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.

PECAN Predicts Patterns of Cancer Cell Cytostatic Activity of Natural Products Using Deep Learning.

Many machine learning techniques are used as drug discovery tools with the intent to speed characterization by determining relationships between compound structure and biological function. However, particularly in anticancer drug discovery, these models often make only binary decisions about the biological activity for a narrow scope of drug targets. We present a feed-forward neural network, PECAN (Prediction Engine for the Cytostatic Activity of Natural product-like compounds), that simultaneously classifies the potential antiproliferative activity of compounds against 59 cancer cell lines. It predicts the activity to be one of six categories, indicating not only if activity is present but the degree of activity. Using an independent subset of NCI data as a test set, we show that PECAN can reach 60.1% accuracy in a six-way classification and present further evidence that it classifies based on useful structural features of compounds using a "within-one" measure that reaches 93.0% accuracy.

The Kavaratamides: Discovery of Linear Lipodepsipeptides from the Marine Cyanobacterium Moorena bouillonii Using a Comparative Chemogeographic Analysis.

Kavaratamide A (1), a new linear lipodepsipeptide possessing an unusual isopropyl-O-methylpyrrolinone moiety, was discovered from the tropical marine filamentous cyanobacterium Moorena bouillonii collected from Kavaratti, India. A comparative chemogeographic analysis of M. bouillonii collected from six different geographical regions led to the prioritized isolation of this metabolite from India as distinctive among our data sets. AI-based structure annotation tools, including SMART 2.1 and DeepSAT, accelerated the structure elucidation by providing useful structural clues, and the full planar structure was elucidated based on comprehensive HRMS, MS/MS fragmentation, and NMR data interpretation. Subsequently, the absolute configuration of 1 was determined using advanced Marfey's analysis, modified Mosher's ester derivatization, and chiral-phase HPLC. The structures of kavaratamides B (2) and C (3) are proposed based on a detailed analysis of their MS/MS fragmentations. The biological activity of kavaratamide A was also investigated and found to show moderate cytotoxicity to the D283-medullablastoma cell line.

Development of Potent and Highly Selective Epoxyketone-Based Plasmodium Proteasome Inhibitors.

Here, we present remarkable epoxyketone-based proteasome inhibitors with low nanomolar in vitro potency for blood-stage Plasmodium falciparum and low cytotoxicity for human cells. Our best compound has more than 2,000-fold greater selectivity for erythrocytic-stage P. falciparum over HepG2 and H460 cells, which is largely driven by the accommodation of the parasite proteasome for a D-amino acid in the P3 position and the preference for a difluorobenzyl group in the P1 position. We isolated the proteasome from P. falciparum cell extracts and determined that the best compound is 171-fold more potent at inhibiting the ß5 subunit of P. falciparum proteasome when compared to the same subunit of the human constitutive proteasome. These compounds also significantly reduce parasitemia in a P. berghei mouse infection model and prolong survival of animals by an average of 6 days. The current epoxyketone inhibitors are ideal starting compounds for orally bioavailable anti-malarial drugs.

Structure and Candidate Biosynthetic Gene Cluster of a Manumycin-Type Metabolite from Salinispora pacifica.

A new manumycin-type natural product named pacificamide (1) and its candidate biosynthetic gene cluster (pac) were discovered from the marine actinobacterium Salinispora pacifica CNT-855. The structure of the compound was determined using NMR, electronic circular dichroism, and bioinformatic predictions. The pac gene cluster is unique to S. pacifica and found in only two of the 119 Salinispora genomes analyzed across nine species. Comparative analyses of biosynthetic gene clusters encoding the production of related manumycin-type compounds revealed genetic differences in accordance with the unique pacificamide structure. Further queries of manumycin-type gene clusters from public databases revealed their limited distribution across the phylum Actinobacteria and orphan diversity that suggests additional products remain to be discovered in this compound class. Production of the known metabolite triacsin D is also reported for the first time from the genus Salinispora. This study adds two classes of compounds to the natural product collective isolated from the genus Salinispora, which has proven to be a useful model for natural product research.

Total Synthesis of Laucysteinamide A, a Monomeric Congener of Somocystinamide A.

Laucysteinamide A (4) is a marine natural product isolated from the cyanobacterium Caldora penicillata and contains structural motifs found in promising cancer drug leads. The first total synthesis of 4 and its analogues was achieved, which also enabled a concise formal synthesis of somocystinamide A (3), a dimeric congener of 4 that previously showed extremely potent antiproliferative activities. This work provides further insights on structure-activity relationships in this class of natural products.

