Leptochelins A-C, Cytotoxic Metallophores Produced by Geographically Dispersed Leptothoe Strains of Marine Cyanobacteria.

Metals are important cofactors in the metabolic processes of cyanobacteria, including photosynthesis, cellular respiration, DNA replication, and the biosynthesis of primary and secondary metabolites. In adaptation to the marine environment, cyanobacteria use metallophores to acquire trace metals when necessary as well as to reduce potential toxicity from excessive metal concentrations. Leptochelins A-C were identified as structurally novel metallophores from three geographically dispersed cyanobacteria of the genus Leptothoe. Determination of the complex structures of these metabolites presented numerous challenges, but they were ultimately solved using integrated data from NMR, mass spectrometry and deductions from the biosynthetic gene cluster. The leptochelins are comprised of halogenated linear NRPS-PKS hybrid products with multiple heterocycles that have potential for hexadentate and tetradentate coordination with metal ions. The genomes of the three leptochelin producers were sequenced, and retrobiosynthetic analysis revealed one candidate biosynthetic gene cluster (BGC) consistent with the structure of leptochelin. The putative BGC is highly homologous in all three Leptothoe strains, and all possess genetic signatures associated with metallophores. Postcolumn infusion of metals using an LC-MS metabolomics workflow performed with leptochelins A and B revealed promiscuous binding of iron, copper, cobalt, and zinc, with greatest preference for copper. Iron depletion and copper toxicity experiments support the hypothesis that leptochelin metallophores may play key ecological roles in iron acquisition and in copper detoxification. In addition, the leptochelins possess significant cytotoxicity against several cancer cell lines.

Hippo pathway regulation by phosphatidylinositol transfer protein and phosphoinositides.

The Hippo pathway plays a key role in development, organ size control and tissue homeostasis, and its dysregulation contributes to cancer. The LATS tumor suppressor kinases phosphorylate and inhibit the YAP/TAZ transcriptional co-activators to suppress gene expression and cell growth. Through a screen of marine natural products, we identified microcolin B (MCB) as a Hippo activator that preferentially kills YAP-dependent cancer cells. Structure-activity optimization yielded more potent MCB analogs, which led to the identification of phosphatidylinositol transfer proteins α and ß (PITPα/ß) as the direct molecular targets. We established a critical role of PITPα/ß in regulating LATS and YAP. Moreover, we showed that PITPα/ß influence the Hippo pathway via plasma membrane phosphatidylinositol-4-phosphate. This study uncovers a previously unrecognized role of PITPα/ß in Hippo pathway regulation and as potential cancer therapeutic targets.

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

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

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

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

Enzymatic Halogenation of Terminal Alkynes.

The biosynthetic installation of halogen atoms is largely performed by oxidative halogenases that target a wide array of electron-rich substrates, including aromatic compounds and conjugated systems. Halogenated alkyne-containing molecules are known to occur in Nature; however, halogen atom installation on the terminus of an alkyne has not been demonstrated in enzyme catalysis. Herein, we report the discovery and characterization of an alkynyl halogenase in natural product biosynthesis. We show that the flavin-dependent halogenase from the jamaicamide biosynthetic pathway, JamD, is not only capable of terminal alkyne halogenation on a late-stage intermediate en route to the final natural product but also has broad substrate tolerance for simple to complex alkynes. Furthermore, JamD is specific for terminal alkynes over other electron-rich aromatic substrates and belongs to a newly identified family of halogenases from marine cyanobacteria, indicating its potential as a chemoselective biocatalyst for the formation of haloalkynes.

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

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

Kalkitoxin: A Potent Suppressor of Distant Breast Cancer Metastasis.

Bone metastasis resulting from advanced breast cancer causes osteolysis and increases mortality in patients. Kalkitoxin (KT), a lipopeptide toxin derived from the marine cyanobacterium Moorena producens (previously Lyngbya majuscula), has an anti-metastatic effect on cancer cells. We verified that KT suppressed cancer cell migration and invasion in vitro and in animal models in the present study. We confirmed that KT suppressed osteoclast-soup-derived MDA-MB-231 cell invasion in vitro and induced osteolysis in a mouse model, possibly enhancing/inhibiting metastasis markers. Furthermore, KT inhibits CXCL5 and CXCR2 expression, suppressing the secondary growth of breast cancer cells on the bone, brain, and lungs. The breast-cancer-induced osteolysis in the mouse model further reveals that KT plays a protective role, judging by micro-computed tomography and immunohistochemistry. We report for the first time the novel suppressive effects of KT on cancer cell migration and invasion in vitro and on MDA-MB-231-induced bone loss in vivo. These results suggest that KT may be a potential therapeutic drug for the treatment of breast cancer metastasis.

