35 Years of Marine Natural Product Research in Sweden: Cool Molecules and Models from Cold Waters.

Currents efforts in marine biodiscovery have essentially focused on temperate to tropical shallow water organisms. With more than 6000 species of marine plants and animals, the Kosterfjord area has the richest marine biodiversity in Swedish waters, but it remains understudied. The overall objective of our marine pharmacognosy research is to explore and reveal the pharmacological potential of organisms from this poorly explored region. More generally, we wish to understand aspects of structure-activity relationships of chemical interactions in cold-water marine environment (shallow and deep). Our strategy is based on ecologically guided search for compounds through studies of physiology and organism interactions coupled to identification of bioactive molecules guided by especially in vivo assays. The research programme originated in the beginning of the 1980s with a broad screening of Swedish marine organisms using both in vitro and in vivo assays, resulting in isolation and identification of several different bioactive molecules. Two congenerous cyclopeptides, i.e. barettin and 8,9-dihydrobarettin, were isolated from the deep-sea sponge Geodia barretti, and structurally elucidated, guided by their antifouling activity and their affinity to a selection of human serotonin receptors. To optimize the activity a number of analogues of barettin were synthezised and tested for antifouling activity. Within the EU project BlueGenics, two larger homologous peptides, barrettides A and B, were isolated from G. baretti. Also, metabolic fingerprinting combined with sponge systematics was used to further study deep-sea natural product diversity in the genus Geodia. Finally, the chemical property space model 'ChemGPS-NP' has been developed and used in our research group, enabling a more efficient use of obtained compounds and exploration of possible biological activities and targets. Another approach is the broad application of phylogenetic frameworks, which can be used in prediction of where-in which organisms-to search for novel molecules or better sources of known molecules in marine organisms. In a further perspective, the deeper understanding of evolution and development of life on Earth can also provide answers to why marine organisms produce specific molecules.

Isolation, Characterization, and Synthesis of the Barrettides: Disulfide-Containing Peptides from the Marine Sponge Geodia barretti.

Two disulfide-containing peptides, barrettides A (1) and B (2), from the cold-water marine sponge Geodia barretti are described. Those 31 amino acid residue long peptides were sequenced using mass spectrometry methods and structurally characterized using NMR spectroscopy. The structure of 1 was confirmed by total synthesis using the solid-phase peptide synthesis approach that was developed. The two peptides were found to differ only at a single position in their sequence. The three-dimensional structure of 1 revealed that these peptides possess a unique fold consisting of a long ß-hairpin structure that is cross-braced by two disulfide bonds in a ladder-like arrangement. The peptides are amphipathic in nature with the hydrophobic and charged residues clustered on separate faces of the molecule. The barrettides were found not to inhibit the growth of either Escherichia coli or Staphylococcus aureus but displayed antifouling activity against barnacle larvae (Balanus improvisus) without lethal effects in the concentrations tested.

Bioassays in natural product research - strategies and methods in the search for anti-inflammatory and antimicrobial activity.

INTRODUCTION: Identifying bioactive molecules from complex biomasses requires careful selection and execution of relevant bioassays in the various stages of the discovery process of potential leads and targets. OBJECTIVE: The aim of this review is to share our long-term experience in bioassay-guided isolation, and mechanistic studies, of bioactive compounds from different organisms in nature with emphasis on anti-inflammatory and antimicrobial activity. METHODS: In the search for anti-inflammatory activity, in vivo and in vitro model combinations with enzymes and cells involved in the inflammatory process have been used, such as cyclooxygenases, human neutrophils and human cancer cell lines. Methods concerning adsorption and perforation of bacteria, fungi, human cells and model membranes, have been developed and optimised, with emphasis on antimicrobial peptides and their interaction with the membrane target, in particular their ability to distinguish host from pathogen. RESULTS: A long-term research has provided experience of selection and combination of bioassay models, which has led to an increased understanding of ethnopharmacological and ecological observations, together with in-depth knowledge of mode of action of isolated compounds. CONCLUSION: A more multidisciplinary approach and a higher degree of fundamental research in development of bioassays are often necessary to identify and to fully understand the mode of action of bioactive molecules with novel structure-activity relationships from natural sources.

