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Pye, CR, Bertin MJ, Lokey SR, Gerwick WH, Linington RG.  2017.  Retrospective analysis of natural products provides insights for future discovery trends. Proceedings of the National Academy of Sciences.   10.1073/pnas.1614680114   Abstract

Understanding of the capacity of the natural world to produce secondary metabolites is important to a broad range of fields, including drug discovery, ecology, biosynthesis, and chemical biology, among others. Both the absolute number and the rate of discovery of natural products have increased significantly in recent years. However, there is a perception and concern that the fundamental novelty of these discoveries is decreasing relative to previously known natural products. This study presents a quantitative examination of the field from the perspective of both number of compounds and compound novelty using a dataset of all published microbial and marine-derived natural products. This analysis aimed to explore a number of key questions, such as how the rate of discovery of new natural products has changed over the past decades, how the average natural product structural novelty has changed as a function of time, whether exploring novel taxonomic space affords an advantage in terms of novel compound discovery, and whether it is possible to estimate how close we are to having described all of the chemical space covered by natural products. Our analyses demonstrate that most natural products being published today bear structural similarity to previously published compounds, and that the range of scaffolds readily accessible from nature is limited. However, the analysis also shows that the field continues to discover appreciable numbers of natural products with no structural precedent. Together, these results suggest that the development of innovative discovery methods will continue to yield compounds with unique structural and biological properties.

Dhaneesha, M, Naman CB, Krishnan KP, Sinha RK, Jayesh P, Joseph V, Singh ISB, Gerwick WH, Sajeevan TP.  2017.  Streptomyces artemisiae MCCB 248 isolated from Arctic fjord sediments has unique PKS and NRPS biosynthetic genes and produces potential new anticancer natural products. 3 Biotech. 7   10.1007/s13205-017-0610-3   AbstractWebsite

After screening marine actinomycetes isolated from sediment samples collected from the Arctic fjord Kongsfjorden for potential anticancer activity, an isolate identified as Streptomyces artemisiae MCCB 248 exhibited promising results against the NCI-H460 human lung cancer cell line. H460 cells treated with the ethyl acetate extract of strain MCCB 248 and stained with Hoechst 33342 showed clear signs of apoptosis, including shrinkage of the cell nucleus, DNA fragmentation and chromatin condensation. Further to this treated cells showed indications of early apoptotic cell death, including a significant proportion of Annexin V positive staining and evidence of DNA damage as observed in the TUNEL assay. Amplified PKS 1 and NRPS genes involved in secondary metabolite production showed only 82% similarity to known biosynthetic genes of Streptomyces, indicating the likely production of a novel secondary metabolite in this extract. Additionally, chemical dereplication efforts using LC-MS/MS molecular networking suggested the presence of a series of undescribed tetraene polyols. Taken together, these results revealed that this Arctic S. artemisiae strain MCCB 248 is a promising candidate for natural products drug discovery and genome mining for potential anticancer agents.

Leao, T, Castelao G, Korobeynikov A, Monroe EA, Podell S, Glukhov E, Allen EE, Gerwick WH, Gerwick L.  2017.  Comparative genomics uncovers the prolific and distinctive metabolic potential of the cyanobacterial genus Moorea. Proceedings of the National Academy of Sciences of the United States of America. 114:3198-3203.   10.1073/pnas.1618556114   AbstractWebsite

Cyanobacteria are major sources of oxygen, nitrogen, and carbon in nature. In addition to the importance of their primary metabolism, some cyanobacteria are prolific producers of unique and bioactive secondary metabolites. Chemical investigations of the cyanobacterial genus Moorea have resulted in the isolation of over 190 compounds in the last two decades. However, preliminary genomic analysis has suggested that genome-guided approaches can enable the discovery of novel compounds from even well-studied Moorea strains, highlighting the importance of obtaining complete genomes. We report a complete genome of a filamentous tropical marine cyanobacterium, Moorea producens PAL, which reveals that about one-fifth of its genome is devoted to production of secondary metabolites, an impressive four times the cyanobacterial average. Moreover, possession of the complete PAL genome has allowed improvement to the assembly of three other Moorea draft genomes. Comparative genomics revealed that they are remarkably similar to one another, despite their differences in geography, morphology, and secondary metabolite profiles. Gene cluster networking highlights that this genus is distinctive among cyanobacteria, not only in the number of secondary metabolite pathways but also in the content of many pathways, which are potentially distinct from all other bacterial gene clusters to date. These findings portend that future genome-guided secondary metabolite discovery and isolation efforts should be highly productive.

