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Abstract The proteasome is a proteolytic enzyme complex essential for protein homeostasis in mammalian cells and protozoan parasites like Trichomonas vaginalis (Tv), the cause of the most common, non-viral sexually transmitted disease. Tv and other protozoan 20S proteasomes have been validated as druggable targets for antimicrobials. However, low yields and purity of the native proteasome have hindered studies of the Tv 20S proteasome (Tv20S). We address this challenge by creating a recombinant protozoan proteasome by expressing all seven α and seven β subunits of Tv20S alongside the Ump-1 chaperone in insect cells. The recombinant Tv20S displays biochemical equivalence to its native counterpart, confirmed by various assays. Notably, the marizomib (MZB) inhibits all catalytic subunits of Tv20S, while the peptide inhibitor carmaphycin-17 (CP-17) specifically targets β2 and β5. Cryo-electron microscopy (cryo-EM) unveils the structures of Tv20S bound to MZB and CP-17 at 2.8 Å. These findings explain MZB’s low specificity for Tv20S compared to the human proteasome and demonstrate CP-17’s higher specificity. Overall, these data provide a structure-based strategy for the development of specific Tv20S inhibitors to treat trichomoniasis.

Abstract Protein kinases are key signaling nodes that regulate fundamental biological and disease processes. Illuminating kinase signaling from multiple angles can provide deeper insights into disease mechanisms and improve therapeutic targeting. While fluorescent biosensors are powerful tools for visualizing live-cell kinase activity dynamics in real time, new molecular tools are needed that enable recording of transient signaling activities for post hoc analysis and targeted manipulation. Here, we develop a light-gated kinase activity coupled transcriptional integrator (KINACT) that converts dynamic kinase signals into “permanent” fluorescent marks. KINACT enables robust monitoring of kinase activity across scales, accurately recording subcellular PKA activity, highlighting PKA activity distribution in 3D cultures, and identifying PKA activators and inhibitors in high-throughput screens. We further leverage the ability of KINACT to drive signaling effector expression to allow feedback manipulation of the balance of Gαs R201C-induced PKA and ERK activation and dissect the mechanisms of oncogenic G protein signaling.

Abstract Despite the increasing availability of tandem mass spectrometry (MS/MS) community spectral libraries for untargeted metabolomics over the past decade, the majority of acquired MS/MS spectra remain uninterpreted. To further aid in interpreting unannotated spectra, we created a nearest neighbor suspect spectral library, consisting of 87,916 annotated MS/MS spectra derived from hundreds of millions of MS/MS spectra originating from published untargeted metabolomics experiments. Entries in this library, or “suspects,” were derived from unannotated spectra that could be linked in a molecular network to an annotated spectrum. Annotations were propagated to unknowns based on structural relationships to reference molecules using MS/MS-based spectrum alignment. We demonstrate the broad relevance of the nearest neighbor suspect spectral library through representative examples of propagation-based annotation of acylcarnitines, bacterial and plant natural products, and drug metabolism. Our results also highlight how the library can help to better understand an Alzheimer’s brain phenotype. The nearest neighbor suspect spectral library is openly available for download or for data analysis through the GNPS platform to help investigators hypothesize candidate structures for unknown MS/MS spectra in untargeted metabolomics data.

Abstract The identification of molecular structure is essential for understanding chemical diversity and for developing drug leads from small molecules. Nevertheless, the structure elucidation of small molecules by Nuclear Magnetic Resonance (NMR) experiments is often a long and non-trivial process that relies on years of training. To achieve this process efficiently, several spectral databases have been established to retrieve reference NMR spectra. However, the number of reference NMR spectra available is limited and has mostly facilitated annotation of commercially available derivatives. Here, we introduce DeepSAT, a neural network-based structure annotation and scaffold prediction system that directly extracts the chemical features associated with molecular structures from their NMR spectra. Using only the 1H-13C HSQC spectrum, DeepSAT identifies related known compounds and thus efficiently assists in the identification of molecular structures. DeepSAT is expected to accelerate chemical and biomedical research by accelerating the identification of molecular structures.