Antioxidative dietary compounds modulate gene expression associated with apoptosis, DNA repair, inhibition of cell proliferation and migration.

Many dietary compounds are known to have health benefits owing to their antioxidative and anti-inflammatory properties. To determine the molecular mechanism of these food-derived compounds, we analyzed their effect on various genes related to cell apoptosis, DNA damage and repair, oxidation and inflammation using in vitro cell culture assays. This review further tests the hypothesis proposed previously that downstream products of COX-2 (cyclooxygenase-2) called electrophilic oxo-derivatives induce antioxidant responsive elements (ARE), which leads to cell proliferation under antioxidative conditions. Our findings support this hypothesis and show that cell proliferation was inhibited when COX-2 was down-regulated by polyphenols and polysaccharides. Flattened macrophage morphology was also observed following the induction of cytokine production by polysaccharides extracted from viili, a traditional Nordic fermented dairy product. Coix lacryma-jobi (coix) polysaccharides were found to reduce mitochondrial membrane potential and induce caspase-3- and 9-mediated apoptosis. In contrast, polyphenols from blueberries were involved in the ultraviolet-activated p53/Gadd45/MDM2 DNA repair system by restoring the cell membrane potential. Inhibition of hypoxia-inducible factor-1 by saponin extracts of ginsenoside (Ginsen) and Gynostemma and inhibition of S100A4 by coix polysaccharides inhibited cancer cell migration and invasion. These observations suggest that antioxidants and changes in cell membrane potential are the major driving forces that transfer signals through the cell membrane into the cytosol and nucleus, triggering gene expression, changes in cell proliferation and the induction of apoptosis or DNA repair.

Gambogic acid is cytotoxic to cancer cells through inhibition of the ubiquitin-proteasome system.

Gambogic acid (GA), displays cytotoxicity towards a wide variety of tumor cells and has been shown to affect many important cell-signaling pathways. In the present work, we investigated the mechanism of action of GA by analysis of drug-induced changes in gene expression profiles and identified GA and the derivative dihydro GA as possible inhibitors of the ubiquitin-proteasome system (UPS). Both GA and dihydro GA inhibited proteasome function in cells resulting in the accumulation of polyubiquitin complexes. In vitro experiments showed that both GA and dihydro GA inhibited 20S chymotrypsin activity and the inhibitory effects of GA and dihydro GA on proteasome function corresponded with apoptosis induction and cell death. In conclusion, our results show that GA and dihydro GA exert their cytotoxic activity through inhibition of the UPS, specifically by acting as inhibitors of the chymotrypsin activity of the 20S proteasome.

Two brominated cyclic dipeptides released by the coldwater marine sponge Geodia barretti act in synergy as chemical defense.

The current work shows that two structurally similar cyclodipeptides, barettin (1) and 8,9-dihydrobarettin (2), produced by the coldwater marine sponge Geodia barretti Bowerbank act in synergy to deter larvae of surface settlers and may also be involved in defense against grazers. Previously, 1 and 2 were demonstrated to bind specifically to serotonergic 5-HT receptors. It may be suggested that chemical defense in G. barretti involves a synergistic action where one of the molecular targets is a 5-HT receptor. A mixture of 1 and 2 lowered the EC(50) of larval settlement as compared to the calculated theoretical additive effect of the two compounds. Moreover, an in situ sampling at 120 m depth using a remotely operated vehicle revealed that the sponge releases these two compounds to the ambient water. Thus, it is suggested that the synergistic action of 1 and 2 may benefit the sponge by reducing the expenditure of continuous production and release of its chemical defense substances. Furthermore, a synergistic action between structurally closely related compounds produced by the same bioenzymatic machinery ought to be the most energy effective for the organism and, thus, is more common than synergy between structurally indistinct compounds.

Evaluation of the cyclooxygenase inhibiting effects of six major cannabinoids isolated from Cannabis sativa.

