Leptochelins A-C, Cytotoxic Metallophores Produced by Geographically Dispersed Leptothoe Strains of Marine Cyanobacteria.

Metals are important cofactors in the metabolic processes of cyanobacteria, including photosynthesis, cellular respiration, DNA replication, and the biosynthesis of primary and secondary metabolites. In adaptation to the marine environment, cyanobacteria use metallophores to acquire trace metals when necessary as well as to reduce potential toxicity from excessive metal concentrations. Leptochelins A-C were identified as structurally novel metallophores from three geographically dispersed cyanobacteria of the genus Leptothoe. Determination of the complex structures of these metabolites presented numerous challenges, but they were ultimately solved using integrated data from NMR, mass spectrometry and deductions from the biosynthetic gene cluster. The leptochelins are comprised of halogenated linear NRPS-PKS hybrid products with multiple heterocycles that have potential for hexadentate and tetradentate coordination with metal ions. The genomes of the three leptochelin producers were sequenced, and retrobiosynthetic analysis revealed one candidate biosynthetic gene cluster (BGC) consistent with the structure of leptochelin. The putative BGC is highly homologous in all three Leptothoe strains, and all possess genetic signatures associated with metallophores. Postcolumn infusion of metals using an LC-MS metabolomics workflow performed with leptochelins A and B revealed promiscuous binding of iron, copper, cobalt, and zinc, with greatest preference for copper. Iron depletion and copper toxicity experiments support the hypothesis that leptochelin metallophores may play key ecological roles in iron acquisition and in copper detoxification. In addition, the leptochelins possess significant cytotoxicity against several cancer cell lines.

Portobelamides A and B and Caciqueamide, Cytotoxic Peptidic Natural Products from a Caldora sp. Marine Cyanobacterium.

Three new compounds, portobelamides A and B (1 and 2), 3-amino-2-methyl-7-octynoic acid (AMOYA) and hydroxyisovaleric acid (Hiva) containing cyclic depsipeptides, and one long chain lipopeptide caciqueamide (3), were isolated from a field-collection of a Caldora sp. marine cyanobacterium obtained from Panama as part of the Panama International Cooperative Biodiversity Group Program. Their planar structures were elucidated through analysis of 2D NMR and MS data, especially high resolution (HR) MS2/MS3 fragmentation methods. The absolute configurations of compounds 1 and 2 were deduced by traditional hydrolysis, derivative formation, and chromatographic analyses compared with standards. Portobelamide A (1) showed good cytotoxicity against H-460 human lung cancer cells (33% survival at 0.9 µM).

MetaMiner: A Scalable Peptidogenomics Approach for Discovery of Ribosomal Peptide Natural Products with Blind Modifications from Microbial Communities.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are an important class of natural products that contain antibiotics and a variety of other bioactive compounds. The existing methods for discovery of RiPPs by combining genome mining and computational mass spectrometry are limited to discovering specific classes of RiPPs from small datasets, and these methods fail to handle unknown post-translational modifications. Here, we present MetaMiner, a software tool for addressing these challenges that is compatible with large-scale screening platforms for natural product discovery. After searching millions of spectra in the Global Natural Products Social (GNPS) molecular networking infrastructure against just eight genomic and metagenomic datasets, MetaMiner discovered 31 known and seven unknown RiPPs from diverse microbial communities, including human microbiome and lichen microbiome, and microorganisms isolated from the International Space Station.

Cytotoxic Microcolin Lipopeptides from the Marine Cyanobacterium Moorea producens.

Nine new linear lipopeptides, microcolins E-M (1-9), together with four known related compounds, microcolins A-D (10-13), were isolated from the marine cyanobacterium Moorea producens using bioassay-guided and LC-MS/MS molecular networking approaches. Catalytic hydrogenation of microcolins A-D (10-13) yielded two known compounds, 3,4-dihydromicrocolins A and B (14, 15), and two new derivatives, 3,4-dihydromicrocolins C and D (16, 17), respectively. The structures of these new compounds were determined by a combination of spectroscopic and advanced Marfey's analysis. Structurally unusual amino acid units, 4-methyl-2-(methylamino)pent-3-enoic (Mpe) acid and 2-amino-4-methylhexanoic acid (N-Me-homoisoleucine), in compounds 1-3 and 8, respectively, are rare residues in naturally occurring peptides. These metabolites showed significant cytotoxic activity against H-460 human lung cancer cells with IC50 values ranging from 6 nM to 5.0 µM. The variations in structure and attendant biological activities provided fresh insights concerning structure-activity relationships for the microcolin class of lipopeptides.

