Metabolomics-Guided Discovery of Microginin Peptides from Cultures of the Cyanobacterium Microcystis aeruginosa.

We report a mass-spectrometry-based metabolomics study of a laboratory-cultured strain of Microcystis aeruginosa (UTEX LB2385), which has led to the discovery of five peptides (1-5) belonging to the microginin class of linear cyanopeptides. The structures and configurations of these peptides were determined by spectroscopic analyses and chemical derivitization. The microginin peptides described herein are the first reported derivatives containing N-methyl methionine (1, 5) and N-methyl methionine sulfoxide (2-4). The two tripeptide microginin analogues (4, 5) identified represent the smallest members of this peptide family. Their angiotensin-converting enzyme (ACE) inhibitory activity was also investigated. Microginin 527 (4) was the most potent of the group, with an IC50 of 31 µM.

Biosynthetic Studies of 13-Desmethylspirolide C Produced by Alexandrium ostenfeldii (= A. peruvianum): Rationalization of the Biosynthetic Pathway Following Incorporation of (13)C-Labeled Methionine and Application of the Odd-Even Rule of Methylation.

Understanding the biosynthesis of dinoflagellate polyketides presents many unique challenges. Because of the remaining hurdles to dinoflagellate genome sequencing, precursor labeling studies remain the only viable way to investigate dinoflagellate biosynthesis. However, prior studies have shown that polyketide chain assembly does not follow any of the established processes. Additionally, acetate, the common precursor for polyketides, is frequently scrambled, thus compromising interpretation. These factors are further compounded by low production yields of the compounds of interest. A recent report on the biosynthesis of spirolides, a group belonging to the growing class of toxic spiroimines, provided some insight into the polyketide assembly process based on acetate labeling studies, but many details were left uncertain. By feeding (13)C methyl-labeled methionine to cultures of Alexandrium ostenfeldii, the producing organism of 13-desmethylspirolide C, and application of the odd-even methylation rule, the complete biosynthetic pathway has been established.

Monacyclinones, New Angucyclinone Metabolites Isolated from Streptomyces sp. M7_15 Associated with the Puerto Rican Sponge Scopalina ruetzleri.

During an investigation of new actinomycete species from Caribbean sponges for novel bioactive natural products, frigocyclinone (1), dimethyldehydrorabelomycin (3) and six new angucyclinone derivatives were isolated from Streptomyces sp. strain M7_15 associated with the sponge Scopalina ruetzleri. Of these, monacyclinones A-B (4-5) contain the core ring structure of dehydrorabelomycin (2) with the aminodeoxysugar found in frigocyclinone (1). Monacyclinone C (6) is a hydroxylated variant of frigocyclinone (1) and monacyclinone D (7) is a Baeyer Villiger derivative of (6) which also exists as the open chain hydrolysis product monacyclinone E (8). Monacyclinone F (9) contains two unique epoxide rings attached to the angucyclinone moiety and an additional aminodeoxysugar attached through an angular oxygen bond. All structures were confirmed through spectral analyses. Activity against rhabdomycosarcoma cancer cells (SJCRH30) after 48 h of treatment was observed with frigocyclinone (1; EC50 = 5.2 µM), monacyclinone C (6; 160 µM), monacyclinone E (8; 270 µM), and monacyclinone F (9; 0.73 µM). The strongest bioactivity against rhabdomycosarcoma cancer cells and gram-positive bacteria was exhibited by compound 9, suggesting that the extra aminodeoxysugar subunit is important for biological activity.

Polyketide biosynthesis in dinoflagellates: what makes it different?

Dinoflagellates produce unique polyketides characterized by their size and complexity. The biosynthesis of a limited number of such metabolites has been reported, with studies largely hampered by the low yield of compounds and the severe scrambling of label in the isotopically-labeled precursors. Nonetheless, of the successful biosynthetic experiments that have been reported, many surprising and unique processes have been discovered. This knowledge has been accessed through a series of biochemical labeling studies, and while limited molecular genetic data has been amassed, it is still in the early stages of development. In an attempt to meet this challenge, this review has compared some of the biosynthetic processes with similar ones identified in other microbes such as bacteria and myxobacteria, with the idea that similar genes and enzymes are employed by dinoflagellates.

Homoseongomycin, a compound isolated from marine actinomycete bacteria K3-1, is a potent inhibitor of encephalitic alphaviruses.