A Multi-Omics Characterization of the Natural Product Potential of Tropical Filamentous Marine Cyanobacteria.

Microbial natural products are important for the understanding of microbial interactions, chemical defense and communication, and have also served as an inspirational source for numerous pharmaceutical drugs. Tropical marine cyanobacteria have been highlighted as a great source of new natural products, however, few reports have appeared wherein a multi-omics approach has been used to study their natural products potential (i.e., reports are often focused on an individual natural product and its biosynthesis). This study focuses on describing the natural product genetic potential as well as the expressed natural product molecules in benthic tropical cyanobacteria. We collected from several sites around the world and sequenced the genomes of 24 tropical filamentous marine cyanobacteria. The informatics program antiSMASH was used to annotate the major classes of gene clusters. BiG-SCAPE phylum-wide analysis revealed the most promising strains for natural product discovery among these cyanobacteria. LCMS/MS-based metabolomics highlighted the most abundant molecules and molecular classes among 10 of these marine cyanobacterial samples. We observed that despite many genes encoding for peptidic natural products, peptides were not as abundant as lipids and lipopeptides in the chemical extracts. Our results highlight a number of highly interesting biosynthetic gene clusters for genome mining among these cyanobacterial samples.

A Convolutional Neural Network-Based Approach for the Rapid Annotation of Molecularly Diverse Natural Products.

This report describes the first application of the novel NMR-based machine learning tool "Small Molecule Accurate Recognition Technology" (SMART 2.0) for mixture analysis and subsequent accelerated discovery and characterization of new natural products. The concept was applied to the extract of a filamentous marine cyanobacterium known to be a prolific producer of cytotoxic natural products. This environmental Symploca extract was roughly fractionated, and then prioritized and guided by cancer cell cytotoxicity, NMR-based SMART 2.0, and MS2-based molecular networking. This led to the isolation and rapid identification of a new chimeric swinholide-like macrolide, symplocolide A, as well as the annotation of swinholide A, samholides A-I, and several new derivatives. The planar structure of symplocolide A was confirmed to be a structural hybrid between swinholide A and luminaolide B by 1D/2D NMR and LC-MS2 analysis. A second example applies SMART 2.0 to the characterization of structurally novel cyclic peptides, and compares this approach to the recently appearing "atomic sort" method. This study exemplifies the revolutionary potential of combined traditional and deep learning-assisted analytical approaches to overcome longstanding challenges in natural products drug discovery.

Pagoamide A, a Cyclic Depsipeptide Isolated from a Cultured Marine Chlorophyte, Derbesia sp., Using MS/MS-Based Molecular Networking.

A thiazole-containing cyclic depsipeptide with 11 amino acid residues, named pagoamide A (1), was isolated from laboratory cultures of a marine Chlorophyte, Derbesia sp. This green algal sample was collected from America Samoa, and pagoamide A was isolated using guidance by MS/MS-based molecular networking. Cultures were grown in a light- and temperature-controlled environment and harvested after several months of growth. The planar structure of pagoamide A (1) was characterized by detailed 1D and 2D NMR experiments along with MS and UV analysis. The absolute configurations of its amino acid residues were determined by advanced Marfey's analysis following chemical hydrolysis and hydrazinolysis reactions. Two of the residues in pagoamide A (1), phenylalanine and serine, each occurred twice in the molecule, once in the d- and once in the l-configuration. The biosynthetic origin of pagoamide A (1) was considered in light of other natural products investigations with coenocytic green algae.

An anti-inflammatory isoflavone from soybean inoculated with a marine fungus Aspergillus terreus C23-3.

A new isoflavone derivative compound 1 (psoralenone) was isolated from soybean inoculated with a marine fungus Aspergillus terreus C23-3, together with seven known compounds including isoflavones 2-6, butyrolactone I (7) and blumenol A (8). Their structures were elucidated by MS, NMR, and ECD. Psoralenone displayed moderate in vitro anti-inflammatory activity in the LPS-induced RAW264.7 cell model. Compound 2 (genistein) showed moderate acetylcholinesterase (AChE) inhibitory activity whereas compounds 2, 5 (biochanin A), 6 (psoralenol), and 7 exhibited potent larvicidal activity against brine shrimp. Compounds 3 (daidzein), 4 (4'-hydroxy-6,7-dimethoxyisoflavone), and 5-7 showed broad-spectrum anti-microbial activity, and compound 7 also showed moderate 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity.

Applying a Chemogeographic Strategy for Natural Product Discovery from the Marine Cyanobacterium Moorena bouillonii.