Naman, CB, Rattan R, Nikoulina SE, Lee J, Miller BW, Moss NA, Armstrong L, Boudreau PD, Debonsi HM, Valeriote FA, Dorrestein PC, Gerwick WH.  2017.  Integrating molecular networking and biological assays to target the isolation of a cytotoxic cyclic octapeptide, Samoamide A, from an American Samoan marine cyanobacterium. Journal of Natural Products. 80:625-633.   10.1021/acsjnatprod.6600907   AbstractWebsite

Integrating LC-MS/MS molecular networking and bioassay-guided fractionation enabled the targeted isolation of a new and bioactive cyclic octapeptide, samoamide A (1), from a sample of cf. Symploca sp. collected in American Samoa. The structure of 1 was established by detailed 1D and 2D NMR experiments, HRESIMS data, and chemical degradation/chromatographic (e.g., Marfey's analysis) studies. Pure compound 1 was shown to have in vitro cytotoxic activity against several human cancer cell lines in both traditional cell culture and zone inhibition bioassays. Although there was no particular selectivity between the cell lines tested for samoamide A, the most potent activity was observed against H460 human non-small-cell lung cancer cells (IC50 = 1.1 mu M). Molecular modeling studies suggested that one possible mechanism of action for 1 is the inhibition of the enzyme dipeptidyl peptidase (CD26, DPP4) at a reported allosteric binding site, which could lead to many downstream pharmacological effects. However, this interaction was moderate when tested in vitro at up to 10 mu M and only resulted in about 16% peptidase inhibition. Combining bioassay screening with the cheminformatics strategy of LC-MS/MS molecular networking as a discovery tool expedited the targeted isolation of a natural product possessing both a novel chemical structure and a desired biological activity.

Sabry, OM, Goeger DE, Gerwick WH.  2017.  Biologically active new metabolites from a Florida collection of Moorea producens. Natural Product Research. 31:555-561.   10.1080/14786419.2016.1207074   AbstractWebsite

A bioassay-guided investigation (cancer cell cytotoxicity) of a Moorea producens collection from Key West, Florida, led to the discovery of two new bioactive natural products [(+)-malyngamide Y and a cyclic depsipeptide, (+)-floridamide]. Their planar structures were deduced through extensive analysis of 1D and 2D NMR spectroscopic data and supported by HRFAB mass spectrometry. The new cyclic depsipeptide contains four amino acids units, including N- methyl phenylalanine, proline, valine and alanine, beside the unique unit, 2,2-dimethyl-3hydroxy- octanoic acid. In addition to the discovery of these two new compounds, two previously reported metabolites were also isolated and identified from this cyanobacterial collection; (-)-C-12 lyngbic acid and the antibacterial agent (-)-malyngolide.

Sabry, OMM, Goeger DE, Valeriote FA, Gerwick WH.  2017.  Cytotoxic halogenated monoterpenes from Plocamium cartilagineum. Natural Product Research. 31:261-267.   10.1080/14786419.2016.1230115   AbstractWebsite

As a result of our efforts to identify bioactive agents from marine algae, we have isolated and identified one new halogenated monoterpene 1 [(-)-(5E,7Z)-348-trichloro-7-dichloromethyl-3-methyl-157-octatriene] in addition to three known compounds (2, 3 and 4) from the red alga Plocamium cartilagineum collected by hand from the eastern coast of South Africa. Compound 1 was found to be active as a cytotoxic agent in human lung cancer (NCI-H460) and mouse neuro-2a cell lines (IC50 4g/mL). Two of these compounds (3 and 4) were found to have cytotoxic activity in other cell line assays, especially against human leukaemia and human colon cancers (IC50 1.3g/mL). None of these metabolites were active as sodium channel blockers or activators. All structures were determined by spectroscopic methods (UV, IR, LRMS, HRMS, 1D NMR and 2D NMR). 1D and 2D NOE experiments were carried out on these compounds to confirm the geometry of the double bonds. [GRAPHICS] .

Skiba, MA, Sikkema AP, Fiers WD, Gerwick WH, Sherman DH, Aldrich CC, Smith JL.  2016.  Domain Organization and Active Site Architecture of a Polyketide Synthase C-methyltransferase. Acs Chemical Biology. 11:3319-3327.   10.1021/acschembio.6b00759   AbstractWebsite

Polyketide metabolites produced by modular type I polyketide synthases (PKS) acquire their chemical diversity through the variety of catalytic domains within modules of the pathway. Methyltransferases are among the least characterized of the catalytic domains common to PKS systems. We determined the domain boundaries and characterized the activity of a PKS C-methyltransferase (C-MT) from the curacin A biosynthetic pathway. The C-MT catalyzes S-adenosylmethionine-dependent methyl transfer to the alpha-position of beta-ketoacyl substrates linked to aryl carrier protein (ACP) or a small-molecule analog but does not act on beta-hydroxyacyl substrates or malonyl-ACP. Key catalytic residues conserved in both bacterial and fungal PKS C-MTs were identified in a 2 angstrom crystal structure and validated biochemically. Analysis of the structure and the sequences bordering the C-MT provides insight into the positioning of this domain within complete PKS modules.