Marine microorganisms have been a resource for novel therapeutic drugs for decades. In addition to anticancer drugs, the drug acyclovir, derived from a marine sponge, is FDA-approved for the treatment of human herpes simplex virus-1 infections. Most alphaviruses that are infectious to terrestrial animals and humans, such as Venezuelan and eastern equine encephalitis viruses (VEEV and EEEV), lack efficient antiviral drugs and it is imperative to develop these remedies. To push the discovery and development of anti-alphavirus compounds forward, this study aimed to isolate and screen for potential antiviral compounds from cultured marine microbes originating from the marine environment. Compounds from marine microbes were of interest as they are prolific producers of bioactive compounds across the spectrum of human diseases and infections. Homoseongomycin, an actinobacteria isolated from a marine sponge displayed impressive activity against VEEV from a total of 76 marine bioactive products. The 50% effective concentration (EC50) for homoseongomycin was 8.6 µM for suppressing VEEV TC-83 luciferase reporter virus replication. Homoseongomycin was non-toxic up to 50 µM and partially rescued cells from VEEV induced cell death. Homoseongomycin exhibited highly efficient antiviral activity with a reduction of VEEV infectious titers by 8 log10 at 50 µM. It also inhibited EEEV replication with an EC50 of 1.2 µM. Mechanism of action studies suggest that homoseongomycin affects both early and late stages of the viral life cycle. Cells treated with 25 µM of homoseongomycin had a ~90% reduction in viral entry. In comparison, later stages showed a more robust reduction in infectious titers (6 log10) and VEEV extracellular viral RNA levels (4 log10), but a lesser impact on intracellular viral RNA levels (1.5 log10). In sum, this work demonstrates that homoseongomycin is a potential anti-VEEV and anti-EEEV compound due to its low cytotoxicity and potent antiviral activity.

Structure and biosynthesis of amphidinol 17, a hemolytic compound from Amphidinium carterae.

Amphidinol 17 (AM17; 1), a novel amphidinol, has been isolated from a Bahamas strain of Amphidinium carterae. This new congener contains the signature hairpin region and a Delta(6) polyene arm, whereas the polyol arm is distinct from those of other amphidinols. The pattern of acetate incorporation in 1 was directly determined by feeding a single labeled substrate, [2-(13)C]acetate. While the highly conserved regions within the amphidinol family of AM17 have exhibited identical occurrences of cleaved acetates to other amphidinols for which the biosynthesis has been explored, the polyol arm for AM17 displays a higher degree of nascent chain processing that shows similarities to amphidinolide biosynthesis. AM17 exhibited an EC(50) of 4.9 microM in a hemolytic assay using human red blood cells but displayed no detectable antifungal activity.

Structure and relative potency of several karlotoxins from Karlodinium veneficum.

The karlotoxins are a family of amphidinol-like compounds that play roles in avoiding predation and in prey capture for the toxic dinoflagellate Karlodinium veneficum. The first member of the toxin group to be reported was KmTx 1 (1), and here we report an additional five new members of this family (3-7) from the same strain. Of these additional compounds, KmTx 3 (3) differs from KmTx 1 (1) in having one less methylene group in the saturated portion of its lipophilic arm. In addition, 64-E-chloro-KmTx 3 (4) and 10-O-sulfo-KmTx 3 (5) were identified. Likewise, 65-E-chloro-KmTx 1 (6) and 10-O-sulfo-KmTx 1 (7) were also isolated. Comparison of the hemolytic activities of the newly isolated compounds to that of KmTx 1 shows that potency correlates positively with the length of the lipophilic arm and is disrupted by sulfonation of the polyol arm.

Absolute configuration of brevisamide and brevisin: confirmation of a universal biosynthetic process for Karenia brevis polyethers.

The discovery of brevisin, the first example of an "interrupted" polycyclic ether, obtained from the dinoflagellate Karenia brevis, posed some important questions regarding the mechanism of the cyclization process. Consequently, we have established absolute configurations of brevisin and its related metabolite brevisamide using a modified Mosher's esterification method. For brevisin, analysis was carried out on both the 31-monokis- and the 10,31-bis-MTPA esters. The results suggest that both metabolites, like other polyethers from K. brevis, result from polyepoxide precursors with uniform (S, S) configurations for all epoxides and provide further support for a universal stereochemical model for dinoflagellate polyether formation.

Brevisin: an aberrant polycyclic ether structure from the dinoflagellate Karenia brevis and its implications for polyether assembly.

