Rapid Unambiguous Structure Elucidation of Streptnatamide A, a New Cyclic Peptide Isolated from A Marine-derived Streptomyces sp.

Cyclic peptides have been excellent source of drug leads. With the advances in discovery platforms, the pharmaceutical industry has a growing interest in cyclic peptides and has pushed several into clinical trials. However, structural complexity of cyclic peptides brings extreme challenges for structure elucidation efforts. Isotopic fine structure analysis, Nuclear magnetic resonance (NMR), and detailed tandem mass spectrometry rapidly provided peptide sequence for streptnatamide A, a cyclic peptide isolated from a marine-derived Streptomyces sp. Marfey's analysis determined the stereochemistry of all amino acids, enabling the unambiguous structure determination of this compound. A non-ribosomal peptide synthetase biosynthetic gene cluster (stp) was tentatively identified and annotated for streptnatamide A based on the in silico analysis of whole genome sequencing data. These analytical tools will be powerful tools to overcome the challenges for cyclic peptide structure elucidation and accelerate the development of bioactive cyclic peptides.

Metabolomics and Genomics Enable the Discovery of a New Class of Nonribosomal Peptidic Metallophores from a Marine Micromonospora.

Although microbial genomes harbor an abundance of biosynthetic gene clusters, there remain substantial technological gaps that impair the direct correlation of newly discovered gene clusters and their corresponding secondary metabolite products. As an example of one approach designed to minimize or bridge such gaps, we employed hierarchical clustering analysis and principal component analysis (hcapca, whose sole input is MS data) to prioritize 109 marine Micromonospora strains and ultimately identify novel strain WMMB482 as a candidate for in-depth "metabologenomics" analysis following its prioritization. Highlighting the power of current MS-based technologies, not only did hcapca enable the discovery of one new, nonribosomal peptide bearing an incredible diversity of unique functional groups, but metabolomics for WMMB482 unveiled 16 additional congeners via the application of Global Natural Product Social molecular networking (GNPS), herein named ecteinamines A-Q (1-17). The ecteinamines possess an unprecedented skeleton housing a host of uncommon functionalities including a menaquinone pathway-derived 2-naphthoate moiety, 4-methyloxazoline, the first example of a naturally occurring Ψ[CH2NH] "reduced amide", a methylsulfinyl moiety, and a d-cysteinyl residue that appears to derive from a unique noncanonical epimerase domain. Extensive in silico analysis of the ecteinamine (ect) biosynthetic gene cluster and stable isotope-feeding experiments helped illuminate the novel enzymology driving ecteinamine assembly as well the role of cluster collaborations or "duets" in producing such structurally complex agents. Finally, ecteinamines were found to bind nickel, cobalt, zinc, and copper, suggesting a possible biological role as broad-spectrum metallophores.

Genome Mining and Metabolomics Unveil Pseudonochelin: A Siderophore Containing 5-Aminosalicylate from a Marine-Derived Pseudonocardia sp. Bacterium.

Pseudonochelin (1), a siderophore from a marine-derived Pseudonocardia sp. bacterium, was discovered using genome mining and metabolomics technologies. A 5-aminosalicylic acid (5-ASA) unit, not previously found in siderophore natural products, was identified in 1. Annotation of a putative psn biosynthetic gene cluster combined with bioinformatics and isotopic enrichment studies enabled us to propose the biosynthesis of 1. Moreover, 1 was found to display in vitro and in vivo antibacterial activity in an iron-dependent fashion.

Bacillimidazoles A-F, Imidazolium-Containing Compounds Isolated from a Marine Bacillus.

Chemical investigations of a marine sponge-associated Bacillus revealed six new imidazolium-containing compounds, bacillimidazoles A-F (1-6). Previous reports of related imidazolium-containing natural products are rare. Initially unveiled by timsTOF (trapped ion mobility spectrometry) MS data, extensive HRMS and 1D and 2D NMR analyses enabled the structural elucidation of 1-6. In addition, a plausible biosynthetic pathway to bacillimidazoles is proposed based on isotopic labeling experiments and invokes the highly reactive glycolytic adduct 2,3-butanedione. Combined, the results of structure elucidation efforts, isotopic labeling studies and bioinformatics suggest that 1-6 result from a fascinating intersection of primary and secondary metabolic pathways in Bacillus sp. WMMC1349. Antimicrobial assays revealed that, of 1-6, only compound six displayed discernible antibacterial activity, despite the close structural similarities shared by all six natural products.