A healthy ocean is essential for human health, and yet the links between the ocean and human health are often overlooked. By providing new medicines, technologies, energy, foods, recreation, and inspiration, the ocean has the potential to enhance human health and wellbeing. However, climate change, pollution, biodiversity loss, and inequity threaten both ocean and human health. Sustainable realisation of the ocean’s health benefits will require overcoming these challenges through equitable partnerships, enforcement of laws and treaties, robust monitoring, and use of metrics that assess both the ocean’s natural capital and human wellbeing. Achieving this will require an explicit focus on human rights, equity, sustainability, and social justice. In addition to highlighting the potential unique role of the healthcare sector, we offer science-based recommendations to protect both ocean health and human health, and we highlight the unique potential of the healthcare sector tolead this effort.

Therapeutics discovery and development for Alzheimer’s disease (AD) has been an area of intense research to alleviate memory loss and the underlying pathogenic processes. Recent drug discovery approaches have utilized in silico computational strategies for drug candidate selection which has opened the door to repurposing drugs for AD. Computational analysis of gene expression signatures of patients stratified by the APOE4 risk allele of AD led to the discovery of the FDA-approved drug bumetanide as a top candidate agent that reverses APOE4 transcriptomic brain signatures and improves memory deficits in APOE4 animal models of AD. Bumetanide is a loop diuretic which inhibits the kidney Na+-K+-2Cl− cotransporter isoform, NKCC2, for the treatment of hypertension and edema in cardiovascular, liver, and renal disease. Electronic health record data revealed that patients exposed to bumetanide have lower incidences of AD by 35%–70%. In the brain, bumetanide has been proposed to antagonize the NKCC1 isoform which mediates cellular uptake of chloride ions. Blocking neuronal NKCC1 leads to a decrease in intracellular chloride and thus promotes GABAergic receptor mediated hyperpolarization, which may ameliorate disease conditions associated with GABAergic-mediated depolarization. NKCC1 is expressed in neurons and in all brain cells including glia (oligodendrocytes, microglia, and astrocytes) and the vasculature. In consideration of bumetanide as a repurposed drug for AD, this review evaluates its pharmaceutical properties with respect to its estimated brain levels across doses that can improve neurologic disease deficits of animal models to distinguish between NKCC1 and non-NKCC1 mechanisms. The available data indicate that bumetanide efficacy may occur at brain drug levels that are below those required for inhibition of the NKCC1 transporter which implicates non-NKCC1 brain mechansims for improvement of brain dysfunctions and memory deficits. Alternatively, peripheral bumetanide mechanisms may involve cells outside the central nervous system (e.g., in epithelia and the immune system). Clinical bumetanide doses for improved neurological deficits are reviewed. Regardless of mechanism, the efficacy of bumetanide to improve memory deficits in the APOE4 model of AD and its potential to reduce the incidence of AD provide support for clinical investigation of bumetanide as a repurposed AD therapeutic agent.

Bioactivity-guided isolation of specialized metabolites is an iterative process. Here, the authors demonstrate a native metabolomics approach that allows for fast screening of complex metabolite extracts against a protein of interest and simultaneous structure annotation.

Lake Avernus is a volcanic lake located in southern Italy. Since ancient times, it has inspired numerous myths and legends due to the occurrence of singular phenomena, such as coloring events. Only recently has an explanation been found for them, i.e., the recurring color change over time is due to the alternation of cyanobacterial blooms that are a consequence of natural nutrient inputs as well as pollution resulting from human activities. This current report specifically describes the red coloring event that occurred on Lake Avernus in March 2022, the springtime season in this region of Italy. Our innovative multidisciplinary approach, the ‘Fast Detection Strategy’ (FDS), was devised to monitor cyanobacterial blooms and their toxins. It integrates remote sensing data from satellites and drones, on-site sampling, and analytical/bioinformatics analyses into a cohesive information flow. Thanks to FDS, we determined that the red color was attributable to a bloom of Planktothrix rubescens, a toxin-producing cyanobacterium. Here, we report the detection and identification of 14 anabenopeptins from this P. rubescens strain, seven of which are known and seven are newly reported herein. Moreover, we explored the mechanisms and causes behind this cyclic phenomenon, confirming cyanobacteria’s role as reliable indicators of environmental changes. This investigation further validates FDS’s effectiveness in detecting and characterizing cyanobacterial blooms and their associated toxins, expanding its potential applications.