Cyclooxygenase enzymes (COX-1 and COX-2) catalyse the production of prostaglandins from arachidonic acid. Prostaglandins are important mediators in the inflammatory process and their production can be reduced by COX-inhibitors. Endocannabinoids, endogenous analogues of the plant derived cannabinoids, occur normally in the human body. The Endocannabinoids are structurally similar to arachidonic acid and have been suggested to interfere with the inflammatory process. They have also been shown to inhibit cancer cell proliferation. Anti-inflammatory effects of cannabinoids and endocannabinoids have been observed, however the mode of action is not yet clarified. Anti-inflammatory activity (i.e., inhibition of COX-2) is proposed to play an important role in the development of colon cancer, which makes this subject interesting to study further. In the present work, the six cannabinoids tetrahydrocannabinol (Δ9-THC), tetrahydrocannabinolic acid (Δ9-THC-A), cannabidiol (CBD), cannabidiolic acid (CBDA), cannabigerol (CBG) and cannabigerolic acid (CBGA), isolated from Cannabis sativa, were evaluated for their effects on prostaglandin production. For this purpose an in vitro enzyme based COX-1/COX-2 inhibition assay and a cell based prostaglandin production radioimmunoassay were used. Cannabinoids inhibited cyclooxygenase enzyme activity with IC50 values ranging from 1.7·10⁻³ to 2.0·10⁻4 M.

Antimicrobial and antineoplastic activities of agelasine analogs modified in the purine 2-position.

Agelasines are 7,9-dialkylpurinium salts found in marine sponges (Agelas sp.), which display a variety of antimicrobial and cytotoxic effects. We have synthesized simplified agelasine analogs modified in the purine 2-position and examined their antimicrobial and anticancer activities. The compounds were screened against Staphylococcus aureus, Escherichia coli, Mycobacterium tuberculosis, Candida krusei, and Candida albicans, protozoa causing tropical diseases (Plasmodium falciparum, Leishmania infantum, Trypanosoma cruzi, and Trypanosoma brucei), a panel of human cancer cell lines (U-937 GTB, RPMI 8226/s, CEM/s, and ACHN) as well as VERO and/or MRC-5 cells. The results indicate that the introduction of a methyl group in the purine 2-position is beneficial for antimycobacterial and antiprotozoal activity, and that amino groups may enhance activity against several cancer cell lines.

Natural products in modern life science.

With a realistic threat against biodiversity in rain forests and in the sea, a sustainable use of natural products is becoming more and more important. Basic research directed against different organisms in Nature could reveal unexpected insights into fundamental biological mechanisms but also new pharmaceutical or biotechnological possibilities of more immediate use. Many different strategies have been used prospecting the biodiversity of Earth in the search for novel structure-activity relationships, which has resulted in important discoveries in drug development. However, we believe that the development of multidisciplinary incentives will be necessary for a future successful exploration of Nature. With this aim, one way would be a modernization and renewal of a venerable proven interdisciplinary science, Pharmacognosy, which represents an integrated way of studying biological systems. This has been demonstrated based on an explanatory model where the different parts of the model are explained by our ongoing research. Anti-inflammatory natural products have been discovered based on ethnopharmacological observations, marine sponges in cold water have resulted in substances with ecological impact, combinatory strategy of ecology and chemistry has revealed new insights into the biodiversity of fungi, in depth studies of cyclic peptides (cyclotides) has created new possibilities for engineering of bioactive peptides, development of new strategies using phylogeny and chemography has resulted in new possibilities for navigating chemical and biological space, and using bioinformatic tools for understanding of lateral gene transfer could provide potential drug targets. A multidisciplinary subject like Pharmacognosy, one of several scientific disciplines bridging biology and chemistry with medicine, has a strategic position for studies of complex scientific questions based on observations in Nature. Furthermore, natural product research based on intriguing scientific questions in Nature can be of value to increase the attraction for young students in modern life science.

Isolation, characterization, and bioactivity of cyclotides from the Micronesian plant Psychotria leptothyrsa.

