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Tao, YW, Li PL, Zhang DJ, Glukhov E, Gerwick L, Zhang C, Murray TF, Gerwick WH.  2018.  Samholides, swinholide-related metabolites from a marine cyanobacterium cf. Phormidium sp. Journal of Organic Chemistry. 83:3034-3046.   10.1021/acs.joc.8b00028   AbstractWebsite

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.

Shao, CL, Mou XF, Cao F, Spadafora C, Glukhov E, Gerwick L, Wang CY, Gerwick WH.  2018.  Bastimolide B, an antimalarial 24-membered marine macrolide possessing a tert-butyl group. Journal of Natural Products. 81:211-215.   10.1021/acs.jnatprod.7b00917   AbstractWebsite

We reported previously the discovery of the potent antimalarial 40-membered macrolide bastimolide A (1) from the tropical marine cyanobacterium Okeania hirsute. Continued investigation has led to the discovery of a new analogue, bastimolide B (2), a 24-membered polyhydroxy macrolide with a long aliphatic chain and unique terminal tertbutyl group. Its complete structure was determined by a combination of extensive spectroscopic methods and comparative analysis of its methanolysis products with those of bastimolide A. A methanolysis mechanism for bastimolide A is proposed, and one unexpected isomerization product of the C2-C3 double bond, 2-(E)-bastimolide A (3), was obtained. Bastimolide B (2) showed strong antimalarial activity against chloroquine-sensitive Plasmodium falciparum strain HB3. A preliminary investigation of the structure activity relationship based on six analogues revealed the importance of the double bond as well as the 1,3-diol and 1,3,5-triol functionalities.

Skiba, MA, Sikkema AP, Moss NA, Tran CL, Sturgis RM, Gerwick L, Gerwick WH, Sherman DH, Smith JL.  2017.  A mononuclear iron-dependent methyltransferase catalyzes initial steps in assembly of the apratoxin A polyketide starter unit. Acs Chemical Biology. 12:3039-3048.   10.1021/acschembio.7b00746   AbstractWebsite

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" (GCNS-related N-acetyltransferase) loading modules of several modular biosynthetic pathways with propionyl, isobutyryt 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 alpha-carbon. The GNAT domain is truncated relative to functional homologues. These results afford an expanded understanding of MT1-GNAT structure and activity arid permit the functional annotation of homologous GNAT loading modules both with and without methyltransferases, additionally revealing their rapid evolutionary adaptation in different biosynthetic contexts.

Zhang, C, Idelbayev Y, Roberts N, Tao YW, Nannapaneni Y, Duggan BM, Min J, Lin EC, Gerwick EC, Cottrell GW, Gerwick WH.  2017.  Small Molecule Accurate Recognition Technology (SMART) to Enhance Natural Products Research. Scientific Reports. 7   10.1038/s41598-017-13923-x   AbstractWebsite

Various algorithms comparing 2D NMR spectra have been explored for their ability to dereplicate natural products as well as determine molecular structures. However, spectroscopic artefacts, solvent effects, and the interactive effect of functional group(s) on chemical shifts combine to hinder their effectiveness. Here, we leveraged Non-Uniform Sampling (NUS) 2D NMR techniques and deep Convolutional Neural Networks (CNNs) to create a tool, SMART, that can assist in natural products discovery efforts. First, an NUS heteronuclear single quantum coherence (HSQC) NMR pulse sequence was adapted to a state-of-the-art nuclear magnetic resonance (NMR) instrument, and data reconstruction methods were optimized, and second, a deep CNN with contrastive loss was trained on a database containing over 2,054 HSQC spectra as the training set. To demonstrate the utility of SMART, several newly isolated compounds were automatically located with their known analogues in the embedded clustering space, thereby streamlining the discovery pipeline for new natural products.

Gerwick, WH.  2017.  The Face of a Molecule. Journal of Natural Products. 80:2583-2588.   10.1021/acs.jnatprod.7b00624   AbstractWebsite

Recent technological advances in mass spectrometry and NMR spectroscopy have enabled new approaches for the rapid and insightful profiling of natural product mixtures. MALDI-MS with the provision of biosynthetic heavy-isotope labeled precursors can be a powerful method by which to interrogate a natural product metabolome and to gain insight into its unique constituents; this is illustrated herein by the detection, isolation, and characterization of cryptomaldamide. MS/MS-based Molecular Networks, facilitated by the Global Natural Products Social (GNPS) platform, is rapidly changing the way in which we dereplicate known natural products in mixtures, find new analogues in desired structure classes, and identify fundamentally new chemical entities. This method can be linked to genomic information to assist in genome-driven natural products discovery and is illustrated here with the characterization of the columbamides. Similarly, algorithmic interpretation of NMR data is facilitating the automatic identification or classification of new natural products. We developed such a tool named the Small Molecule Accurate Recognition Technology (SMART), which employs a convolutional neural network to classify HSQC spectra of organic molecules using pattern recognition principles. The discovery and rapid classification of several new peptides from a marine cyanobacterium as members of the viequeamide class provides an example of its utility in natural products research. These three illustrations represent different methods by which to look at the external features of a chemical substance and derive valuable insights into its identity or, as described herein, the "face of a molecule".