Turbinmicin inhibits Candida biofilm growth by disrupting fungal vesicle-mediated trafficking.

The emergence of drug-resistant fungi has prompted an urgent threat alert from the US Centers for Disease Control (CDC). Biofilm assembly by these pathogens further impairs effective therapy. We recently identified an antifungal, turbinmicin, that inhibits the fungal vesicle-mediated trafficking pathway and demonstrates broad-spectrum activity against planktonically growing fungi. During biofilm growth, vesicles with unique features play a critical role in the delivery of biofilm extracellular matrix components. As these components are largely responsible for the drug resistance associated with biofilm growth, we explored the utility of turbinmicin in the biofilm setting. We found that turbinmicin disrupted extracellular vesicle (EV) delivery during biofilm growth and that this impaired the subsequent assembly of the biofilm matrix. We demonstrated that elimination of the extracellular matrix rendered the drug-resistant biofilm communities susceptible to fungal killing by turbinmicin. Furthermore, the addition of turbinmicin to otherwise ineffective antifungal therapy potentiated the activity of these drugs. The underlying role of vesicles explains this dramatic activity and was supported by phenotype reversal with the addition of exogenous biofilm EVs. This striking capacity to cripple biofilm assembly mechanisms reveals a new approach to eradicating biofilms and sheds light on turbinmicin as a promising anti-biofilm drug.

Bacillibactins E and F from a Marine Sponge-Associated Bacillus sp.

Chemical investigation of a marine sponge-associated Bacillus sp. led to the discovery of bacillibactins E and F (1 and 2). Despite containing the well-established cyclic triester core of iron-binding natural products such as enterobactin, bacillibactins E and F (1 and 2) are the first bacterial siderophores that contain nicotinic and benzoic acid moieties. The structures of the new compounds, including their absolute configurations, were determined by extensive spectroscopic analyses and Marfey's method. A plausible biosynthetic pathway to 1 and 2 is proposed; this route bears great similarity to other previously established bacillibactin-like pathways but appears to differentiate itself by a promiscuous DhbE, which likely installs the nicotinic moiety of 1 and the benzoic acid group of 2.

A marine microbiome antifungal targets urgent-threat drug-resistant fungi.

New antifungal drugs are urgently needed to address the emergence and transcontinental spread of fungal infectious diseases, such as pandrug-resistant Candida auris. Leveraging the microbiomes of marine animals and cutting-edge metabolomics and genomic tools, we identified encouraging lead antifungal molecules with in vivo efficacy. The most promising lead, turbinmicin, displays potent in vitro and mouse-model efficacy toward multiple-drug-resistant fungal pathogens, exhibits a wide safety index, and functions through a fungal-specific mode of action, targeting Sec14 of the vesicular trafficking pathway. The efficacy, safety, and mode of action distinct from other antifungal drugs make turbinmicin a highly promising antifungal drug lead to help address devastating global fungal pathogens such as C. auris.

Draft Genome Sequence of Pseudenhygromyxa sp. Strain WMMC2535, a Marine Ascidian-Associated Bacterium.

Pseudenhygromyxa WMMC2535, a representative of the myxobacteria (family Nannocystaceae), was isolated from a ragged sea hare in the Florida Keys, and its genome was sequenced using PacBio technology. The WMMC2535 genome sequence is the first of this genus and validates the notion that myxobacteria represent outstanding sources of structurally diverse natural products.

hcapca: Automated Hierarchical Clustering and Principal Component Analysis of Large Metabolomic Datasets in R.

