LCMS-Metabolomic Profiling and Genome Mining of Delftia lacustris DSM 21246 Revealed Lipophilic Delftibactin Metallophores.

Bacteria have evolved various strategies to combat heavy metal stress, including the secretion of small molecules, known as metallophores. These molecules hold a potential role in the mitigation of toxic metal contamination from the environment (bioremediation). Herein, we employed combined comparative metabolomic and genomic analyses to study the metallophores excreted by Delftia lacustris DSM 21246. LCMS-metabolomic analysis of this bacterium cultured under iron limitation led to a suite of lipophilic metallophores exclusively secreted in response to iron starvation. Additionally, we conducted genome sequencing of the DSM 21246 strain using nanopore sequencing technology and employed antiSMASH to mine the genome, leading to the identification of a biosynthetic gene cluster (BGC) matching the known BGC responsible for delftibactin A production. The isolated suite of amphiphilic metallophores, termed delftibactins C-F (1-4), was characterized using various chromatographic, spectroscopic, and bioinformatic techniques. The planar structure of these compounds was elucidated through 1D and 2D NMR analyses, as well as LCMS/MS-based fragmentation studies. Notably, their structures differed from previously known delftibactins due to the presence of a lipid tail. Marfey's and bioinformatic analyses were employed to determine the absolute configuration of the peptide scaffold. Delftibactin A, a previously identified metallophore, has exhibited a gold biomineralizing property; compound 1 was tested for and also demonstrated this property.

Structure and Biosynthesis of Hectoramide B, a Linear Depsipeptide from Marine Cyanobacterium Moorena producens JHB Discovered via Coculture with Candida albicans.

The tropical marine cyanobacterium Moorena producens JHB is a prolific source of secondary metabolites with potential biomedical utility. Previous studies on this strain led to the discovery of several novel compounds such as hectochlorins and jamaicamides. However, bioinformatic analyses of its genome indicate the presence of numerous cryptic biosynthetic gene clusters that have yet to be characterized. To potentially stimulate the production of novel compounds from this strain, it was cocultured with Candida albicans. From this experiment, we observed the increased production of a new compound that we characterize here as hectoramide B. Bioinformatic analysis of the M. producens JHB genome enabled the identification of a putative biosynthetic gene cluster responsible for hectoramide B biosynthesis. This work demonstrates that coculture competition experiments can be a valuable method to facilitate the discovery of novel natural products from cyanobacteria.

Comparative metabolomic profiling of Cupriavidus necator B-4383 revealed production of cupriachelin siderophores, one with activity against Cryptococcus neoformans.

Cupriavidus necator H16 is known to be a rich source of linear lipopeptide siderophores when grown under iron-depleted conditions; prior literature termed these compounds cupriachelins. These small molecules bear ß-hydroxyaspartate moieties that contribute to a photoreduction of iron when bound as ferric cupriachelin. Here, we present structural assignment of cupriachelins from C. necator B-4383 grown under iron limitation. The characterization of B-4383 cupriachelins is based on MS/MS fragmentation analysis, which was confirmed by 1D- and 2D-NMR for the most abundant analog (1). The cupriachelin congeners distinguish these two strains with differences in the preferred lipid tail; however, our rigorous metabolomic investigation also revealed minor analogs with changes in the peptide core, hinting at a potential mechanism by which these siderophores may reduce biologically unavailable ferric iron (4-6). Antifungal screening of the C. necator B-4383 supernatant extract and the isolated cupriachelin analog (1) revealed inhibitory activity against Cryptococcus neoformans, with IC50 values of 16.6 and 3.2 µg/mL, respectively. This antifungal activity could be explained by the critical role of the iron acquisition pathway in the growth and pathogenesis of the C. neoformans fungal pathogen.

Structure and Biosynthesis of Hectoramide B, a Linear Depsipeptide from the Marine Cyanobacterium Moorena producens JHB Discovered via Co-culture with Candida albicans.