Sun, P, Yu Q, Li J, Riccio R, Lauro G, Bifulco G, Kurtan T, Mandi A, Tang H, Li TJ, Zhuang CL, Gerwick WH, Zhang W.  2016.  Bissubvilides A and B, cembrane-capnosane heterodimers from the soft coral Sarcophyton subviride. Journal of Natural Products. 79:2552-2558.   10.1021/acs.jnatprod.6b00453   AbstractWebsite

Two new biscembranoid-like compounds, bissubvilides A (1) and B (2), were isolated together with sarsolilide B (3), the proposed biogenetic precursor to 1, from the soft coral Sarcophyton subviride. The structures and absolute configurations were solved by spectroscopic analysis and TDDFT/ECD and DFT/NMR calculations. The bissubvilides represent a novel biscembranoid-like skeleton presumed to derive from a cembrane-type diene and a capnosane-type dienophile via a Diels-Alder reaction. These two molecules exerted no cytotoxicity against MG-63 or A549 tumor cells or HuH7 tumor stem cells.

Maloney, FP, Gerwick L, Gerwick WH, Sherman DH, Smith JL.  2016.  Anatomy of the beta-branching enzyme of polyketide biosynthesis and its interaction with an acyl-ACP substrate. Proceedings of the National Academy of Sciences of the United States of America. 113:10316-10321.   10.1073/pnas.1607210113   AbstractWebsite

Alkyl branching at the beta position of a polyketide intermediate is an important variation on canonical polyketide natural product biosynthesis. The branching enzyme, 3-hydroxy-3-methylglutaryl synthase (HMGS), catalyzes the aldol addition of an acyl donor to a beta-keto-polyketide intermediate acceptor. HMGS is highly selective for two specialized acyl carrier proteins (ACPs) that deliver the donor and acceptor substrates. The HMGS from the curacin A biosynthetic pathway (CurD) was examined to establish the basis for ACP selectivity. The donor ACP (CurB) had high affinity for the enzyme (K-d = 0.5 mu M) and could not be substituted by the acceptor ACP. High-resolution crystal structures of HMGS alone and in complex with its donor ACP reveal a tight interaction that depends on exquisite surface shape and charge complementarity between the proteins. Selectivity is explained by HMGS binding to an unusual surface cleft on the donor ACP, in a manner that would exclude the acceptor ACP. Within the active site, HMGS discriminates between pre-and postreaction states of the donor ACP. The free phosphopantetheine (Ppant) cofactor of ACP occupies a conserved pocket that excludes the acetyl-Ppant substrate. In comparison with HMG-CoA (CoA) synthase, the homologous enzyme from primary metabolism, HMGS has several differences at the active site entrance, including a flexible-loop insertion, which may account for the specificity of one enzyme for substrates delivered by ACP and the other by CoA.

Videau, P, Wells KN, Singh AJ, Gerwick WH, Philmus B.  2016.  Assessment of Anabaena sp strain PCC 7120 as a heterologous expression host for cyanobacterial natural products: production of lyngbyatoxin A. Acs Synthetic Biology. 5:978-988.   10.1021/acssynbio.6b00038   AbstractWebsite

Cyanobacteria are well-known producers of natural products of highly varied structure and biological properties. However, the long doubling times, difficulty in establishing genetic methods for marine cyanobacteria, and low compound titers have hindered research into the biosynthesis of their secondary metabolites. While a few attempts to heterologously express cyanobacterial natural products have occurred, the results have been of varied success. Here, we report the first steps in developing the model freshwater cyanobacterium Anabaena sp. strain PCC 7120 (Anabaena 7120) as a general heterologous expression host for cyanobacterial secondary metabolites. We show that Anabaena 7120 can heterologously synthesize lyngbyatoxin A in yields comparable to those of the native producer, Moorea producens, and detail the design and use of replicative plasmids for compound production. We also demonstrate that Anabaena 7120 recognizes promoters from various biosynthetic gene clusters from both free-living and obligate symbiotic marine cyanobacteria. Through simple genetic manipulations, the titer of lyngbyatoxin A can be improved up to 13-fold. The development of Anabaena 7120 as a general heterologous expression host enables investigation of interesting cyanobacterial biosynthetic reactions and genetic engineering of their biosynthetic pathways.