Microbial natural product discovery programs face two main challenges today: rapidly prioritizing strains for discovering new molecules and avoiding the rediscovery of already known molecules. Typically, these problems have been tackled using biological assays to identify promising strains and techniques that model variance in a dataset such as PCA to highlight novel chemistry. While these tools have shown successful outcomes in the past, datasets are becoming much larger and require a new approach. Since PCA models are dependent on the members of the group being modeled, large datasets with many members make it difficult to accurately model the variance in the data. Our tool, hcapca, first groups strains based on the similarity of their chemical composition, and then applies PCA to the smaller sub-groups yielding more robust PCA models. This allows for scalable chemical comparisons among hundreds of strains with thousands of molecular features. As a proof of concept, we applied our open-source tool to a dataset with 1046 LCMS profiles of marine invertebrate associated bacteria and discovered three new analogs of an established anticancer agent from one promising strain.

Pyridine-2,6-Dithiocarboxylic Acid and Its Metal Complexes: New Inhibitors of New Delhi Metallo ß-Lactamase-1.

Keywords: NDM-1 inhibitors; marine-derived Streptomyces sp.; carbapenem-resistant Enterobacteriaceae; metal chelators.

MS-Derived Isotopic Fine Structure Reveals Forazoline A as a Thioketone-Containing Marine-Derived Natural Product.

Forazoline A is a structurally complex PKS-NRPS hybrid produced by marine-derived Actinomadura sp. During the course of studies highlighting the application of IFS analysis as a powerful tool for natural products analysis, we were alerted to an earlier misinterpretation with respect to forazoline A structure elucidation. In particular, IFS reveals that forazoline A contains a thioketone moiety rarely seen in secondary metabolites and, thus, constitutes an even more intriguing structure than originally thought.

Phallusialides A-E, Pyrrole-Derived Alkaloids Discovered from a Marine-Derived Micromonospora sp. Bacterium Using MS-Based Metabolomics Approaches.

Integrating MS-based metabolomics approaches, LC-MS-PCA and molecular networking enabled the targeted isolation of five new pyrrole-derived alkaloids, phallusialides A-E (1-5), from a marine-derived Micromonospora sp. bacterium. The structures of 1-5 were elucidated by analysis of their HRMS, MS/MS, and NMR spectroscopic data. The absolute configuration of phallusialide A (1) was determined on the basis of comparisons of experimental and theoretically calculated ECD spectra. Compounds 1 and 2 exhibited antibacterial activity against methicillin resistant S. aureus (MRSA) and E. coli, with MIC values of 32 and 64 µg/mL, respectively, whereas 3-5 showed no antibacterial activity even at 256 µg/mL, yielding important SAR insights for this class of compounds.

Enhypyrazinones A and B, Pyrazinone Natural Products from a Marine-Derived Myxobacterium Enhygromyxa sp.

To date, studies describing myxobacterial secondary metabolites have been relatively scarce in comparison to those addressing actinobacterial secondary metabolites. This realization suggests the immense potential of myxobacteria as an intriguing source of secondary metabolites with unusual structural features and a wide array of biological activities. Marine-derived myxobacteria are especially attractive due to their unique biosynthetic gene clusters, although they are more difficult to handle than terrestrial myxobacteria. Here, we report the discovery of two new pyrazinone-type molecules, enhypyrazinones A and B, from a marine-derived myxobacterium Enhygromyxa sp. Their structures were elucidated by HRESIMS and comprehensive NMR data analyses. Compounds 1 and 2, which contain a rare trisubstituted-pyrazinone core, represent a unique class of molecules from Enhygromyxa sp.

Bulbiferates A and B: Antibacterial Acetamidohydroxybenzoates from a Marine Proteobacterium, Microbulbifer sp.

Here we report the discovery of two new 3-acetamido-4-hydroxybenzoate esters, bulbiferates A (1) and B (2), isolated from Microbulbifer sp. cultivated from the marine tunicate Ecteinascidia turbinata. The structures of 1 and 2 were determined by analysis of 2D NMR and MS data. Additionally, three synthetic analogues (3-5), differing in ester sizes/lengths, were prepared for the purposes of evaluating potential structure-activity relationships; no clear correlations tying ester lengths to activity were evident. Bulbiferates A (1) and B (2) demonstrated antibacterial activity against both Escherichia coli (E. coli) and methicillin-sensitive Staphylococcus aureus (MSSA), whereas the synthetic analogues 3 and 4 displayed activity only against MSSA.