Samholides, Swinholide-Related Metabolites from a Marine Cyanobacterium cf. Phormidium sp.

Cancer cell cytotoxicity was used to guide the isolation of nine new swinholide-related compounds, named samholides A-I (1-9), from an American Samoan marine cyanobacterium cf. Phormidium sp. Their structures were determined by extensive analysis of 1D and 2D NMR spectroscopic data. The new compounds share an unusual 20-demethyl 44-membered lactone ring composed of two monomers, and they demonstrate structural diversity arising from geometric isomerization of double bonds, sugar units with unique glyceryl moieties and varied methylation patterns. All of the new samholides were potently active against the H-460 human lung cancer cell line with IC50 values ranging from 170 to 910 nM. The isolation of these new swinholide-related compounds from a marine cyanobacterium reinvigorates questions concerning the evolution and biosynthetic origin of these natural products.

Marine Natural Product Honaucin A Attenuates Inflammation by Activating the Nrf2-ARE Pathway.

The cyanobacterial marine natural product honaucin A inhibits mammalian innate inflammation in vitro and in vivo. To decipher its mechanism of action, RNA sequencing was used to evaluate differences in gene expression of cultured macrophages following honaucin A treatment. This analysis led to the hypothesis that honaucin A exerts its anti-inflammatory activity through activation of the cytoprotective nuclear erythroid 2-related factor 2 (Nrf2)-antioxidant response element/electrophile response element (ARE/EpRE) signaling pathway. Activation of this pathway by honaucin A in cultured human MCF7 cells was confirmed using an Nrf2 luciferase reporter assay. In vitro alkylation experiments with the natural product and N-acetyl-l-cysteine suggest that honaucin A activates this pathway through covalent interaction with the sulfhydryl residues of the cytosolic repressor protein Keap1. Honaucin A presents a potential therapeutic lead for diseases with an inflammatory component modulated by Nrf2-ARE.

A Mononuclear Iron-Dependent Methyltransferase Catalyzes Initial Steps in Assembly of the Apratoxin A Polyketide Starter Unit.

Natural product biosynthetic pathways contain a plethora of enzymatic tools to carry out difficult biosynthetic transformations. Here, we discover an unusual mononuclear iron-dependent methyltransferase that acts in the initiation steps of apratoxin A biosynthesis (AprA MT1). Fe3+-replete AprA MT1 catalyzes one or two methyl transfer reactions on the substrate malonyl-ACP (acyl carrier protein), whereas Co2+, Fe2+, Mn2+, and Ni2+ support only a single methyl transfer. MT1 homologues exist within the "GNAT" (GCN5-related N-acetyltransferase) loading modules of several modular biosynthetic pathways with propionyl, isobutyryl, or pivaloyl starter units. GNAT domains are thought to catalyze decarboxylation of malonyl-CoA and acetyl transfer to a carrier protein. In AprA, the GNAT domain lacks both decarboxylation and acyl transfer activity. A crystal structure of the AprA MT1-GNAT di-domain with bound Mn2+, malonate, and the methyl donor S-adenosylmethionine (SAM) reveals that the malonyl substrate is a bidentate metal ligand, indicating that the metal acts as a Lewis acid to promote methylation of the malonyl α-carbon. The GNAT domain is truncated relative to functional homologues. These results afford an expanded understanding of MT1-GNAT structure and activity and permit the functional annotation of homologous GNAT loading modules both with and without methyltransferases, additionally revealing their rapid evolutionary adaptation in different biosynthetic contexts.

Development of a Potent Inhibitor of the Plasmodium Proteasome with Reduced Mammalian Toxicity.