Brevisin is an unprecedented polycyclic ether isolated from the dinoflagellate Karenia brevis, an organism well-known to produce complex polycyclic ethers. The structure of brevisin was determined by detailed analyses of MS and 2D NMR spectra and is remarkable in that it consists of two separate fused polyether ring assemblies linked by a methylene group. One of the polycyclic moieties contains a conjugated aldehyde side chain similar to that recently observed in other K. brevis metabolites, though the "interrupted" polyether structure of brevisin is novel and provides further insight into the biogenesis of such fused-ring polyether systems. On the basis of the unusual structure of brevisin, principles underlying the initiation of polyether assemblies are proposed. Brevisin was found to inhibit the binding of [(3)H]-PbTx-3 to its binding site on the voltage-sensitive sodium channels in rat brain synaptosomes.

Further studies on the chemistry of the flustra alkaloids from the bryozoan Flustra foliacea.

Since 1980, over a dozen novel brominated alkaloids, named flustramines, have been isolated from Scandinavian and Canadian collections of the marine bryozoan Flustra foliacea. This paper describes the reisolation of the known compound dihydroflustramine C (1) and the isolation of 11 new flustramines (2-4, 6-13), including two dimers (12, 13) that may be isolation artifacts. Together these compounds, some with an unexpected aryl substitution pattern, reveal an intricate network of metabolites present in the extracts of the bryozoan. The structures of these metabolites were solved using a variety of spectroscopic techniques and chemical derivatization and modification. This work also led to the recognition of an unusual rearrangement reaction that occurred slowly over a number of years.

Identification of Portimine B, a New Cell Permeable Spiroimine That Induces Apoptosis in Oral Squamous Cell Carcinoma.

Spiroimines are a class of compounds produced by marine dinoflagellates with a wide range of toxicity and therapeutic potential. The smallest of the cyclic imines, portimine, is far less toxic than other known members in several animal models. Portimine has also been shown to induce apoptosis and reduce the growth of a variety of cancer cell lines at low nanomolar concentrations. In an effort to discover new spiroimines, the current study undertook a metabolomic analysis of cultures of cyclic imine-producing dinoflagellates, and a new analog of portimine was discovered in which the five-membered cyclic ether is open. Further scrutiny with human oral cavity squamous cell carcinoma (OCSCC) cell lines revealed that the open ring congener was less potent than portimine A but could still lead to the accumulation of apoptotic gene transcripts, fragment genomic DNA, and reduce cancer cell proliferation in the range of 100-200 nM.

Flavones and flavone glycosides from Halophila johnsonii.

Halophila johnsonii Eiseman is a shallow-water marine angiosperm which contains UV-absorbing metabolites. Studies on methanol extracts of H. johnsonii by means of HPLC-UV, NMR, HPLC-MS resulted in isolation and identification of seven previously unknown flavone glycosides: 5,6,7,3',4',5'-hexahydroxyflavone-7-O-beta-glucopyranoside (1), 5,6,7,3',4',5'-hexahydroxyflavone-7-O-(6''-O-acetyl)-beta-glucopyranoside (2), 6-hydroxyluteolin-7-O-(6''-O-acetyl)-beta-glucopyranoside (3), 6-hydroxyapigenin-7-O-(6''-O-acetyl)-beta-glucopyranoside (4), 6-hydroxyapigenin-7-O-(6''-O-[E]-coumaroyl)-beta-glucopyranoside (5), 6-hydroxyapigenin-7-O-(6''-O-[E]-caffeoyl)-beta-glucopyranoside (6) and 6-hydroxyluteolin-7-O-(6''-O-[E]-coumaroyl)-beta-glucopyranoside (7). Also isolated were three known flavone glycosides, 6-hydroxyluteolin 7-O-beta-glucopyranoside (8), scutellarein-7-O-beta-glucopyranoside (9), and spicoside (10), and five known flavones, pedalitin (11), ladanetin (12), luteolin (13), apegenin (14) and myricetin (15). Qualitative comparison of the flavonoid distribution in the leaf and rhizome-root portions of the plant was also investigated, with the aim of establishing the UV-protecting roles that flavonoids played in the sea grass.

Isolation and characterization of karlotoxin 1, a new amphipathic toxin from Karlodinium veneficum.