Omics Technologies to Understand Activation of a Biosynthetic Gene Cluster in Micromonospora sp. WMMB235: Deciphering Keyicin Biosynthesis.

DNA sequencing of a large collection of bacterial genomes reveals a wealth of orphan biosynthetic gene clusters (BGCs) with no identifiable products. BGC silencing, for those orphan clusters that are truly silent, rather than those whose products have simply evaded detection and cluster correlation, is postulated to result from transcriptional inactivation of these clusters under standard laboratory conditions. Here, we employ a multi-omics approach to demonstrate how interspecies interactions modulate the keyicin producing kyc cluster at the transcriptome level in cocultures of kyc-bearing Micromonospora sp. and a Rhodococcus sp. We further correlate coculture dependent changes in keyicin production to changes in transcriptomic and proteomic profiles and show that these changes are attributable to small molecule signaling consistent with a quorum sensing pathway. In piecing together the various elements underlying keyicin production in coculture, this study highlights how omics technologies can expedite future efforts to understand and exploit silent BGCs.

Biemamides A-E, Inhibitors of the TGF-ß Pathway That Block the Epithelial to Mesenchymal Transition.

Screening of a marine natural products library for inhibitors of TGF-ß revealed five pyrimidinedione derivatives, biemamides A-E (1-5). The structures were determined by 2D NMR and HRMS experiments; absolute configurations were established by advanced Marfey's analysis and ECD calculations. Biemamides A-E specifically inhibited in vitro TGF-ß induced epithelial to mesenchymal transition in NMuMG cells. Additionally, using Caenorhabditis elegans, selected biemmamides were found to influence in vivo developmental processes related to body size regulation in a dose-dependent manner.

Draft Genome Sequence of Micromonospora sp. Strain WMMA1996, a Marine Sponge-Associated Bacterium.

Micromonospora sp. strain WMMA1996 was isolated in 2013 off the coast of the Florida Keys, United States, from a marine sponge as part of bacterial coculture-based drug discovery initiatives. Analysis of the ∼6.44-Mb genome reveals this microbe's potential role in the discovery of new drugs.

Complete Genome Sequence of Dietzia sp. Strain WMMA184, a Marine Coral-Associated Bacterium.

Dietzia sp. strain WMMA184 was isolated from the marine coral Montastraea faveolata as part of ongoing drug discovery efforts. Analysis of the 4.16-Mb genome provides information regarding interspecies interactions as it pertains to the regulation of secondary metabolism and natural product biosynthesis potential.

Coculture of Marine Invertebrate-Associated Bacteria and Interdisciplinary Technologies Enable Biosynthesis and Discovery of a New Antibiotic, Keyicin.

Advances in genomics and metabolomics have made clear in recent years that microbial biosynthetic capacities on Earth far exceed previous expectations. This is attributable, in part, to the realization that most microbial natural product (NP) producers harbor biosynthetic machineries not readily amenable to classical laboratory fermentation conditions. Such "cryptic" or dormant biosynthetic gene clusters (BGCs) encode for a vast assortment of potentially new antibiotics and, as such, have become extremely attractive targets for activation under controlled laboratory conditions. We report here that coculturing of a Rhodococcus sp. and a Micromonospora sp. affords keyicin, a new and otherwise unattainable bis-nitroglycosylated anthracycline whose mechanism of action (MOA) appears to deviate from those of other anthracyclines. The structure of keyicin was elucidated using high resolution MS and NMR technologies, as well as detailed molecular modeling studies. Sequencing of the keyicin BGC (within the Micromonospora genome) enabled both structural and genomic comparisons to other anthracycline-producing systems informing efforts to characterize keyicin. The new NP was found to be selectively active against Gram-positive bacteria including both Rhodococcus sp. and Mycobacterium sp. E. coli-based chemical genomics studies revealed that keyicin's MOA, in contrast to many other anthracyclines, does not invoke nucleic acid damage.