Cummings, SL, Barbe D, Leao TF, Korobeynikov A, Engene N, Glukhov E, Gerwick WH, Gerwick L.  2016.  A novel uncultured heterotrophic bacterial associate of the cyanobacterium Moorea producens JHB. Bmc Microbiology. 16   10.1186/s12866-016-0817-1   AbstractWebsite

Background: Filamentous tropical marine cyanobacteria such as Moorea producens strain JHB possess a rich community of heterotrophic bacteria on their polysaccharide sheaths; however, these bacterial communities have not yet been adequately studied or characterized. Results and discussion: Through efforts to sequence the genome of this cyanobacterial strain, the 5.99 MB genome of an unknown bacterium emerged from the metagenomic information, named here as Mor1. Analysis of its genome revealed that the bacterium is heterotrophic and belongs to the phylum Acidobacteria, subgroup 22; however, it is only 85 % identical to the nearest cultured representative. Comparative genomics further revealed that Mor1 has a large number of genes involved in transcriptional regulation, is completely devoid of transposases, is not able to synthesize the full complement of proteogenic amino acids and appears to lack genes for nitrate uptake. Mor1 was found to be present in lab cultures of M. producens collected from various locations, but not other cyanobacterial species. Diverse efforts failed to culture the bacterium separately from filaments of M. producens JHB. Additionally, a co-culturing experiment between M. producens JHB possessing Mor1 and cultures of other genera of cyanobacteria indicated that the bacterium was not transferable. Conclusion: The data presented support a specific relationship between this novel uncultured bacterium and M. producens, however, verification of this proposed relationship cannot be done until the "uncultured" bacterium can be cultured.

Wang, MX, Carver JJ, Phelan VV, Sanchez LM, Garg N, Peng Y, Nguyen DD, Watrous J, Kapono CA, Luzzatto-Knaan T et al..  2016.  Sharing and community curation of mass spectrometry data with Global Natural Products Social Molecular Networking. Nature Biotechnology. 34:828-837.   10.1038/nbt.3597   AbstractWebsite

The potential of the diverse chemistries present in natural products (NP) for biotechnology and medicine remains untapped because NP databases are not searchable with raw data and the NP community has no way to share data other than in published papers. Although mass spectrometry (MS) techniques are well-suited to high-throughput characterization of NP, there is a pressing need for an infrastructure to enable sharing and curation of data. We present Global Natural Products Social Molecular Networking (GNPS;, an open-access knowledge base for community-wide organization and sharing of raw, processed or identified tandem mass (MS/MS) spectrometry data. In GNPS, crowdsourced curation of freely available community-wide reference MS libraries will underpin improved annotations. Data-driven social-networking should facilitate identification of spectra and foster collaborations. We also introduce the concept of 'living data' through continuous reanalysis of deposited data.

Huang, KC, Chen ZH, Jiang YM, Akare S, Kolber-Simonds D, Condon K, Agoulnik S, TenDyke K, Shen YC, Wu KM, Mathieu S, Choi HW, Zhu XJ, Shimizu H, Kotake Y, Gerwick WH, Uenaka T, Woodall-Jappe M, Nomoto K.  2016.  Apratoxin A shows novel pancreas-targeting activity through the binding of Sec 61. Molecular Cancer Therapeutics. 15:1208-1216.   10.1158/1535-7163.mct-15-0648   AbstractWebsite

Apratoxin A is a natural product with potent antiproliferative activity against many human cancer cell lines. However, we and other investigators observed that it has a narrow therapeutic window in vivo. Previous mechanistic studies have suggested its involvement in the secretory pathway as well as the process of chaperone-mediated autophagy. Still the link between the biologic activities of apratoxin A and its in vivo toxicity has remained largely unknown. A better understanding of this relationship is critically important for any further development of apratoxin A as an anticancer drug. Here, we describe a detailed pathologic analysis that revealed a specific pancreas-targeting activity of apratoxin A, such that severe pancreatic atrophy was observed in apratoxin A-treated animals. Follow-up tissue distribution studies further uncovered a unique drug distribution profile for apratoxin A, showing high drug exposure in pancreas and salivary gland. It has been shown previously that apratoxin A inhibits the protein secretory pathway by preventing cotranslational translocation. However, the molecule targeted by apratoxin A in this pathway has not been well defined. By using a H-3-labeled apratoxin A probe and specific Sec 61 alpha/beta antibodies, we identified that the Sec 61 complex is the molecular target of apratoxin A. We conclude that apratoxin A in vivo toxicity is likely caused by pancreas atrophy due to high apratoxin A exposure. (C)2016 AACR.