Naturally derived chemical compounds are the foundation of much of our pharmacopeia, especially in antiproliferative and anti-infective drug classes. Here, we report that a naturally derived molecule called carmaphycin B is a potent inhibitor against both the asexual and sexual blood stages of malaria infection. Using a combination of in silico molecular docking and in vitro directed evolution in a well-characterized drug-sensitive yeast model, we determined that these compounds target the ß5 subunit of the proteasome. These studies were validated using in vitro inhibition assays with proteasomes isolated from Plasmodium falciparum. As carmaphycin B is toxic to mammalian cells, we synthesized a series of chemical analogs that reduce host cell toxicity while maintaining blood-stage and gametocytocidal antimalarial activity and proteasome inhibition. This study describes a promising new class of antimalarial compound based on the carmaphycin B scaffold, as well as several chemical structural features that serve to enhance antimalarial specificity.

Spongosine production by a Vibrio harveyi strain associated with the sponge Tectitethya crypta.

Spongosine (1), deoxyspongosine (2), spongothymidine (Ara T) (3), and spongouridine (Ara U) were isolated from the Caribbean sponge Tectitethya crypta and given the general name "spongonucleosides". Spongosine, a methoxyadenosine derivative, has demonstrated a diverse bioactivity profile including anti-inflammatory activity and analgesic and vasodilation properties. Investigations into unusual nucleoside production by T. crypta-associated microorganisms using mass spectrometric techniques have identified a spongosine-producing strain of Vibrio harveyi and several structurally related compounds from multiple strains.

Polyketide genes in the marine sponge Plakortis simplex: a new group of mono-modular type I polyketide synthases from sponge symbionts.

Sponge symbionts are a largely unexplored source of new and unusual metabolic pathways. Insights into the distribution and function of metabolic genes of sponge symbionts are crucial to dissect and exploit their biotechnological potential. Screening of the metagenome of the marine sponge Plakortis simplex led to the discovery of the swf family, a new group of mono-modular type I polyketide synthase/fatty acid synthase (PKS/FAS) specifically associated with sponge symbionts. Two different examples of the swf cluster were present in the metagenome of P. simplex. A third example of the cluster is present in the previously sequenced genome of a poribacterium from the sponge Aplysina aerophoba but was formerly considered orthologous to the wcb/rkp cluster. The swf cluster was also found in six additional species of sponges. Therefore, the swf cluster represents the second group of mono-modular PKS, after the supA family, to be widespread in marine sponges. The putative swf operon consists of swfA (type I PKS/FAS), swfB (reductase and sulphotransferase domains) and swfC (radical S-adenosylmethionine, or radical SAM). Activation of the acyl carrier protein (ACP) domain of the SwfA protein to its holo-form by co-expression with Svp is the first functional proof of swf type genes in marine sponges. However, the precise biosynthetic role of the swf clusters remains unknown.

Examination of the mode of action of the almiramide family of natural products against the kinetoplastid parasite Trypanosoma brucei.

Almiramide C is a marine natural product with low micromolar activity against Leishmania donovani, the causative agent of leishmaniasis. We have now shown that almiramide C is also active against the related parasite Trypanosoma brucei, the causative agent of human African trypanosomiasis. A series of activity-based probes have been synthesized to explore both the molecular target of this compound series in T. brucei lysates and site localization through epifluorescence microscopy. These target identification studies indicate that the almiramides likely perturb glycosomal function through disruption of membrane assembly machinery. Glycosomes, which are organelles specific to kinetoplastid parasites, house the first seven steps of glycolysis and have been shown to be essential for parasite survival in the bloodstream stage. There are currently no reported small-molecule disruptors of glycosome function, making the almiramides unique molecular probes for this understudied parasite-specific organelle. Additionally, examination of toxicity in an in vivo zebrafish model has shown that these compounds have little effect on organism development, even at high concentrations, and has uncovered a potential side effect through localization of fluorescent derivatives to zebrafish neuromast cells. Combined, these results further our understanding of the potential value of this lead series as development candidates against T. brucei.

Structural basis of functional group activation by sulfotransferases in complex metabolic pathways.