The karlotoxins (KmTxs) are a family of compounds produced by the dinoflagellate Karlodinium veneficum that cause membrane permeabilization. The structure of KmTx 1, determined using extensive 2D NMR spectroscopy, is very similar to the amphidinols and related compounds, though KmTx 1 features unique structural modifications of the conserved core region. The structure of KmTx 1 differs from that reported for KmTx 2, the only other reported karlotoxin to date, in lacking chlorination at its terminal alkene and possessing a hydrophobic arm that is two carbons longer.

A brief review of innovative uses for local anesthetics.

PURPOSE OF REVIEW: Local anesthetics are drugs which have many potentially beneficial actions, especially when used as a continuous intravenous infusion. The following review gives an update on the most recent literature along with some interesting and promising areas where systemic local anesthetics are being implemented. RECENT FINDINGS: Surprisingly little research has been conducted with the use of systemic local anesthetics when considering their cost effectiveness, good side effect profile when used in low-dose infusions, and promising results in the available literature. The in-vivo studies, ranging from case reports to randomized controlled trials, have small sample sizes, yet most have found that low-dose systemic infusions are efficacious for various conditions. SUMMARY: Local anesthetics have many beneficial properties, and promising results have been seen when low-dose infusions are implemented. This review briefly describes the anti-inflammatory properties of local anesthetics and discusses the benefits seen when used systemically for neuroprotection, postoperative ileus, decompression sickness, and glaucoma.

The biosynthesis of 15N-labeled microcystins and the comparative MS/MS fragmentation of natural abundance and their 15N-labeled congeners using LC-MS/MS.

The global need for accurate and sensitive quantitation of microcystins (MCs) persists as incidents of cyanobacterial harmful algal blooms continue to rise and recent research reveals an underestimation of the human health implications of these toxins. An optimal approach for their accurate quantitation relies on the availability of stable isotope-labeled MC standards for use in stable isotope dilution analysis (SIDA) strategies involving liquid chromatography tandem mass spectrometry (LC-MS/MS). Due to the dearth of isotopically labeled MCs, ten different 15N-enriched MCs were biosynthesized from producing cultures and fully characterized. This involved the comparative MS/MS fragmentation of natural abundance or unlabeled metabolites with their 15N-labeled congeners for improved confidence in product ion annotation. These results revealed a series of incorrect annotations described previously in the literature. In this manuscript, the biosynthesis of labeled microcystin is detailed, and their complete analytical characterization for prospective use in targeted SIDA applications, such as routine water testing is described.

Sulfated diesters of okadaic acid and DTX-1: Self-protective precursors of diarrhetic shellfish poisoning (DSP) toxins.

Many toxic secondary metabolites used for defense are also toxic to the producing organism. One important way to circumvent toxicity is to store the toxin as an inactive precursor. Several sulfated diesters of the diarrhetic shellfish poisoning (DSP) toxin okadaic acid have been reported from cultures of various dinoflagellate species belonging to the genus Prorocentrum. It has been proposed that these sulfated diesters are a means of toxin storage within the dinoflagellate cell, and that a putative enzyme mediated two-step hydrolysis of sulfated diesters such as DTX-4 and DTX-5 initially leads to the formation of diol esters and ultimately to the release of free okadaic acid. However, only one diol ester and no sulfated diesters of DTX-1, a closely related DSP toxin, have been isolated leading some to speculate that this toxin is not stored as a sulfated diester and is processed by some other means. DSP components in organic extracts of two large scale Prorocentrum lima laboratory cultures have been investigated. In addition to the usual suite of okadaic acid esters, as well as the free acids okadaic acid and DTX-1, a group of corresponding diol- and sulfated diesters of both okadaic acid and DTX-1 have now been isolated and structurally characterized, confirming that both okadaic acid and DTX-1 are initially formed in the dinoflagellate cell as the non-toxic sulfated diesters.

Identification and Characterization of Pectenotoxin (PTX) 4 and PTX7 as Spiroketal Stereoisomers of Two Previously Reported Pectenotoxins.

Pectenotoxins (PTXs) isolated from the scallop Patinopecten yessoensis were shown to be involved in an episode of diarrhetic shellfish poisoning (DSP). A total of eight analogues (PTX1-PTX7 and PTX10) have been isolated to date, and the structures of four of these analogues (PTX1, PTX2, PTX3, and PTX6) have already been elucidated. Here, we report the characterization of PTX4 and PTX7 as 7-epi-PTX1 and 7-epi-PTX6, respectively, on the basis of NMR data and an acid-catalyzed chemical interconversion. The structures of two new artifacts, PTX8 and PTX9, produced following this treatment are also reported.