Fang, F, Zhao JY, Ding LJ, Huang CH, Naman CB, He S, Wu B, Zhu P, Luo QJ, Gerwick WH, Yan XJ, Wang QW, Zhang ZJ, Cui W.  2017.  5-Hydroxycyclopenicillone, a New beta-Amyloid Fibrillization Inhibitor from a Sponge-Derived Fungus Trichoderma sp HPQJ-34. Marine Drugs. 15   10.3390/md15080260   AbstractWebsite

A new cyclopentenone, 5-hydroxycyclopeni cillone (1), was isolated together with three known compounds, ar-turmerone (2), citreoisocoumarin (3), and 6-O-methyl-citreoisocoumarin (4), from a culture of the sponge-derived fungus Trichoderma sp. HPQJ-34. The structures of 1-4 were characterized using comprehensive spectroscopic analyses. The absolute configuration of 1 was determined by comparison of electronic circular dichroism (ECD) spectra with literature values used for the reported analogue, cyclopenicillone (5), which was not isolated in this research. Compound 1 was shown to scavenge 2,2-diphenyl-1-picrylhydrazyl free radicals, and decrease beta-amyloid (A beta) fibrillization in vitro. Moreover, 1 significantly reduced H2O2-induced neurotoxicity in SH-SY5Y cells. These findings suggested that compound 1, a newly discovered cyclopentenone, has moderate anti-oxidative, anti-A fi fibrillization properties and neuroprotective effects, and might be a good free radical scavenger.

Naman, CB, Almaliti J, Armstrong L, Caro-Diaz EJ, Pierce ML, Glukhov E, Fenner A, Spadafora C, Debonsi HM, Dorrestein PC, Murray TF, Gerwick WH.  2017.  Discovery and Synthesis of Caracolamide A, an Ion Channel Modulating Dichlorovinylidene Containing Phenethylamide from a Panamanian Marine Cyanobacterium cf. Symploca Species. Journal of Natural Products. 80:2328-2334.   10.1021/acs.jnatprod.7b00367   AbstractWebsite

A recent untargeted metabolomics investigation into the chemical profile of 10 organic extracts from cf. Symploca spp. revealed several interesting chemical leads for further natural product drug discovery. Subsequent target directed isolation efforts with one of these, a Panamanian marine cyanobacterium cf. Symploca sp., yielded a phenethylamide metabolite that terminates in a relatively rare gemdichlorovinylidene moiety, caracolarnide A (1), along with a known isotactic polymethoxy-1-alkene (2). Detailed NMR and HRESIMS analyses were used to determine the structures of these molecules, and compound 1 was confirmed by a three step synthesis. Pure compound 1 was shown to have in vitro calcium influx and calcium channel oscillation modulatory activity when tested as low as 10 pM using cultured murine cortical neurons, but was not cytotoxic to NCI-H460 human non-small-cell lung cancer cells in vitro (IC50 > 10 mu M).

Almaliti, J, Malloy KL, Glukhov E, Spadafora C, Gutierrez M, Gerwick WH.  2017.  Dudawalamides A-D, antiparasitic cyclic depsipeptides from the marine cyanobacterium Moorea producens. Journal of Natural Products. 80:1827-1836.   10.1021/acs.jnatprod.7b00034   AbstractWebsite

A family of 2,2-dimethyl-3-hydroxy-7-octynoic acid (Dhoya)-containing cyclic depsipeptides, named dudawalamides A-D (1-4), was isolated from a Papua New Guinean field collection of the cyanobacterium Moorea producens using bioassay-guided and spectroscopic approaches. The planar structures of dudawalamides A-D were determined by a combination of 1D and 2D NMR experiments and MS analysis, whereas the absolute configurations were determined by X-ray crystallography, modified Marfey's analysis, chiral-phase GCMS, and chiral-phase HPLC. Dudawalamides A-D possess a broad spectrum of antiparasitic activity with minimal mammalian cell cytotoxicity. Comparative analysis of the Dhoya-containing class of lipopeptides reveals intriguing structure-activity relationship features of these NRPS-PKS-derived metabolites and their derivatives.