Ottilie et al. employ an experimental evolution approach to investigate the role of transcription factors in yeast chemical resistance. Most emergent mutations in resistant strains were enriched in transcription factor coding genes, highlighting their importance in drug resistance.

Tyrosinase, an important oxidase involved in the primary immune response in humans, can sometimes become problematic as it can catalyze undesirable oxidation reactions. Therefore, for decades there has been a strong pharmaceutical interest in the discovery of novel inhibitors of this enzyme. Recent studies have also indicated that tyrosinase inhibitors can potentially be used in the treatment of melanoma cancer. Over the years, many new tyrosinase inhibitors have been discovered from various natural sources; however, marine natural products (MNPs) have contributed only a small number of promising candidates. Therefore, in this study we focused on the discovery of new MNP tyrosinase inhibitors of marine cyanobacterial and algal origins. A colorimetric tyrosinase inhibitory assay was used to screen over 4,500 marine extracts against mushroom tyrosinase (A. bisporus). Our results revealed that scytonemin monomer (ScyM), a pure compound from our compound library and also the monomeric last-step precursor in the biosynthesis of the well-known cyanobacterial sunscreen pigment “scytonemin,” consistently showed the highest tyrosinase inhibitory score. Determination of the half maximal inhibitory concentration (IC50) further indicated that ScyM is more potent than the commonly used commercial inhibitor standard “kojic acid” (KA; IC50 of ScyM: 4.90 μM vs. IC50 of KA: 11.31 μM). After a scaled-up chemical synthesis of ScyM as well as its O-methyl analog (ScyM-OMe), we conducted a series of follow-up studies on their structures, inhibitory properties, and mode of inhibition. Our results supported ScyM as the second case ever of a novel tyrosinase inhibitory compound based on a marine cyanobacterial natural product. The excellent in vitro performance of ScyM makes it a promising candidate for applications such as a skin-whitening agent or an adjuvant therapy for melanoma cancer treatment.

The dinoflagellate Ostreopsis cf. ovata produces several families of toxic polyketides. Despite only a few field measurements of these phycotoxins in seawater and aerosols, they are believed to be responsible for dermatitis and the toxic inhalations reported during blooms of this species. Therefore, the stability of these compounds in seawater is essential to understanding the causes of these symptoms, however, this has never been assessed. In the current study, the optimization of a solid phase extraction (SPE) procedure was first performed to ensure the most efficient extraction of all phycotoxins known to be produced by this strain, including the recently described liguriatoxins. The SPE cartridge SDBL® under non acidified conditions offered the best option. The stability of the ovatoxins and the liguriatoxins under biotic and abiotic stress was assessed by exposing the spent medium of a culture of Ostreopsis cf. ovata to its bacterial consortium and natural sunlight. A rapid biotic transformation was detected for both families of compounds. When exposed to bacteria, the half-lives of the ovatoxins were reached before 10 h and at 36 h, 97% of these toxins had been transformed. The half-lives of the liguriatoxins were 10 h under these conditions. Photolysis (abiotic degradation) of the ovatoxins (T1/2 < 36 h) was faster than for the liguriatoxins (T1/2 > 62 h). Although none of the catabolites of these phycotoxins were thoroughly identified, an untargeted metabolomics approach combined with molecular networking highlighted the presence of several compounds exhibiting structural similarities with the ovatoxins. Additional work should confirm the preliminary findings on these potential ovatoxins’ catabolites and their biological properties. The rapid transformation of O. cf. ovata’s phycotoxins introduces questions concerning their presence in seawater and their dispersion in the sea spray aerosols. The compounds involved in the toxic inhalations and dermatitis often experienced by beachgoers may stem from the catabolites of these toxins or even unrelated and as yet unidentified compounds.

The fungus Inonotus obliquus has historically been used in traditional medicine in Europe and Asia. A melanin fraction and six triterpenoids were obtained from I. obliquus sclerotia, and evaluated in various bioassays including for immunomodulatory, cytotoxicity and enzyme-interacting properties. The water-soluble, nitrogen-free melanin fraction and the triterpenoids 3β-hydroxy-8,24-dien-21-al (1) and inotodiol (2) displayed potent activity in a human complement assay. The melanin fraction inhibited the complement cascade, whereas 1 and 2 activated the same cascade. Compound 2, as well as betulinic acid (3) and betulin (4) had anti-proliferative properties against the colon adenocarcinoma cell line HT29-MTX. Further, the melanin fraction and betulin-3-O-caffeate (6) reduced nitric oxide production in primary murine macrophages. Furthermore, the metabolites were nontoxic against the common gut bacteria E. coli and B. subtilis. The results demonstrate the anti-inflammatory and immunomodulatory effects of I. obliquus melanin and triterpenoids, which could potentially justify the consumption of this increasingly popular “edible” fungus.