The tropical marine cyanobacterium Moorena bouillonii occupies a large geographic range across the Indian and Western Tropical Pacific Oceans and is a prolific producer of structurally unique and biologically active natural products. An ensemble of computational approaches, including the creation of the ORCA (Objective Relational Comparative Analysis) pipeline for flexible MS1 feature detection and multivariate analyses, were used to analyze various M. bouillonii samples. The observed chemogeographic patterns suggested the production of regionally specific natural products by M. bouillonii. Analyzing the drivers of these chemogeographic patterns allowed for the identification, targeted isolation, and structure elucidation of a regionally specific natural product, doscadenamide A (1). Analyses of MS2 fragmentation patterns further revealed this natural product to be part of an extensive family of herein annotated, proposed natural structural analogs (doscadenamides B-J, 2-10); the ensemble of structures reflect a combinatorial biosynthesis using nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) components. Compound 1 displayed synergistic in vitro cancer cell cytotoxicity when administered with lipopolysaccharide (LPS). These discoveries illustrate the utility in leveraging chemogeographic patterns for prioritizing natural product discovery efforts.

Comparative genomics uncovers the prolific and distinctive metabolic potential of the cyanobacterial genus Moorea.

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.

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.

Integrated Genomic and Metabolomic Approach to the Discovery of Potential Anti-Quorum Sensing Natural Products from Microbes Associated with Marine Samples from Singapore.

With 70% of the Earth's surface covered in water, the marine ecosystem offers immense opportunities for drug discovery and development. Due to the decreasing rate of novel natural product discovery from terrestrial sources in recent years, many researchers are beginning to look seaward for breakthroughs in new therapeutic agents. As part of an ongoing marine drug discovery programme in Singapore, an integrated approach of combining metabolomic and genomic techniques were initiated for uncovering novel anti-quorum sensing molecules from bacteria associated with subtidal samples collected in the Singapore Strait. Based on the culture-dependent method, a total of 102 marine bacteria strains were isolated and the identities of selected strains were established based on their 16S rRNA gene sequences. About 5% of the marine bacterial organic extracts showed quorum sensing inhibitory (QSI) activity in a dose-dependent manner based on the Pseudomonas aeruginosa QS reporter system. In addition, the extracts were subjected to mass spectrometry-based molecular networking and the genome of selected strains were analysed for known as well as new biosynthetic gene clusters. This study revealed that using integrated techniques, coupled with biological assays, can provide an effective and rapid prioritization of marine bacterial strains for downstream large-scale culturing for the purpose of isolation and structural elucidation of novel bioactive compounds.

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.

Bastimolide B, an Antimalarial 24-Membered Marine Macrolide Possessing a tert-Butyl Group.

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 tert-butyl 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.

MS/MS-Based Molecular Networking Approach for the Detection of Aplysiatoxin-Related Compounds in Environmental Marine Cyanobacteria.

Certain strains of cyanobacteria produce a wide array of cyanotoxins, such as microcystins, lyngbyatoxins and aplysiatoxins, that are associated with public health issues. In this pilot study, an approach combining LC-MS/MS and molecular networking was employed as a rapid analytical method to detect aplysiatoxins present in four environmental marine cyanobacterial samples collected from intertidal areas in Singapore. Based on 16S-ITS rRNA gene sequences, these filamentous cyanobacterial samples collected from Pulau Hantu were determined as Trichodesmium erythraeum, Oscillatoria sp. PAB-2 and Okeania sp. PNG05-4. Organic extracts were prepared and analyzed on LC-HRMS/MS and Global Natural Product Social Molecular Networking (GNPS) for the presence of aplysiatoxin-related molecules. From the molecular networking, six known compounds, debromoaplysiatoxin (1), anhydrodebromoaplysiatoxin (2), 3-methoxydebromoaplysiatoxin (3), aplysiatoxin (4), oscillatoxin A (5) and 31-noroscillatoxin B (6), as well as potential new analogues, were detected in these samples. In addition, differences and similarities in molecular networking clusters related to the aplysiatoxin molecular family were observed in extracts of Trichodesmium erythraeum collected from two different locations and from different cyanobacterial species found at Pulau Hantu, respectively.

Dudawalamides A-D, Antiparasitic Cyclic Depsipeptides from the Marine Cyanobacterium Moorea producens.

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.

Discovery and Synthesis of Caracolamide A, an Ion Channel Modulating Dichlorovinylidene Containing Phenethylamide from a Panamanian Marine Cyanobacterium cf. Symploca Species.

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 gem-dichlorovinylidene moiety, caracolamide 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 µM).