity against HSV.

Polyunsaturated fatty acid-derived chromones exhibiting potent antioxidant activity.

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.