Sulfated molecules with diverse functions are common in biology, but sulfonation as a method to activate a metabolite for chemical catalysis is rare. Catalytic activity was characterized and crystal structures were determined for two such "activating" sulfotransferases (STs) that sulfonate ß-hydroxyacyl thioester substrates. The CurM polyketide synthase (PKS) ST domain from the curacin A biosynthetic pathway of Moorea producens and the olefin synthase (OLS) ST from a hydrocarbon-producing system of Synechococcus PCC 7002 both occur as a unique acyl carrier protein (ACP), ST, and thioesterase (TE) tridomain within a larger polypeptide. During pathway termination, these cyanobacterial systems introduce a terminal double bond into the ß-hydroxyacyl-ACP-linked substrate by the combined action of the ST and TE. Under in vitro conditions, CurM PKS ST and OLS ST acted on ß-hydroxy fatty acyl-ACP substrates; however, OLS ST was not reactive toward analogues of the natural PKS ST substrate bearing a C5-methoxy substituent. The crystal structures of CurM ST and OLS ST revealed that they are members of a distinct protein family relative to other prokaryotic and eukaryotic sulfotransferases. A common binding site for the sulfonate donor 3'-phosphoadenosine-5'-phosphosulfate was visualized in complexes with the product 3'-phosphoadenosine-5'-phosphate. Critical functions for several conserved amino acids in the active site were confirmed by site-directed mutagenesis, including a proposed glutamate catalytic base. A dynamic active-site flap unique to the "activating" ST family affects substrate selectivity and product formation, based on the activities of chimeras of the PKS and OLS STs with exchanged active-site flaps.

Honaucins A-C, potent inhibitors of inflammation and bacterial quorum sensing: synthetic derivatives and structure-activity relationships.

Honaucins A-C were isolated from the cyanobacterium Leptolyngbya crossbyana which was found overgrowing corals on the Hawaiian coast. Honaucin A consists of (S)-3-hydroxy-γ-butyrolactone and 4-chlorocrotonic acid, which are connected via an ester linkage. Honaucin A and its two natural analogs exhibit potent inhibition of both bioluminescence, a quorum-sensing-dependent phenotype, in Vibrio harveyi BB120 and lipopolysaccharide-stimulated nitric oxide production in the murine macrophage cell line RAW264.7. The decrease in nitric oxide production was accompanied by a decrease in the transcripts of several proinflammatory cytokines, most dramatically interleukin-1ß. Synthesis of honaucin A, as well as a number of analogs, and subsequent evaluation in anti-inflammation and quorum-sensing inhibition bioassays revealed the essential structural features for activity in this chemical class and provided analogs with greater potency in both assays.

Single cell genome amplification accelerates identification of the apratoxin biosynthetic pathway from a complex microbial assemblage.

Filamentous marine cyanobacteria are extraordinarily rich sources of structurally novel, biomedically relevant natural products. To understand their biosynthetic origins as well as produce increased supplies and analog molecules, access to the clustered biosynthetic genes that encode for the assembly enzymes is necessary. Complicating these efforts is the universal presence of heterotrophic bacteria in the cell wall and sheath material of cyanobacteria obtained from the environment and those grown in uni-cyanobacterial culture. Moreover, the high similarity in genetic elements across disparate secondary metabolite biosynthetic pathways renders imprecise current gene cluster targeting strategies and contributes sequence complexity resulting in partial genome coverage. Thus, it was necessary to use a dual-method approach of single-cell genomic sequencing based on multiple displacement amplification (MDA) and metagenomic library screening. Here, we report the identification of the putative apratoxin. A biosynthetic gene cluster, a potent cancer cell cytotoxin with promise for medicinal applications. The roughly 58 kb biosynthetic gene cluster is composed of 12 open reading frames and has a type I modular mixed polyketide synthase/nonribosomal peptide synthetase (PKS/NRPS) organization and features loading and off-loading domain architecture never previously described. Moreover, this work represents the first successful isolation of a complete biosynthetic gene cluster from Lyngbya bouillonii, a tropical marine cyanobacterium renowned for its production of diverse bioactive secondary metabolites.

Malyngamide 2, an oxidized lipopeptide with nitric oxide inhibiting activity from a Papua New Guinea marine cyanobacterium.