The tropical marine cyanobacterium Moorena producens JHB is a prolific source of secondary metabolites with potential biomedical utility. Previous studies of this strain led to the discovery of several novel compounds such as the hectochlorins and jamaicamides; however, bioinformatic analyses of its genome suggested that there were many more cryptic biosynthetic gene clusters yet to be characterized. To potentially stimulate the production of novel compounds from this strain, it was co-cultured with Candida albicans. From this experiment, we observed the increased production of a new compound that we characterize here as hectoramide B. Bioinformatic analysis of the M. producens JHB genome enabled the identification of a putative biosynthetic gene cluster responsible for hectoramide B biosynthesis. This work demonstrates that co-culture competition experiments can be a valuable method to facilitate the discovery of novel natural products from cyanobacteria.

Luquilloamides, Cytotoxic Lipopeptides from a Puerto Rican Collection of the Filamentous Marine Cyanobacterium Oscillatoria sp.

Luquilloamides A-G (1-7) were isolated from a small environmental collection of a marine cyanobacterium found growing on eelgrass (Zostera sp.) near Luquillo, Puerto Rico. Structure elucidation of the luquilloamides was accomplished via detailed NMR and MS analyses, and absolute configurations were determined using a combination of advanced Mosher's method, J-based configuration analysis, semisynthetic fragment analysis derived from ozonolysis, methylation, Baeyer-Villiger oxidation, Mosher's esterification, specific rotations, and ECD data. Except for 2, the luquilloamides share a characteristic tert-butyl-containing polyketide fragment, ß-alanine, and a proposed highly modified polyketide extension. While compound 1 is a linear lipopeptide with two α-methyl branches and a vinyl chloride functionality in the polyketide portion, compounds 4, 6, and 7 possess a cyclohexanone structure with methylation on the α- or ß-positions of the polyketide as well as an acetyl group. Interestingly, the absolute configuration at C-5 and C-6 on the cyclohexanone unit in 7 is opposite to that of 4-6. Compound 3 was revealed to have a tert-butyl-containing polyketide, ß-alanine, and a PKS/NRPS-derived γ-isopropyl pyrrolinone. Compound 2 may be a hydrolysis product of 3. Of the seven new compounds, 1 showed the most potent cytotoxicity to human H-460 lung cancer cells.

Design of Gallinamide A Analogs as Potent Inhibitors of the Cysteine Proteases Human Cathepsin L and Trypanosoma cruzi Cruzain.

Gallinamide A, originally isolated with a modest antimalarial activity, was subsequently reisolated and characterized as a potent, selective, and irreversible inhibitor of the human cysteine protease cathepsin L. Molecular docking identified potential modifications to improve binding, which were synthesized as a suite of analogs. Resultingly, this current study produced the most potent gallinamide analog yet tested against cathepsin L (10, Ki = 0.0937 ± 0.01 nM and kinact/Ki = 8 730 000). From a protein structure and substrate preference perspective, cruzain, an essential Trypanosoma cruzi cysteine protease, is highly homologous. Our investigations revealed that gallinamide and its analogs potently inhibit cruzain and are exquisitely toxic toward T. cruzi in the intracellular amastigote stage. The most active compound, 5, had an IC50 = 5.1 ± 1.4 nM, but was relatively inactive to both the epimastigote (insect stage) and the host cell, and thus represents a new candidate for the treatment of Chagas disease.

Reactivity of an Unusual Amidase May Explain Colibactin's DNA Cross-Linking Activity.

Certain commensal and pathogenic bacteria produce colibactin, a small-molecule genotoxin that causes interstrand cross-links in host cell DNA. Although colibactin alkylates DNA, the molecular basis for cross-link formation is unclear. Here, we report that the colibactin biosynthetic enzyme ClbL is an amide bond-forming enzyme that links aminoketone and ß-keto thioester substrates in vitro and in vivo. The substrate specificity of ClbL strongly supports a role for this enzyme in terminating the colibactin NRPS-PKS assembly line and incorporating two electrophilic cyclopropane warheads into the final natural product scaffold. This proposed transformation was supported by the detection of a colibactin-derived cross-linked DNA adduct. Overall, this work provides a biosynthetic explanation for colibactin's DNA cross-linking activity and paves the way for further study of its chemical structure and biological roles.

The human gut bacterial genotoxin colibactin alkylates DNA.