Briand, E, Humbert JF, Tambosco K, Bormans M, Gerwick WH.  2016.  Role of bacteria in the production and degradation of Microcystis cyanopeptides. Microbiologyopen. 5:469-478.   10.1002/mbo3.343   AbstractWebsite

The freshwater cyanobacteria, Microcystis sp., commonly form large colonies with bacteria embedded in their mucilage. Positive and negative interactions between Microcystis species and their associated bacteria have been reported. However, the potential role of bacteria in the production and degradation of cyanobacterial secondary metabolites has not been investigated. In this study, a Microcystis-associated bacterial community was isolated and added to the axenic M. aeruginosaPCC7806 liquid culture. After 3years of cocultivation, we studied the bacterial genetic diversity adapted to the PCC7806 strain and compared the intra- and extracellular concentration of major cyanopeptides produced by the cyanobacterial strain under xenic and axenic conditions. Mass spectrometric analyses showed that the intracellular concentration of peptides was not affected by the presence of bacteria. Interestingly, the produced peptides were detected in the axenic media but could not be found in the xenic media. This investigation revealed that a natural bacterial community, dominated by Alpha-proteobacteria, was able to degrade a wide panel of structurally varying cyclic cyanopeptides.

Torres-Mendoza, D, Gonzalez Y, Gomez-Reyes JF, Guzman HM, Lopez-Perez JL, Gerwick WH, Fernandez PL, Gutierrez M.  2016.  Uprolides N, O and P from the Panamanian Octocoral Eunicea succinea. Molecules. 21   10.3390/molecules21060819   AbstractWebsite

Three new diterpenes, uprolide N (1), uprolide O (2), uprolide P (3) and a known one, dolabellane (4), were isolated from the CH2Cl2-MeOH extract of the gorgonian octocoral Eunicea succinea, collected from Bocas del Toro, on the Caribbean coast of Panama. Their structures were determined using spectroscopic analyses, including 1D and 2D NMR and high-resolution mass spectrometry (HRMS) together with molecular modeling studies. Compounds 1-3 displayed anti-inflammatory properties by inhibiting production of Tumor Necrosis Factor (TNF) and Interleukin (IL)-6 induced by lipopolysaccharide (LPS) in murine macrophages.

Paatero, AO, Kellosalo J, Dunyak BM, Almaliti J, Gestwicki JE, Gerwick WH, Taunton J, Paavilainen VO.  2016.  Apratoxin kills cells by direct blockade of the sec61 protein translocation channel. Cell Chemical Biology. 23:561-566.   10.1016/j.chembiol.2016.04.008   AbstractWebsite

Apratoxin A is a cytotoxic natural product that prevents the biogenesis of secretory and membrane proteins. Biochemically, apratoxin A inhibits cotranslational translocation into the ER, but its cellular target and mechanism of action have remained controversial. Here, we demonstrate that apratoxin A prevents protein translocation by directly targeting Sec61 alpha, the central subunit of the protein translocation channel. Mutagenesis and competitive photo-crosslinking studies indicate that apratoxin A binds to the Sec61 lateral gate in a manner that differs from cotransin, a substrate-selective Sec61 inhibitor. In contrast to cotransin, apratoxin A does not exhibit a substrate-selective inhibitory mechanism, but blocks ER translocation of all tested Sec61 clients with similar potency. Our results suggest that multiple structurally unrelated natural products have evolved to target overlapping but non-identical binding sites on Sec61, thereby producing distinct biological outcomes.

Kunz, HH, Park J, Mevers E, Garcia AV, Highhouse S, Gerwick WH, Parker JE, Schroeder JI.  2016.  Small molecule DFPM derivative-activated plant resistance protein signaling in roots is unaffected by EDS1 subcellular targeting signal and chemical genetic isolation of victr r-protein mutants. Plos One. 11   10.1371/journal.pone.0155937   AbstractWebsite