A Papua New Guinea collection of the marine cyanobacterium cf. Lyngbya sordida yielded three known compounds as well as a new PKS-NRPS-derived malyngamide with anti-inflammatory and cytotoxic activity. Malyngamide 2 features an extensively oxidized cyclohexanone ring. Resolution of the ring core as a 6,8,9-triol rather then a 7,8,9-triol and relative configuration was based on chemical shift and bond geometry modeling in conjunction with homonuclear and heteronuclear coupling constants, NOE and ROE correlations, and other structural information. Malyngamide 2 exhibited anti-inflammatory activity in LPS-induced RAW macrophage cells (IC(50) = 8.0 µM) with only modest cytotoxicity to the mammalian cell line.

Marine natural product drug discovery: Leads for treatment of inflammation, cancer, infections, and neurological disorders.

Natural products, secondary metabolites, isolated from plants, animals and microbes are important sources for bioactive molecules that in many cases have been developed into treatments for diseases. This review will focus on describing the potential for finding new treatments from marine natural products for inflammation, cancer, infections, and neurological disorders. Historically terrestrial natural products have been studied to a greater extent and such classic drugs as aspirin, vincristine and many of the antibiotics are derived from terrestrial natural products. The need for new therapeutics in the four areas mentioned is dire. Within the last 30 years marine natural products, with their unique structures and high level of halogenation, have shown many promising activities against the inflammatory response, cancer, infections and neurological disorders. The review will outline examples of such compounds and activities.

Polyketide decarboxylative chain termination preceded by o-sulfonation in curacin a biosynthesis.

Biosynthetic innovation in natural product systems is driven by the recruitment of new genes and enzymes into these complex pathways. Here, an unprecedented decarboxylative chain termination mechanism is described for the polyketide synthase of curacin A, an anticancer lead compound isolated from the marine cyanobacterium Lyngbya majuscula. The unusual chain termination module containing adjacent sulfotransferase (ST) and thioesterase (TE) catalytic domains embedded in CurM was biochemically characterized. The TE was proved to catalyze a hydrolytic chain release of the polyketide chain elongation intermediate. Moreover, a selective ST-mediated sulfonation of the (R)-beta-hydroxyl group was found to precede TE-mediated hydrolysis, triggering a successive decarboxylative elimination and resulting in the formation of a rare terminal olefin in the final metabolite.

Use of a rainbow trout oligonucleotide microarray to determine transcriptional patterns in aflatoxin B1-induced hepatocellular carcinoma compared to adjacent liver.

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide, and its occurrence is associated with a number of environmental factors including ingestion of the dietary contaminant aflatoxin B(1) (AFB(1)). Research over the last 40 years has revealed rainbow trout (Oncorhynchus mykiss) to be an excellent research model for study of AFB(1)-induced hepatocarcinogenesis; however, little is known about changes at the molecular level in trout tumors. We have developed a rainbow trout oligonucleotide array containing 1672 elements representing over 1400 genes of known or probable relevance to toxicology, comparative immunology, carcinogenesis, endocrinology, and stress physiology. In this study, we applied microarray technology to examine gene expression of AFB(1)-induced HCC in the rainbow trout tumor model. Carcinogenesis was initiated in trout embryos with 50 ppb AFB(1), and after 13 months control livers, tumors, and tumor-adjacent liver tissues were isolated from juvenile fish. Global gene expression was determined in histologically confirmed HCCs compared to noncancerous adjacent tissue and sham-initiated control liver. We observed distinct gene regulation patterns in HCC compared to noncancerous tissue including upregulation of genes important for cell cycle control, transcription, cytoskeletal formation, and the acute phase response and downregulation of genes involved in drug metabolism, lipid metabolism, and retinol metabolism. Interestingly, the expression profiles observed in trout HCC are similar to the transcriptional signatures found in human and rodent HCC, further supporting the validity of the model. Overall, these findings contribute to a better understanding of the mechanism of AFB(1)-induced hepatocarcinogenesis in trout and identify conserved genes important for carcinogenesis in species separated evolutionarily by more than 400 million years.