Certain Escherichia coli strains residing in the human gut produce colibactin, a small-molecule genotoxin implicated in colorectal cancer pathogenesis. However, colibactin's chemical structure and the molecular mechanism underlying its genotoxic effects have remained unknown for more than a decade. Here we combine an untargeted DNA adductomics approach with chemical synthesis to identify and characterize a covalent DNA modification from human cell lines treated with colibactin-producing E. coli Our data establish that colibactin alkylates DNA with an unusual electrophilic cyclopropane. We show that this metabolite is formed in mice colonized by colibactin-producing E. coli and is likely derived from an initially formed, unstable colibactin-DNA adduct. Our findings reveal a potential biomarker for colibactin exposure and provide mechanistic insights into how a gut microbe may contribute to colorectal carcinogenesis.

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

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

Integrating Molecular Networking and Biological Assays To Target the Isolation of a Cytotoxic Cyclic Octapeptide, Samoamide A, from an American Samoan Marine Cyanobacterium.

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 µM). 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 µM 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.

Sharing and community curation of mass spectrometry data with Global Natural Products Social Molecular Networking.

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; http://gnps.ucsd.edu), 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.

Synthesis of Highly Substituted Quinolines via a Tandem Ynamide Benzannulation/Iodocyclization Strategy.

A two-stage "tandem strategy" for the regiocontrolled synthesis of very highly substituted quinolines is described. Benzannulation based on the reaction of cyclobutenones or diazo ketones with N-propargyl-substituted ynamides proceeds via a cascade of several pericyclic reactions to generate multiply substituted aniline derivatives. In the second stage of the tandem strategy, triflate derivatives of the phenolic benzannulation products undergo Larock cyclization upon exposure to iodine to form products that are further elaborated by methods such as palladium-catalyzed coupling to generate quinolines that can be substituted at every position of the bicyclic system.

Expanding the Described Metabolome of the Marine Cyanobacterium Moorea producens JHB through Orthogonal Natural Products Workflows.

Moorea producens JHB, a Jamaican strain of tropical filamentous marine cyanobacteria, has been extensively studied by traditional natural products techniques. These previous bioassay and structure guided isolations led to the discovery of two exciting classes of natural products, hectochlorin (1) and jamaicamides A (2) and B (3). In the current study, mass spectrometry-based 'molecular networking' was used to visualize the metabolome of Moorea producens JHB, and both guided and enhanced the isolation workflow, revealing additional metabolites in these compound classes. Further, we developed additional insight into the metabolic capabilities of this strain by genome sequencing analysis, which subsequently led to the isolation of a compound unrelated to the jamaicamide and hectochlorin families. Another approach involved stimulation of the biosynthesis of a minor jamaicamide metabolite by cultivation in modified media, and provided insights about the underlying biosynthetic machinery as well as preliminary structure-activity information within this structure class. This study demonstrated that these orthogonal approaches are complementary and enrich secondary metabolomic coverage even in an extensively studied bacterial strain.

Microbiota of healthy corals are active against fungi in a light-dependent manner.

Coral reefs are intricate ecosystems that harbor diverse organisms, including 25% of all marine fish. Healthy corals exhibit a complex symbiosis between coral polyps, endosymbiotic alga, and an array of microorganisms, called the coral holobiont. Secretion of specialized metabolites by coral microbiota is thought to contribute to the defense of this sessile organism against harmful biotic and abiotic factors. While few causative agents of coral diseases have been unequivocally identified, fungi have been implicated in the massive destruction of some soft corals worldwide. Because corals are nocturnal feeders, they may be more vulnerable to fungal infection at night, and we hypothesized that the coral microbiota would have the capability to enhance their defenses against fungi in the dark. A Pseudoalteromonas sp. isolated from a healthy octocoral displayed light-dependent antifungal properties when grown adjacent to Penicillium citrinum (P. citrinum) isolated from a diseased Gorgonian octocoral. Microbial MALDI-imaging mass spectrometry (IMS) coupled with molecular network analyses revealed that Pseudoalteromonas produced higher levels of antifungal polyketide alteramides in the dark than in the light. The alteramides were inactivated by light through a photoinduced intramolecular cyclization. Further NMR studies led to a revision of the stereochemical structure of the alteramides. Alteramide A exhibited antifungal properties and elicited changes in fungal metabolite distributions of mycotoxin citrinin and citrinadins. These data support the hypothesis that coral microbiota use abiotic factors such as light to regulate the production of metabolites with specialized functions to combat opportunistic pathogens at night.