Luzzatto-Knaan, T, Garg N, Wang MX, Glukhov E, Peng Y, Ackermann G, Amir A, Duggan BM, Ryazanov S, Gerwick L, Knight R, Alexandov T, Bandeira N, Gerwick WH, Dorrestein PC.  2017.  Digitizing mass spectrometry data to explore the chemical diversity and distribution of marine cyanobacteria and algae. Elife. 6   10.7554/eLife.24214   AbstractWebsite

Natural product screening programs have uncovered molecules from diverse natural sources with various biological activities and unique structures. However, much is yet underexplored and additional information is hidden in these exceptional collections. We applied untargeted mass spectrometry approaches to capture the chemical space and dispersal patterns of metabolites from an in-house library of marine cyanobacterial and algal collections. Remarkably, 86% of the metabolomics signals detected were not found in other available datasets of similar nature, supporting the hypothesis that marine cyanobacteria and algae possess distinctive metabolomes. The data were plotted onto a world map representing eight major sampling sites, and revealed potential geographic locations with high chemical diversity. We demonstrate the use of these inventories as a tool to explore the diversity and distribution of natural products. Finally, we utilized this tool to guide the isolation of a new cyclic lipopeptide, yuvalamide A, from a marine cyanobacterium.

Kinnel, RB, Esquenazi E, Leao T, Moss N, Mevers E, Pereira AR, Monroe EA, Korobeynikov A, Murray TF, Sherman D, Gerwick L, Dorrestein PC, Gerwick WH.  2017.  A maldiisotopic approach to discover natural products: Cryptomaldamide, a hybrid tripeptide from the marine cyanobacterium Moorea producens. Journal of Natural Products. 80:1514-1521.   10.1021/acs.jnatprod.7b00019   AbstractWebsite

Genome sequencing of microorganisms has revealed a greatly increased capacity for natural products biosynthesis than was previously recognized from compound isolation efforts alone. Hence, new methods are needed for the discovery and description of this hidden secondary metabolite potential. Here we show that provision of heavy nitrogen N-15-nitrate to marine cyanobacterial cultures followed by single-filament MALDI analysis over a period of days was highly effective in identifying a new natural product with an exceptionally high nitrogen content. The compound, named cryptomaldamide, was subsequently isolated using MS to guide the purification process, and its structure determined by 2D NMR and other spectroscopic and chromatographic methods. Bioinformatic analysis of the draft genome sequence identified a 28.7 kB gene cluster that putatively encodes for cryptomaldamide biosynthesis. Notably, an amidinotransferase is proposed to initiate the biosynthetic process by transferring an amidino group from arginine to serine to produce the first residue to be incorporated by the hybrid NRPS-PKS pathway. The maldiisotopic approach presented here is thus demonstrated to provide an orthogonal method by which to discover novel chemical diversity from Nature.

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.

Zhang, C, Naman CB, Engene N, Gerwick WH.  2017.  Laucysteinamide A, a hybrid PKS/NRPS metabolite from a Saipan cyanobacterium, cf. Caldora penicillata. Marine Drugs. 15   10.3390/md15040121   AbstractWebsite

A bioactivity guided study of a cf. Caldora penicillata species, collected during a 2013 expedition to the Pacific island of Saipan, Northern Mariana Islands (a commonwealth of the USA), led to the isolation of a new thiazoline-containing alkaloid, laucysteinamide A (1). Laucysteinamide A is a new monomeric analogue of the marine cyanobacterial metabolite, somocystinamide A (2), a disulfide-bonded dimeric compound that was isolated previously from a Fijian marine cyanobacterium. The structure and absolute configuration of laucysteinamide A (1) was determined by a detailed analysis of its NMR, MS, and CD spectra. In addition, the highly bioactive lipid, curacin D (3), was also found to be present in this cyanobacterial extract. The latter compound was responsible for the potent cytotoxicity of this extract to H-460 human non-small cell lung cancer cells in vitro.

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] .

Lin, JJ, Yu J, Zhao JY, Zhang K, Zheng JC, Wang JL, Huang CH, Zhang JR, Yan XJ, Gerwick WH, Wang QW, Cui W, He S.  2017.  Fucoxanthin, a Marine Carotenoid, Attenuates beta-Amyloid Oligomer-Induced Neurotoxicity Possibly via Regulating the PI3K/Akt and the ERK Pathways in SH-SY5Y Cells. Oxidative Medicine and Cellular Longevity.   10.1155/2017/6792543   AbstractWebsite
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.