Abstract Background Short chain oxylipins in plants as the main volatile organic carbon have been speculated to playing an important role for plant innate immunity, however, not yet intensively studied and far away established as the fully recognized algae defense signals. Results The production of 1-octen-3-ol is self-amplified via the fatty acid-oxylipin metabolic cycle through positive feedback loop. Production of 1-octen-3-ol may act as a messenger that induces P. haitanensis to be in a “primed” state and ready for defense by upregulating the synthesis of methyl jasmonic acid, indole-3-acetic acid, and gibberellin A3. Production of these oxylipins also adjust the redox state in cells, resulting in host defense activation. Conclusions We provide the first demonstration that 1-octen-3-ol from P. haitanensis, can act as a self-stimulating community messenger. The multiple effects of 1-octen-3-ol may explain why P. haitanensis, a very ancient lineage within plant kingdom, thrives in the niche of intertidal zones.

ABSTRACT Next-generation sequencing studies of saliva and dental plaque from subjects in both healthy and diseased states have identified bacteria belonging to the Rothia genus as ubiquitous members of the oral microbiota. To gain a deeper understanding of molecular mechanisms underlying the chemical ecology of this unexplored group, we applied a genome mining approach that targets functionally important biosynthetic gene clusters (BGCs). All 45 genomes that were mined, representing Rothia mucilaginosa, Rothia dentocariosa, and Rothia aeria, harbored a catechol-siderophore-like BGC. To explore siderophore production further, we grew the previously characterized R. mucilaginosa ATCC 25296 in liquid cultures, amended with glycerol, which led to the identification of the archetype siderophore enterobactin by using tandem liquid chromatography-mass spectrometry (LC-MS/MS), high-performance liquid chromatography (HPLC), and nuclear magnetic resonance (NMR) spectroscopy. Normally attributed to pathogenic gut bacteria, R. mucilaginosa is the first commensal oral bacterium found to produce enterobactin. Cocultivation studies including R. mucilaginosa or purified enterobactin revealed that enterobactin reduced growth of certain strains of cariogenic Streptococcus mutans and pathogenic strains of Staphylococcus aureus. Commensal oral bacteria were either unaffected, reduced in growth, or induced to grow adjacent to enterobactin-producing R. mucilaginosa or the pure compound. Taken together with Rothia’s known capacity to ferment a variety of carbohydrates and amino acids, our findings of enterobactin production add an additional level of explanation to R. mucilaginosa’s prevalence in the oral cavity. Enterobactin is the strongest Fe(III) binding siderophore known, and its role in oral health requires further investigation. IMPORTANCE The communication language of the human oral microbiota is vastly underexplored. However, a few studies have shown that specialized small molecules encoded by BGCs have critical roles such as in colonization resistance against pathogens and quorum sensing. Here, by using a genome mining approach in combination with compound screening of growth cultures, we identified that the commensal oral community member R. mucilaginosa harbors a catecholate-siderophore BGC, which is responsible for the biosynthesis of enterobactin. The iron-scavenging role of enterobactin is known to have positive effects on the host’s iron pool and negative effects on host immune function; however, its role in oral health remains unexplored. R. mucilaginosa was previously identified as an abundant community member in cystic fibrosis, where bacterial iron cycling plays a major role in virulence development. With respect to iron’s broad biological importance, iron-chelating enterobactin may explain R. mucilaginosa’s colonization success in both health and disease.