Cyclotides, the largest known family of head-to-tail cyclic peptides, have approximately 30 amino acid residues with a complex structure containing a circular peptide backbone and a cystine knot. They are found in plants from the Violaceae and Rubiaceae families and are speculated to function in plant protection. In addition to their insecticidal properties, cyclotides display cytotoxic, anti-HIV, antimicrobial, and inhibition of neurotensin binding activities. Although cyclotides are present in all violaceous species hitherto screened, their distribution and expression in Rubiaceae are not fully understood. In this study, we show that Psychotria leptothyrsa var. longicarpa (Rubiaceae) contains a suite of different cyclotides. The cyclotide fractions were isolated by RP-HPLC, and sequences of six new peptides, named psyles A-F, were determined by MS/MS sequencing. One of these, psyle C, is the first rubiaceous linear variant known. Psyles A, C, and E were analyzed in a fluorometric microculture assay to determine cytotoxicity toward the human lymphoma cell line U937-GTB. The IC(50) values of psyles A, C, and E were 26, 3.50, and 0.76 muM, respectively. This study expands the number of known rubiaceous cyclotides and shows that the linear cyclotide maintains cytotoxicity.

Cytotoxic effects of cardiac glycosides in colon cancer cells, alone and in combination with standard chemotherapeutic drugs.

Cardiac glycosides have been reported to exhibit cytotoxic activity against several different cancer types, but studies against colorectal cancer are lacking. In a screening procedure aimed at identifying natural products with activity against colon cancer, several cardiac glycosides were shown to be of interest, and five of these were further evaluated in different colorectal cancer cell lines and primary cells from patients. Convallatoxin (1), oleandrin (4), and proscillaridin A (5) were identified as the most potent compounds (submicromolar IC50 values), and digitoxin (2) and digoxin (3), which are used in cardiac disease, exhibited somewhat lower activity (IC50 values 0.27-4.1 microM). Selected cardiac glycosides were tested in combination with four clinically relevant cytotoxic drugs (5-fluorouracil, oxaliplatin, cisplatin, irinotecan). The combination of 2 and oxaliplatin exhibited synergism including the otherwise highly drug-resistant HT29 cell line. A ChemGPS-NP application comparing modes of action of anticancer drugs identified cardiac glycosides as a separate cluster. These findings demonstrate that such substances may exhibit significant activity against colorectal cancer cell lines, by mechanisms disparate from currently used anticancer drugs, but at concentrations generally considered not achievable in patient plasma.

Antifouling activity of the sponge metabolite agelasine D and synthesised analogs on Balanus improvisus.

This study reports a screening study for antifouling (AF) activity of the natural compound agelasine D isolated from marine sponges of the genus Agelas and 20 synthesised analogs of agelasines and agelasimines. Agelasine D, together with two of the analogs, ie AV1003A and AKB695, displayed a strong inhibitory effect on settlement of Balanus improvisus cypris larvae. Agelasine D had an EC50 value of 0.11 microM while the two analogs AV1033A and AKB695 had EC50 values of 0.23 and 0.3 microM, respectively. None of these three compounds affected larval mortality as was the case with several of the analogs tested. Moreover, the effect of AV1033A and AKB695 was reversible. When cyprids after 24 h exposure to the compounds were transferred to fresh seawater, the settlement frequency compared with the controls was completely recovered. The properties of the agelasine D analogs AV1003A and AKB695 make them highly attractive candidates as AF agents in future marine coatings.

Antifouling activity of bromotyrosine-derived sponge metabolites and synthetic analogues.