Improvement of vascular dysfunction and blood lipids of insulin-resistant rats by a marine oil-based phytosterol compound.

The syndrome that is characterized by obesity, insulin resistance, and hyperlipidemia is increasingly prevalent in all prosperous societies. It is now recognized as a major contributor to cardiovascular disease. Vascular dysfunction in the form of hypercontractility and impaired nitric oxide-mediated relaxation is a significant component of cardiovascular disease, predisposing to ischemic events. The JCR:LA-cp strain of rats exhibits all major aspects of the obesity/insulin resistance syndrome, including vascular dysfunction and ischemic lesions of the heart. Dietary lipid intake may have a marked effect on plasma lipid levels and, potentially, on vascular disease. We have investigated the effects of a novel preparation, ONC101 (a phytosterol esterified with fish oil), on plasma lipids and vascular function in the insulin-resistant JCR:LA-cp rat. Treatment of obese male rats with ONC101 from 8 to 12 wk of age resulted in no change in plasma lipid concentrations at 0.5 g/kg body weight. At the higher dose of 2.6 g/kg, plasma TG fell 50% (1.26 vs. 2.59 mmol/L, P < 0.002) and cholesterol esters were significantly reduced (1.34 vs. 1.61 mmol/L, P < 0.002). Food intake and body weights were unaffected by ONC101 treatment. At the low dose of 86 mg/kg, the hypercontractility of aortic rings in response to phenylephrine was normalized and the relaxant response to acetylcholine was significantly improved. The results indicate that ONC101 at high doses has significant hypolipidemic effects and, at very low doses, has beneficial effects on endothelial and vascular smooth muscle cell function.

Microcystins and two new micropeptin cyanopeptides produced by unprecedented Microcystis aeruginosa blooms in North Carolina's Cape Fear River.

The Cape Fear River is the largest river system in North Carolina. It is heavily used as a source of drinking water for humans and livestock as well as a source of irrigation water for crops, and production water for industry. It also serves as a major fishery for both commercial and recreational use. In recent years, possibly related to increased eutrophication of the river, massive blooms of cyanobacteria, identified as Microcystis aeruginosa have been observed. Bloom samples collected in 2009 and 2012 were chemically analyzed to determine if they contained cyanobacterial toxins known as microcystins. Both blooms were found to produce microcystins in high yields. Microcystins are potent hepatotoxins that can be bio-accumulated in the food chain. Recent biological studies have also shown a host of other potentially harmful effects of low level microcystin exposure. Detailed chemical analysis of these blooms led us to discover that these blooms produce an additional family of cyanobacterial peptides know as the micropeptins, including two new members named micropeptins 1106 and 1120. The biological activities of these new molecules have not yet been determined, although protease activity has been well documented for this peptide group. These data indicate a need for thorough monitoring of toxin levels especially during bloom events in addition to additional biological testing of other cyanopeptides present in blooms.

Biodiversity of Actinomycetes associated with Caribbean sponges and their potential for natural product discovery.

Marine actinomycetes provide a rich source of structurally unique and bioactive secondary metabolites. Numerous genera of marine actinomycetes have been isolated from marine sediments as well as several sponge species. In this study, 16 different species of Caribbean sponges were collected from four different locations in the coastal waters off Puerto Rico in order to examine diversity and bioactive metabolite production of marine actinomycetes in Caribbean sponges. Sediments were also collected from each location, in order to compare actinomycete communities between these two types of samples. A total of 180 actinomycetes were isolated and identified based on 16S rRNA gene analysis. Phylogenetic analysis revealed the presence of at least 14 new phylotypes belonging to the genera Micromonospora, Verruscosispora, Streptomyces, Salinospora, Solwaraspora, Microbacterium and Cellulosimicrobium. Seventy-eight of the isolates (19 from sediments and 59 from sponges) shared 100 % sequence identity with Micromonospora sp. R1. Despite having identical 16S rRNA sequences, the bioactivity of extracts and subsequent fractions generated from the fermentation of both sponge- and sediment-derived isolates identical to Micromonospora sp. R1 varied greatly, with a marked increase in antibiotic metabolite production in those isolates derived from sponges. These results indicate that the chemical profiles of isolates with high 16S rRNA sequence homology to known strains can be diverse and dependent on the source of isolation. In addition, seven previously reported dihydroquinones produced by five different Streptomyces strains have been purified and characterized from one Streptomyces sp. strain isolated in this study from the Caribbean sponge Agelas sceptrum.