Co-culture is known as an efficient way to explore the metabolic potential of fungal strains for new antibiotics and other therapeutic agents that could counter emerging health issues. To study the effect of co-culture on the secondary metabolites and bioactivities of two marine strains, Aspergillus terreus C23-3 and Aspergillus. unguis DLEP2008001, they were co-cultured in live or inactivated forms successively or simultaneously. The mycelial morphology and high-performance thin layer chromatography (HPTLC) including bioautography of the fermentation extracts were recorded. Furthermore, the agar cup-plate method was used to compare the antimicrobial activity of the extracts. Based on the above, liquid chromatography-photodiode array-tandem mass spectrometry (LC-PDA-MS/MS) together with Global Natural Products Social molecular networking (GNPS) and multiple natural products database mining were used to further analyze their secondary metabolite variations. The comprehensive results showed the following trends: (1) The strain first inoculated will strongly inhibit the growth and metabolism of the latter inoculated one; (2) Autoclaved A. unguis exerted a strong inducing effect on later inoculated A. terreus, while the autoclaved A. terreus showed high stability of its metabolites and still potently suppressed the growth and metabolism of A. unguis; (3) When the two strains are inoculated simultaneously, they both grow and produce metabolites; however, the A. terreus seemed to be more strongly induced by live A. unguis and this inducing effect surpassed that of the autoclaved A. unguis. Under some of the conditions, the extracts showed higher antimicrobial activity than the axenic cultures. Totally, A. unguis was negative in response but potent in stimulating its rival while A. terreus had the opposite effect. Fifteen MS detectable and/or UV active peaks showed different yields in co-cultures vs. the corresponding axenic culture. GNPS analysis assisted by multiple natural products databases mining (PubChem, Dictionary of Natural Products, NPASS, etc.) gave reasonable annotations for some of these peaks, including antimicrobial compounds such as unguisin A, lovastatin, and nidulin. However, some of the peaks were correlated with antagonistic properties and remain as possible novel compounds without mass or UV matching hits from any database. It is intriguing that the two strains both synthesize chemical ‘weapons’ for antagonism, and that these are upregulated when needed in competitive co-culture environment. At the same time, compounds not useful in this antagonistic setting are downregulated in their expression. Some of the natural products produced during antagonism are unknown chlorinated metabolites and deserve further study for their antimicrobial properties. In summary, this study disclosed the different responses of two Aspergillus strains in co-culture, revealed their metabolic variation, and displayed new opportunities for antibiotic discovery.

Ostreopsis cf. ovata is a benthic dinoflagellate known to produce palytoxin (PLTX) and its analogues. Recent investigations suggested the production of unknown toxins by a Mediterranean strain. In the present work, two new families of toxins, potentially novel in their structures, were purified from this same Mediterranean strain of Ostreopsis cf. ovata. The low amount of material isolated only allowed for acquisition of high-resolution mass spectrometry data and the evaluation of their cytotoxicity to human lung cancer cells. Based on their HRMS data, none of these new compounds appear to be close PLTX analogues, although their mass spectra suggest poly-hydroxylated long chain compounds of high molecular weight (1370–2143 Da). The cell cytotoxicity concentrations (CC50) of these new purified toxins ranged between 0.68 and 3.12 µg/mL, and this was enhanced when they were tested as mixtures, suggesting synergistic effects of Ostreopsis toxins. The two families of compounds were named the liguriatoxins (LGTX) and rivieratoxins (RVTX), with each family containing three members. Additional work on purification is needed to fully characterize the structures of these six new dinoflagellate toxins.

Three undescribed 8,3′-neolignans, corynol (1), 3-methoxy-corynol (2) and 3′-deoxy-corynol (3), together with two bergenin derivatives, three flavonoids, two hydrolysable tannins and six simple phenolic compounds, were isolated from the twigs of Corylopsis coreana Uyeki. The structures of the 8,3′-neolignans were elucidated by analyzing their NMR, HRESIMS and ECD spectra. All the isolated compounds were evaluated for their SIRT1 stimulatory activity, and 3′-deoxy-corynol (3) showed SIRT1 stimulation activity. Furthermore, a docking study of 3 was performed with three representative binding pockets of SIRT1.

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.

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.

Leptolyngbya, a well-known genus of cyanobacteria, is found in various ecological habitats including marine, fresh water, swamps, and rice fields. Species of this genus are associated with many ecological phenomena such as nitrogen fixation, primary productivity through photosynthesis and algal blooms. As a result, there have been a number of investigations of the ecology, natural product chemistry, and biological characteristics of members of this genus. In general, the secondary metabolites of cyanobacteria are considered to be rich sources for drug discovery and development. In this review, the secondary metabolites reported in marine Leptolyngbya with their associated biological activities or interesting biosynthetic pathways are reviewed, and new insights and perspectives on their metabolic capacities are gained.