Eighteen brominated sponge-derived metabolites and synthetic analogues were analyzed for antilarval settlement of Balanus improvisus. Only compounds exhibiting oxime substituents including bastadin-3 (4), -4 (1), -9 (2), and -16 (3), hemibastadin-1 (6), aplysamine-2 (5), and psammaplin A (10) turned out to inhibit larval settling at 1 to 10 microM. Analogues of hemibastadin-1 (6) were synthesized and tested for structure activity studies. Debromohemibastadin-1 (8) inhibited settling of B. improvisus, albeit at lower concentrations than hemibastadin-1 (6). Both 6 and 8 also induced cyprid mortality. 5,5'-dibromohemibastadin-1 (7) proved to be nontoxic, but settlement inhibition was observed at 10 microM. Tyrosinyltyramine (9), lacking the oxime function, was not antifouling active and was non-toxic at 100 microM. Hemibastadin-1 (6) and the synthetic products showed no general toxicity when tested against brine shrimp larvae. In contrast to the lipophilic psammaplin A (10), the hydrophilic sulfated psammaplin A derivative (11) showed no antifouling activity even though it contains an oxime group. We therefore hypothesize that the compound needs to cross membranes (probably by diffusion) and that the target for psammaplin A lies intracellularly.

Food for thought.

Do certain kinds of food contain pharmacologically active substances in concentrations that are high enough to have druglike effects when consumed? Are biologically active compounds in food indicative of therapeutic value? Is traditional drug development suitable for testing the merits of food? Is it ethical to test food as a drug on patients? Will dietary disease management remain a pipedream? Is it a fact or fantasy that the Mediterranean diet is beneficial to health? Is a vegetarian diet an elimination therapy, or one of supplementation? What can be learned from animals? Are humans losing the capability of listening to their bodies? In this review, we will address these questions--providing food for thought.

Antifouling activity of synthesized peptide analogs of the sponge metabolite barettin.

Barettin (cyclo [(6-bromo-8-en-tryptophan) arginine]), a diketopiperazine isolated from the marine sponge Geodia barretti, is a potent inhibitor of barnacle larvae settlement with an EC50-value of 0.9 microM. In the present study, 14 analogs of barettin and its structural congener dipodazine were synthezised and tested for their ability to inhibit larval settlement. Two of the analogs have an intact barettin skeleton. The remaining analogs have a dipodazine skeleton (a diketopiperazine where arginine is replaced with glycine). Six of the tested synthetic analogs displayed significant settlement inhibition with the most potent inhibitor being benzo[g]dipodazine, which displayed even stronger activity than barettin (EC50-value 0.034 microM). The effect of benzo[g]dipodazine was also shown to be readily reversible, when cyprids were transferred to filtered seawater (FSW).

Functional versus chemical diversity: is biodiversity important for drug discovery?

Prospecting the full biodiversity of nature to find leads for new drugs is not necessary. Because finding leads is aimed at identifying biological activity, structure is of secondary importance. Furthermore, although natural chemical diversity might be unrivalled, functional diversity is bound to be considerably less. It is likely that many millions of chemically distinct molecules exist in nature but it is inconceivable that the number of different biological functions is near this number. This is corroborated by knowledge obtained from the genome sequences of an increasing number of species. It is unlikely that ligands for specific molecular targets are restricted to one species and even individual compounds are often found in more than one species. Important molecular mechanisms are likely to be ubiquitous and there are no a priori reasons to assume that some are restricted to, for example, tropical rainforests. Thus, there are no obvious advantages of "biodiversity prospecting", which will, possibly, endanger fragile ecosystems in the search for rare species.

Aryl ketones from Acronychia pedunculata with cyclooxygenase-2 inhibitory effects.

1-[2,4-Dihydroxy-6-methoxy-3,5-bis(3-methylbut-2-en-1-yl)phenyl]ethanone (1), and a new aryl ketone, named acrovestenol (2), were isolated as cyclooxygenase-2 (COX-2) inhibitory principles from a CH2Cl2 extract of the bark of Acronychia pedunculata by a bioassay-guided fractionation procedure. Compound 2 inhibited COX-2 with an IC50 value of 142.0+/-2.15 microM, compared to the COX-2 inhibitory reference compound NS-398 with an IC50 value of 11.3+/-1.12 microM. Compound 1 inhibited COX-2-catalyzed PG biosynthesis with 68% at a concentration of 500 microM. The structures were determined by UV, IR, and 1D- and 2D-NMR, including TOCSY, HSQC-DEPT, and HMBC, and MS investigations.