The small molecule DFPM ([5-(3,4-dichlorophenyl)furan-2-yl]-piperidine-1-ylmethanethione) was recently shown to trigger signal transduction via early effector-triggered immunity signaling genes including EDS1 and PAD4 in Arabidopsis thaliana accession Col-0. Chemical genetic analyses of A. thaliana natural variants identified the plant Resistance protein-like Toll/Interleukin1 Receptor (TIR)-Nucleotide Binding (NB)-Leucine-Rich Repeat (LRR) protein VICTR as required for DFPM-mediated root growth arrest. Here a chemical genetic screen for mutants which disrupt DFPM-mediated root growth arrest in the Col-0 accession identified new mutant alleles of the TIR-NB-LRR gene VICTR. One allele, victr-6, carries a Gly216-to-Asp mutation in the Walker A domain supporting an important function of the VICTR nucleotide binding domain in DFPM responses consistent with VICTR acting as a canonical Resistance protein. The essential nucleo-cytoplasmic regulator of TIR-NBLRR-mediated effector-triggered immunity, EDS1, was reported to have both nuclear and cytoplasmic actions in pathogen resistance. DFPM was used to investigate the requirements for subcellular EDS1 localization in DFPM-mediated root growth arrest. EDS1-YFP fusions engineered to localize mainly in the cytoplasm or the nucleus by tagging with a nuclear export signal (NES) or a nuclear localization signal (NLS), respectively, were tested. We found that wild-type EDS1-YFP and both the NES and NLS-tagged EDS1 variants were induced by DFPM treatments and fully complemented eds1 mutant plants in root responses to DFPM, suggesting that enrichment of EDS1 in either compartment could confer DFPM-mediated root growth arrest. We further found that a light and O-2-dependent modification of DFPM is necessary to mediate DFPM signaling in roots. Chemical analyses including Liquid Chromatography-Mass Spectrometry and High-Resolution Atmospheric Pressure Chemical Ionization Mass Spectrometry identified a DFPM modification product that is likely responsible for bioactivity mediating root growth arrest. We propose a chemical structure of this product and a possible reaction mechanism for DFPM modification.

Moss, NA, Bertin MJ, Kleigrewe K, Leao TF, Gerwick L, Gerwick WH.  2016.  Integrating mass spectrometry and genomics for cyanobacterial metabolite discovery. Journal of Industrial Microbiology & Biotechnology. 43:313-324.   10.1007/s10295-015-1705-7   AbstractWebsite

Filamentous marine cyanobacteria produce bioactive natural products with both potential therapeutic value and capacity to be harmful to human health. Genome sequencing has revealed that cyanobacteria have the capacity to produce many more secondary metabolites than have been characterized. The biosynthetic pathways that encode cyanobacterial natural products are mostly uncharacterized, and lack of cyanobacterial genetic tools has largely prevented their heterologous expression. Hence, a combination of cutting edge and traditional techniques has been required to elucidate their secondary metabolite biosynthetic pathways. Here, we review the discovery and refined biochemical understanding of the olefin synthase and fatty acid ACP reductase/aldehyde deformylating oxygenase pathways to hydrocarbons, and the curacin A, jamaicamide A, lyngbyabellin, columbamide, and a trans-acyltransferase macrolactone pathway encoding phormidolide. We integrate into this discussion the use of genomics, mass spectrometric networking, biochemical characterization, and isolation and structure elucidation techniques.

O'Rourke, A, Kremb S, Bader TM, Helfer M, Schmitt-Kopplin P, Gerwick WH, Brack-Werner R, Voolstra CR.  2016.  Alkaloids from the sponge Stylissa carteri present prospective scaffolds for the inhibition of Human Immunodeficiency Virus 1 (HIV-1). Marine Drugs. 14   10.3390/md14020028   AbstractWebsite

The sponge Stylissa carteri is known to produce a number of secondary metabolites displaying anti-fouling, anti-inflammatory, and anti-cancer activity. However, the anti-viral potential of metabolites produced by S. carteri has not been extensively explored. In this study, an S. carteri extract was HPLC fractionated and a cell based assay was used to evaluate the effects of HPLC fractions on parameters of Human Immunodeficiency Virus (HIV-1) infection and cell viability. Candidate HIV-1 inhibitory fractions were then analyzed for the presence of potential HIV-1 inhibitory compounds by mass spectrometry, leading to the identification of three previously characterized compounds, i.e., debromohymenialdisine (DBH), hymenialdisine (HD), and oroidin. Commercially available purified versions of these molecules were re-tested to assess their antiviral potential in greater detail. Specifically, DBH and HD exhibit a 30%-40% inhibition of HIV-1 at 3.1 M and 13 M, respectively; however, both exhibited cytotoxicity. Conversely, oroidin displayed a 50% inhibition of viral replication at 50 M with no associated toxicity. Additional experimentation using a biochemical assay revealed that oroidin inhibited the activity of the HIV-1 Reverse Transcriptase up to 90% at 25 M. Taken together, the chemical search space was narrowed and previously isolated compounds with an unexplored anti-viral potential were found. Our results support exploration of marine natural products for anti-viral drug discovery.