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.

Emerging literature suggests that after a stroke, the peri-infarct region exhibits dynamic changes in excitability. In rodent stroke models, treatments that enhance excitability in the peri-infarct cerebral cortex promote motor recovery. This increase in cortical excitability and plasticity is opposed by increases in tonic GABAergic inhibition in the peri-infarct zone beginning three days after a stroke in a mouse model. Maintenance of a favorable excitatory–inhibitory balance promoting cerebrocortical excitability could potentially improve recovery. Brevetoxin-2 (PbTx-2) is a voltage-gated sodium channel (VGSC) gating modifier that increases intracellular sodium ([Na+]i), upregulates N-methyl-D-aspartate receptor (NMDAR) channel activity and engages downstream calcium (Ca2+) signaling pathways. In immature cerebrocortical neurons, PbTx-2 promoted neuronal structural plasticity by increasing neurite outgrowth, dendritogenesis and synaptogenesis. We hypothesized that PbTx-2 may promote excitability and structural remodeling in the peri-infarct region, leading to improved functional outcomes following a stroke. We tested this hypothesis using epicortical application of PbTx-2 after a photothrombotic stroke in mice. We show that PbTx-2 enhanced the dendritic arborization and synapse density of cortical layer V pyramidal neurons in the peri-infarct cortex. PbTx-2 also produced a robust improvement of motor recovery. These results suggest a novel pharmacologic approach to mimic activity-dependent recovery from stroke.

Marine Cyanobacteria (blue-green algae) have been shown to possess an enormous potential to produce structurally diverse natural products that exhibit a broad spectrum of potent biological activities, including cytotoxic, antifungal, antiparasitic, antiviral, and antibacterial activities. Here, we report the isolation and structure determination of palstimolide A, a complex polyhydroxy macrolide with a 40-membered ring that was isolated from a tropical marine cyanobacterium collected at Palmyra Atoll. NMR-guided fractionation in combination with MS2-based molecular networking and isolation via HPLC yielded 0.7 mg of the pure compound. The small quantity isolated along with the presence of significant signal degeneracy in both the 1H and 13C-NMR spectra complicated the structure elucidation of palstimolide A. Various NMR experiments and solvent systems were employed, including the LR-HSQMBC experiment that allows the detection of long-range 1H–13C correlation data across 4-, 5-, and even 6-bonds. This expanded NMR data set enabled the elucidation of the palstimolide’s planar structure, which is characterized by several 1,5-disposed hydroxy groups as well as a tert-butyl group. The compound showed potent antimalarial activity with an IC50 of 223 nM as well as interesting anti-leishmanial activity with an IC50 of 4.67 µM.

Members of the cyanobacterial genus Trichodesmium are well known for their substantial impact on nitrogen influx in ocean ecosystems and the enormous surface blooms they form in tropical and subtropical locations. However, the secondary metabolite composition of these complex environmental bloom events is not well known, nor the possibility of the production of potent toxins that have been observed in other bloom-forming marine and freshwater cyanobacteria species. In the present work, we aimed to characterize the metabolome of a Trichodesmium bloom utilizing MS/MS-based molecular networking. Furthermore, we integrated cytotoxicity assays in order to identify and ultimately isolate potential cyanotoxins from the bloom. These efforts led to the isolation and identification of several members of the smenamide family, including three new smenamide analogs (1–3) as well as the previously reported smenothiazole A-hybrid polyketide-peptide compounds. Two of these new smenamides possessed cytotoxicity to neuro-2A cells (1 and 3) and their presence elicits further questions as to their potential ecological roles. HPLC profiling and molecular networking of chromatography fractions from the bloom revealed an elaborate secondary metabolome, generating hypotheses with respect to the environmental role of these metabolites and the consistency of this chemical composition across genera, space and time.

