Paul D. Boudreau

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.

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.

Bastimolide A, a Potent Antimalarial Polyhydroxy Macrolide from the Marine Cyanobacterium Okeania hirsuta

Bastimolide A (1), a polyhydroxy macrolide with a 40-membered ring, was isolated from a new genus of the tropical marine cyanobacterium Okeania hirsuta. This novel macrolide was defined by spectroscopy and chemical reactions to possess one 1,3-diol, one 1,3,5-triol, six 1,5-diols, and one tert-butyl group; however, the relationships of these moieties to one another were obscured by a highly degenerate (1)H NMR spectrum. Its complete structure and absolute configuration were therefore unambiguously determined by X-ray diffraction analysis of the nona-p-nitrobenzoate derivative (1d). Pure bastimolide A (1) showed potent antimalarial activity against four resistant strains of Plasmodium falciparum with IC50 values between 80 and 270 nM, although with some toxicity to the control Vero cells (IC50 = 2.1 μM), and thus represents a potentially promising lead for antimalarial drug discovery. Moreover, rigorous establishment of its molecular arrangement gives fresh insight into the structures and biosynthesis of cyanobacterial polyhydroxymacrolides.

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 Penicilliumcitrinum (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.

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., Marfeys 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.

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.

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.

Interkingdom signaling by structurally related cyanobacterial and algal secondary metabolites

Several groups of structurally-related compounds, comprised of either five or six-membered ring structures with attached lipophilic carbon chains and in some cases possessing halogen atoms, have been isolated from various marine algae and filamentous cyanobacteria. The related compounds considered in the present work include the coibacins, laurenciones, honaucins, malyngamides and the tumonoic acids. Members of all of these compound families were assayed and found to inhibit the production of nitric oxide in lipopolysaccharides-stimulated macrophages, indicating their anti-inflammatory potential. In addition, several of these same marine natural products were found to inhibit quorum sensing mediated phenotypes in Vibrio harveyi BB120 and/or Escherichia coli JB525. The mechanism and evolutionary significance for inhibition of these cellular processes in prokaryotic and eukaryotic systems are speculated on and discussed.

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.