Briand, E, Bormans M, Gugger M, Dorrestein PC, Gerwick WH.  2016.  Changes in secondary metabolic profiles of Microcystis aeruginosa strains in response to intraspecific interactions. Environmental Microbiology. 18:384-400.   10.1111/1462-2920.12904   AbstractWebsite

The cyanobacteria Microcystis proliferate in freshwater ecosystems and produce bioactive compounds including the harmful toxins microcystins (MC). These secondary metabolites play an important role in shaping community composition through biotic interactions although their role and mode of regulation are poorly understood. As natural cyanobacterial populations include producing and non-producing strains, we tested if the production of a range of peptides by coexisting cells could be regulated through intraspecific interactions. With an innovative co-culturing chamber together with advanced mass spectrometry (MS) techniques, we monitored the growth and compared the metabolic profiles of a MC-producing as well as two non-MC-producing Microcystis strains under mono- and co-culture conditions. In monocultures, these strains grew comparably; however, the non-MC-producing mutant produced higher concentrations of cyanopeptolins, aerucyclamides and aeruginosins than the wild type. Physiological responses to co-culturing were reflected in a quantitative change in the production of the major peptides. Using a MS/MS-based molecular networking approach, we identified new analogues of known classes of peptides as well as new compounds. This work provides new insights into the factors that regulate the production of MC and other secondary metabolites in cyanobacteria, and suggests interchangeable or complementary functions allowing bloom-forming cyanobacteria to efficiently colonize and dominate in fluctuating aquatic environments.

Bertin, MJ, Demirkiran O, Navarro G, Moss NA, Lee J, Goldgof GM, Vigil E, Winzeler EA, Valeriote FA, Gerwick WH.  2016.  Kalkipyrone B, a marine cyanobacterial gamma-pyrone possessing cytotoxic and anti-fungal activities. Phytochemistry. 122:113-118.   10.1016/j.phytochem.2015.11.011   AbstractWebsite

Bioassay-guided fractionation of two marine cyanobacterial extracts using the H-460 human lung cancer cell line and the OVC-5 human ovarian cancer cell line led to the isolation of three related alpha-methoxy-beta, beta'-dimethyl-gamma-pyrones each containing a modified alkyl chain, one of which was identified as the previously reported kalkipyrone and designated kalkipyrone A. The second compound was an analog designated kalkipyrone B. The third was identified as the recently reported yoshinone A, also isolated from a marine cyanobacterium. Kalkipyrone A and B were obtained from a field-collection of the cyanobacterium Leptolyngbya sp. from Fagasa Bay, American Samoa, while yoshinone A was isolated from a field-collection of cyanobacteria (cf. Schizothrix sp.) from Panama. One-dimensional and twodimensional NMR experiments were used to determine the overall structures and relative configurations of the kalkipyrones, and the absolute configuration of kalkipyrone B was determined by H-1 NMR analysis of diastereomeric Mosher's esters. Kalkipyrone A showed good cytotoxicity to H-460 human lung cancer cells (EC50 = 0.9 mu M), while kalkipyrone B and yoshinone A were less active (EC50 = 9.0 mu M and >10 mu M, respectively). Both kalkipyrone A and B showed moderate toxicity to Saccharomyces cerevisiae ABC16-Monster strain (IC50 = 14.6 and 13.4 mu M, respectively), whereas yoshinone A was of low toxicity to this yeast strain (IC50 = 63.8 mu M). (C) 2015 Elsevier Ltd. All rights reserved.

Fenner, AM, Engene N, Spadafora C, Gerwick WH, Balunas MJ.  2016.  Medusamide A, a Panamanian cyanobacterial depsipeptide with multiple beta-amino acids. Organic Letters. 18:352-355.   10.1021/acs.orglett.5b03110   AbstractWebsite

From a collection of marine cyanobacteria made in the Coiba National Park along the Pacific coast of the Republic of Panama a novel cyclic depsipeptide, given the trivial name medusamide A, has been isolated and fully characterized. Medusamide A contains four contiguous beta-amino acid (2R,3R)-3-amino-2-methylhexanoic acid (Amha) residues. This is the first report of multiple Amha residues and contiguous beta-amino acid residues within a single cyclic peptide-type natural product. Stereochemical assignment of the Amha residues was completed following the synthesis of reference standards for this beta-amino acid and the subsequent derivatization with Marfey's reagent and LC-MS analysis.