The biologically active lipopeptide kalkitoxin was previously isolated from the marine cyanobacterium Moorea producens (Lyngbya majuscula). Kalkitoxin exhibited N-methyl-d-aspartate (NMDA)-mediated neurotoxicity and acted as an inhibitory ligand for voltage-sensitive sodium channels in cultured rat cerebellar granule neurons. Subsequent studies revealed that kalkitoxin generated a delayed form of colon tumor cell cytotoxicity in 7-day clonogenic cell survival assays. Cell line- and exposure time-dependent cytostatic/cytotoxic effects were previously observed with mitochondria-targeted inhibitors of hypoxia-inducible factor-1 (HIF-1). The transcription factor HIF-1 functions as a key regulator of oxygen homeostasis. Therefore, we investigated the ability of kalkitoxin to inhibit hypoxic signaling in human tumor cell lines. Kalkitoxin potently and selectively inhibited hypoxia-induced activation of HIF-1 in T47D breast tumor cells (IC50 5.6 nM). Mechanistic studies revealed that kalkitoxin inhibits HIF-1 activation by suppressing mitochondrial oxygen consumption at electron transport chain (ETC) complex I (NADH-ubiquinone oxidoreductase). Further studies indicate that kalkitoxin targets tumor angiogenesis by blocking the induction of angiogenic factors (i.e., VEGF) in tumor cells.

The frequent occurrence of Moorea producens (formerly Lyngbya majuscula) blooms has been associated with adverse effects on human health. Hoiamide A is a structurally unique cyclic depsipeptide isolated from an assemblage of the marine cyanobacteria M. producens and Phormidium gracile. We examined the influence of hoiamide A on neurite outgrowth in neocortical neurons and found that it suppressed neurite outgrowth with an IC50 value of 4.89 nM. Further study demonstrated that hoiamide A stimulated lactic acid dehydrogenase (LDH) efflux, nuclear condensation and caspase-3 activity with EC50 values of 3.66, 2.55 and 4.33 nM, respectively. These data indicated that hoiamide A triggered a unique neuronal death profile that involves both necrotic and apoptotic mechanisms. The similar potencies and similar time-response relationships between LDH efflux and caspase-3 activation/nuclear condensation suggested that both necrosis and apoptosis may derive from interaction with a common molecular target. The broad-spectrum caspase inhibitor, Z-VAD-FMK completely inhibited hoiamide A-induced neurotoxicity. Additionally, hoiamide A stimulated JNK phosphorylation, and a JNK inhibitor attenuated hoiamide A-induced neurotoxicity. Collectively, these data demonstrate that hoiamide A-induced neuronal death requires both JNK and caspase signaling pathways. The potent neurotoxicity and unique neuronal cell death profile of hoiamide A represents a novel neurotoxic chemotype from marine cyanobacteria.

Four new briarane diterpenoids, junceellolides M–P (1–4), were isolated together with seven known analogs (5–11) from the South China Sea gorgonian, Junceella gemmacea. The structures of these compounds were elucidated by detailed spectroscopic analysis and comparison with the reported data. The absolute configuration of compounds 1–3 were determined based on an ECD experiment, while the absolute configuration of compound 4 was genetically determined. All the compounds were isolated for the first time from J. gemmacea. These compounds showed no growth inhibitory activity against A549, MG63 and SMMC-7721 cell lines in an in vitro bioassay.

The tropical marine cyanobacterium, Moorea bouillonii, has gained recent attention as a rich source of bioactive natural products. Continued chemical investigation of this cyanobacterium, collected from New Britain, Papua New Guinea, yielded a novel cytotoxic cyclic depsipeptide, bouillonamide (1), along with previously reported molecules, ulongamide A and apratoxin A. Planar structure of bouillonamide was established by extensive 1D and 2D NMR experiments, including multi-edited HSQC, TOCSY, HBMC, and ROESY experiments. In addition to the presence of α-amino acid residues, compound 1 contained two unique polyketide-derived moieties, namely a 2-methyl-6-methylamino-hex-5-enoic acid (Mmaha) residue and a unit of 3-methyl-5-hydroxy-heptanoic acid (Mhha). Absolute stereochemistry of the α-amino acid units in bouillonamide was determined mainly by Marfey’s analysis. Compound 1 exhibited mild toxicity with IC50’s of 6.0 µM against the neuron 2a mouse neuroblastoma cells.