Lipopeptides from the tropical marine cyanobacterium Symploca sp.

A collection of the tropical marine cyanobacterium Symploca sp., collected near Kimbe Bay, Papua New Guinea, previously yielded several new metabolites including kimbeamides A-C, kimbelactone A, and tasihalide C. Investigations into a more polar cytotoxic fraction yielded three new lipopeptides, tasiamides C-E (1-3). The planar structures were deduced by 2D NMR spectroscopy and tandem mass spectrometry, and their absolute configurations were determined by a combination of Marfey's and chiral-phase GC-MS analysis. These new metabolites are similar to several previously isolated compounds, including tasiamide (4), grassystatins (5, 6), and symplocin A, all of which were isolated from similar filamentous marine cyanobacteria.

MS/MS-based networking and peptidogenomics guided genome mining revealed the stenothricin gene cluster in Streptomyces roseosporus.

Most (75%) of the anti-infectives that save countless lives and enormously improve quality of life originate from microbes found in nature. Herein, we described a global visualization of the detectable molecules produced from a single microorganism, which we define as the 'molecular network' of that organism, followed by studies to characterize the cellular effects of antibacterial molecules. We demonstrate that Streptomyces roseosporus produces at least four non-ribosomal peptide synthetase-derived molecular families and their gene subnetworks (daptomycin, arylomycin, napsamycin and stenothricin) were identified with different modes of action. A number of previously unreported analogs involving truncation, glycosylation, hydrolysis and biosynthetic intermediates and/or shunt products were also captured and visualized by creation of a map through MS/MS networking. The diversity of antibacterial compounds produced by S. roseosporus highlights the importance of developing new approaches to characterize the molecular capacity of an organism in a more global manner. This allows one to more deeply interrogate the biosynthetic capacities of microorganisms with the goal to streamline the discovery pipeline for biotechnological applications in agriculture and medicine. This is a contribution to a special issue to honor Chris Walsh's amazing career.

Molecular networking as a dereplication strategy.

A major goal in natural product discovery programs is to rapidly dereplicate known entities from complex biological extracts. We demonstrate here that molecular networking, an approach that organizes MS/MS data based on chemical similarity, is a powerful complement to traditional dereplication strategies. Successful dereplication with molecular networks requires MS/MS spectra of the natural product mixture along with MS/MS spectra of known standards, synthetic compounds, or well-characterized organisms, preferably organized into robust databases. This approach can accommodate different ionization platforms, enabling cross correlations of MS/MS data from ambient ionization, direct infusion, and LC-based methods. Molecular networking not only dereplicates known molecules from complex mixtures, it also captures related analogues, a challenge for many other dereplication strategies. To illustrate its utility as a dereplication tool, we apply mass spectrometry-based molecular networking to a diverse array of marine and terrestrial microbial samples, illustrating the dereplication of 58 molecules including analogues.

Viequeamide A, a cytotoxic member of the kulolide superfamily of cyclic depsipeptides from a marine button cyanobacterium.

The viequeamides, a family of 2,2-dimethyl-3-hydroxy-7-octynoic acid (Dhoya)-containing cyclic depsipeptides, were isolated from a shallow subtidal collection of a "button" cyanobacterium (Rivularia sp.) from near the island of Vieques, Puerto Rico. Planar structures of the two major compounds, viequeamide A (1) and viequeamide B (2), were elucidated by 2D-NMR spectroscopy and mass spectrometry, whereas absolute configurations were determined by traditional hydrolysis, derivative formation, and chromatography in comparison with standards. In addition, a series of related minor metabolites, viequeamides C-F (3-6), were characterized by HRMS fragmentation methods. Viequeamide A was found to be highly toxic to H460 human lung cancer cells (IC(50) = 60 ± 10 nM), whereas the mixture of B-F was inactive. From a broader perspective, the viequeamides help to define a "superfamily" of related cyanobacterial natural products, the first of which to be discovered was kulolide. Within the kulolide superfamily, a wide variation in biological properties is observed, and the reported producing strains are also highly divergent, giving rise to several intriguing questions about structure-activity relationships and the evolutionary origins of this metabolite class.