Bertin, MJ, Vulpanovici A, Monroe EA, Korobeynikov A, Sherman DH, Gerwick L, Gerwick WH.  2016.  The phormidolide biosynthetic gene cluster: A trans-at pks pathway encoding a toxic macrocyclic polyketide. Chembiochem. 17:164-173.   10.1002/cbic.201500467   AbstractWebsite

Phormidolide is a polyketide produced by a cultured filamentous marine cyanobacterium and incorporates a 16-membered macrolactone. Its complex structure is recognizably derived from a polyketide synthase pathway, but possesses unique and intriguing structural features that prompted interest in investigating its biosynthetic origin. Stable isotope incorporation experiments confirmed the polyketide nature of this compound. We further characterized the phormidolide gene cluster (phm) through genome sequencing followed by bioinformatic analysis. Two discrete trans-type acyltransferase (trans-AT) ORFs along with KS-AT adaptor regions (ATd) within the polyketide synthase (PKS) megasynthases, suggest that the phormidolide gene cluster is a trans-AT PKS. Insights gained from analysis of the mode of acetate incorporation and ensuing keto reduction prompted our reevaluation of the stereochemistry of phormidolide hydroxy groups located along the linear polyketide chain.

Sapkota, M, Li L, Choi H, Gerwick WH, Soh Y.  2015.  Bromo-honaucin A inhibits osteoclastogenic differentiation in RAW 264.7 cells via Akt and ERK signaling pathways. European Journal of Pharmacology. 769:100-109.   10.1016/j.ejphar.2015.11.003   AbstractWebsite

Osteoclasts are unique bone remodeling cells derived from multinucleated myeloid progenitor cells. They play homeostatic vital roles in skeletal modeling and remodeling but also destroy bone masses in many pathological conditions such as osteoporosis and rheumatoid arthritis. Receptor activation of NF-KB ligand (RANKL) is essential to osteoclastogenesis. In this study, we investigated the effects of bromohonaucin A (Br-H A) isolated from Leptolyngbya crossbyana (cyanobacterium). To investigate the mechanism of the inhibitory effect of Br-H A on osteoclastogenesis, we employed Br-H Ain RANKL-treated murine monocyte/macrophage RAW 264.7 cells for osteoclastic differentiation in-vitro. The inhibitory effects on in-vitro osteoclastogenesis was evaluated by counting the number of Tartarate resistant acid phospatase (TRAP) positive multinucleated cells and by measuring the expression level of osteoclastspecific genes like matrix metalloproteinase 9 (MMP9), cathepsin K (CATH K), GRB2-associated-binding protein 2 (GAB2), c-terminal myc kinase (C-MYC), C-terminal Src kinase (C-SRC) and Microphthalmiaassociated transcription factor (MITE). Moreover, Br-H A blocked the resorbing capacity of RAW 264.7 cells on calcium phosphate-coated plates. Finally, Br-H A clearly decreased the expression of Akt and also decreased the activation of ERK. Thus, the study identifies Br-H A as potent inhibitor potentialin the treatment of diseases involving abnormal bone lysis such as osteoporosis, rheumatoid arthritis, and periodontal bone degradation. (C) 2015 Elsevier B.V. All rights reserved.

Teta, R, Della Sala G, Glukhov E, Gerwick L, Gerwick WH, Mangoni A, Costantino V.  2015.  Combined LC-MS/MS and molecular networking approach reveals new cyanotoxins from the 2014 cyanobacterial bloom in Green Lake, Seattle. Environmental Science & Technology. 49:14301-14310.   10.1021/acs.est.5b04415   AbstractWebsite

Cyanotoxins obtained from a freshwater cyanobacterial collection at Green Lake, Seattle during a cyanobacterial harmful algal bloom in the summer of 2014 were studied using a new approach based on molecular networking analysis of liquid chromatography tandem mass spectrometry (LC-MS/MS) data. This MS networking approach is particularly well-suited for the detection of new cyanotoxin variants and resulted in the discovery of three new cyclic peptides, namely microcystin-MhtyR (6), which comprised about half of the total microcystin content in the bloom, and ferintoic acids C (12) and D (13). Structure elucidation of 6 was aided by a new microscale methylation procedure. Metagenomic analysis of the bloom using the 16S-ITS rRNA region identified Microcystis aeruginosa as the predominant cyanobacterium in the sample. Fragments of the putative biosynthetic genes for the new cyanotoxins were also identified, and their sequences correlated to the structure of the isolated cyanotoxins.