Marine cyanobacteria, notably those from tropical regions, are a rich source of bioactive secondary metabolites. Tropical marine cyanobacteria often grow to high densities in the environment, allowing direct isolation of many secondary metabolites from field-collected material. However, in temperate environments culturing is usually required to produce enough biomass for investigations of their chemical constituents. In this work, we cultured a selection of novel and diverse cyanobacteria isolated from the Portuguese coast, and tested their organic extracts in a series of ecologically-relevant bioassays. The majority of the extracts showed activity in at least one of the bioassays, all of which were run in very small scale. Phylogenetically related isolates exhibited different activity profiles, highlighting the value of microdiversity for bioprospection studies. Furthermore, LC-MS analyses of selected active extracts suggested the presence of previously unidentified secondary metabolites. Overall, the screening strategy employed here, in which previously untapped cyanobacterial diversity was combined with multiple bioassays, proved to be a successful strategy and allowed the selection of several strains for further investigations based on their bioactivity profiles.

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.

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.

Viruses are underrepresented as targets in pharmacological screening efforts, given the difficulties of devising suitable cell-based and biochemical assays. In this study we found that a pre-fractionated organic extract of the Red Sea sponge Amphimedon chloros was able to inhibit the West Nile Virus NS3 protease (WNV NS3). Using liquid chromatography–mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR) spectroscopy, the identity of the bioactive compound was determined as a 3-alkylpyridinium with m/z = 190.16. Diffusion Ordered Spectroscopy (DOSY) NMR and NMR relaxation rate analysis suggest that the bioactive compound forms oligomers of up to 35 kDa. We observed that at 9.4 μg/mL there was up to 40–70% inhibitory activity on WNV NS3 protease in orthogonal biochemical assays for solid phase extracts (SPE) of A. chloros. However, the LC-MS purified fragment was effective at inhibiting the protease up to 95% at an approximate amount of 2 µg/mL with negligible cytotoxicity to HeLa cells based on a High-Content Screening (HCS) cytological profiling strategy. To date, 3-alkylpyridinium type natural products have not been reported to show antiviral activity since the first characterization of halitoxin, or 3-alkylpyridinium, in 1978. This study provides the first account of a 3-alkylpyridinium complex that exhibits a proposed antiviral activity by inhibiting the NS3 protease. We suggest that the here-described compound can be further modified to increase its stability and tested in a cell-based assay to explore its full potential as a potential novel antiviral capable of inhibiting WNV replication.

Abstract: 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 β-amyloid (Aβ) 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-Aβ fibrillization properties and neuroprotective effects, and might be a good free radical scavenger.

Three new diterpenes, uprolide N (1), uprolide O (2), uprolide P (3) and a known one, dolabellane (4), were isolated from the CH2Cl2-MeOH extract of the gorgonian octocoral Eunicea succinea, collected from Bocas del Toro, on the Caribbean coast of Panama. Their structures were determined using spectroscopic analyses, including 1D and 2D NMR and high-resolution mass spectrometry (HRMS) together with molecular modeling studies. Compounds 1–3 displayed anti-inflammatory properties by inhibiting production of Tumor Necrosis Factor (TNF) and Interleukin (IL)-6 induced by lipopolysaccharide (LPS) in murine macrophages.

The sponge Stylissa carteri is known to produce a number of secondary metabolites displaying anti-fouling, anti-inflammatory, and anti-cancer activity. However, the anti-viral potential of metabolites produced by S. carteri has not been extensively explored. In this study, an S. carteri extract was HPLC fractionated and a cell based assay was used to evaluate the effects of HPLC fractions on parameters of Human Immunodeficiency Virus (HIV-1) infection and cell viability. Candidate HIV-1 inhibitory fractions were then analyzed for the presence of potential HIV-1 inhibitory compounds by mass spectrometry, leading to the identification of three previously characterized compounds, i.e., debromohymenialdisine (DBH), hymenialdisine (HD), and oroidin. Commercially available purified versions of these molecules were re-tested to assess their antiviral potential in greater detail. Specifically, DBH and HD exhibit a 30%–40% inhibition of HIV-1 at 3.1 μM and 13 μM, respectively; however, both exhibited cytotoxicity. Conversely, oroidin displayed a 50% inhibition of viral replication at 50 μM with no associated toxicity. Additional experimentation using a biochemical assay revealed that oroidin inhibited the activity of the HIV-1 Reverse Transcriptase up to 90% at 25 μM. Taken together, the chemical search space was narrowed and previously isolated compounds with an unexplored anti-viral potential were found. Our results support exploration of marine natural products for anti-viral drug discovery.