Cyclotides: a novel type of cytotoxic agents.

Cytotoxic activities of three naturally occurring macrocyclic peptides (cyclotides) isolated from the two violets, Viola arvensis Murr. and Viola odorata L., were investigated. A nonclonogenic fluorometric microculture assay was used to examine cytotoxicity in a panel of 10 human tumor cell lines representing defined types of cytotoxic drug resistance. Additionally, primary cultures of tumor cells from patients, and for comparison normal lymphocytes, were used to quantify cytotoxic activity. All three cyclotides, varv A, varv F, and cycloviolacin 02, exhibited strong cytotoxic activities, which varied in a dose-dependent manner. Cycloviolacin 02 was the most potent in all cell lines (IC50 0.1-0.3 microM), followed by varv A (IC50 2.7-6.35 microM) and varv F (IC50 2.6-7.4 microM), respectively. Activity profiles of the cyclotides differed significantly from those of antitumor drugs in clinical use, which may indicate a new mode of action. This, together with the exceptional chemical and biological stability of cyclotides, makes them interesting in particular for their potential as pharmacological tools and possibly as leads to antitumor agents.

Penicillium nalgiovense Laxa isolated from Antarctica is a new source of the antifungal metabolite amphotericin B.

BACKGROUND: The need for new antibiotic drugs increases as pathogenic microorganisms continue to develop resistance against current antibiotics. We obtained samples from Antarctica as part of a search for new antimicrobial metabolites derived from filamentous fungi. This terrestrial environment near the South Pole is hostile and extreme due to a sparsely populated food web, low temperatures, and insufficient liquid water availability. We hypothesize that this environment could cause the development of fungal defense or survival mechanisms not found elsewhere. RESULTS: We isolated a strain of Penicillium nalgiovense Laxa from a soil sample obtained from an abandoned penguin's nest. Amphotericin B was the only metabolite secreted from Penicillium nalgiovense Laxa with noticeable antimicrobial activity, with minimum inhibitory concentration of 0.125 µg/mL against Candida albicans. This is the first time that amphotericin B has been isolated from an organism other than the bacterium Streptomyces nodosus. In terms of amphotericin B production, cultures on solid medium proved to be a more reliable and favorable choice compared to liquid medium. CONCLUSIONS: These results encourage further investigation of the many unexplored sampling sites characterized by extreme conditions, and confirm filamentous fungi as potential sources of metabolites with antimicrobial activity.

Novel strategies for isolation and characterization of cyclotides: the discovery of bioactive macrocyclic plant polypeptides in the Violaceae.

This review focuses on the discovery of cyclotides in the Violaceae, their isolation and their anti-cancer effects. These macrocyclic plant peptides consist of about 30 amino acids, including three conserved disulfide bonds in a cystine knotted arrangement, which renders them a remarkable stability. Their unique structure, combined with a wide array of biological activities, makes them of great interest as possible leads in drug development or as carriers of grafted peptide sequences. Here we describe the work conducted in our laboratory, which started with the overall aim of identifying peptides and small proteins of the size 10-50 amino acid residues in plants with novel chemical structures and biological profiles with a potential for drug development or for use as pharmacological tools. Thus we developed a fractionation protocol to directly address major challenges encountered when dealing with plant material, such as removal of chlorophyll, polyphenols, and low molecular compounds omnipresent in plants. Using this protocol, we then discovered a suite of cyclotides, the varv peptides, from the plant Viola arvensis (Violaceae). Following this, separation methods directly targeting cyclotides were developed, e.g. by adsorption, ion exchange chromatography and solvent-solvent partitioning, which then were used in the isolation of additional cyclotides. To structurally examine cyclotides we have also developed methods based on mass spectrometry for cyclotide sequencing and mapping of disulfide bonds. Finally, to assess structure-activity relationships, regarding their anti-cancer and cytotoxic effects that we focus upon, we have also characterized the three dimensional structure of cyclotides by homology modeling techniques.