Molecular Features of CA-074 pH-Dependent Inhibition of Cathepsin B.

CA-074 is a selective inhibitor of cathepsin B, a lysosomal cysteine protease. CA-074 has been utilized in numerous studies to demonstrate the role of this protease in cellular and physiological functions. Cathepsin B in numerous human disease mechanisms involves its translocation from acidic lysosomes of pH 4.6 to neutral pH 7.2 of cellular locations, including the cytosol and extracellular environment. To gain in-depth knowledge of CA-074 inhibition under these different pH conditions, this study evaluated the molecular features, potency, and selectivity of CA-074 for cathepsin B inhibition under acidic and neutral pH conditions. This study demonstrated that CA-074 is most effective at inhibiting cathepsin B at an acidic pH of 4.6 with nM potency, which was more than 100-fold more potent than its inhibition at a neutral pH of 7.2. The pH-dependent inhibition of CA-074 was abolished by methylation of its C-terminal proline, indicating the requirement for the free C-terminal carboxyl group for pH-dependent inhibition. Under these acidic and neutral pH conditions, CA-074 maintained its specificity for cathepsin B over other cysteine cathepsins, displayed irreversible inhibition, and inhibited diverse cleavages of peptide substrates of cathepsin B assessed by profiling mass spectrometry. Molecular docking suggested that pH-dependent ionic interactions of the C-terminal carboxylate of CA-074 occur with His110 and His111 residues in the S2' subsite of the enzyme at pH 4.6, but these interactions differ at pH 7.2. While high levels of CA-074 or CA-074Me (converted by cellular esterases to CA-074) are used in biological studies to inhibit cathepsin B at both acidic and neutral pH locations, it is possible that adjusted levels of CA-074 or CA-074Me may be explored to differentially affect cathepsin B activity at these different pH values. Overall, the results of this study demonstrate the molecular, kinetic, and protease specificity features of CA-074 pH-dependent inhibition of cathepsin B.

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.

Antillatoxin-Stimulated Neurite Outgrowth Involves the Brain-Derived Neurotrophic Factor (BDNF) - Tropomyosin Related Kinase B (TrkB) Signaling Pathway.

Voltage-gated sodium channel (VGSC) activators promote neurite outgrowth by augmenting intracellular Na+ concentration ([Na+]i) and upregulating N-methyl-d-aspartate receptor (NMDAR) function. NMDAR activation stimulates calcium (Ca2+) influx and increases brain-derived neurotrophic factor (BDNF) release and activation of tropomyosin receptor kinase B (TrkB) signaling. The BDNF-TrkB pathway has been implicated in activity-dependent neuronal development. We have previously shown that antillatoxin (ATX), a novel lipopeptide isolated from the cyanobacterium Moorea producens, is a VGSC activator that produces an elevation of [Na+]i. Here we address the effect of ATX on the synthesis and release of BDNF and determine the signaling mechanisms by which ATX enhances neurite outgrowth in immature cerebrocortical neurons. ATX treatment produced a concentration-dependent release of BDNF. Acute treatment with ATX also resulted in increased synthesis of BDNF. ATX stimulation of neurite outgrowth was prevented by pretreatment with a TrkB inhibitor or transfection with a dominant-negative Trk-B. The ATX activation of TrkB and Akt was blocked by both a NMDAR antagonist (MK-801) and a VGSC blocker (tetrodotoxin). These results suggest that VGSC activators such as the structurally novel ATX may represent a new pharmacological strategy to promote neuronal plasticity through a NMDAR-BDNF-TrkB-dependent mechanism.

Rapid Biotic and Abiotic Transformation of Toxins produced by Ostreopsis. cf. ovata.

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.

SMART-Miner: A convolutional neural network-based metabolite identification from 1 H-13 C HSQC spectra.

The identification of metabolites from complex biofluids and extracts of tissues is an essential process for understanding metabolic profiles. Nuclear magnetic resonance (NMR) spectroscopy is widely used in metabolomics studies for identification and quantification of metabolites. However, the accurate identification of individual metabolites is still a challenging process with higher peak intensity or similar chemical shifts from different metabolites. In this study, we applied a convolutional neural network (CNN) to 1 H-13 C HSQC NMR spectra to achieve accurate peak identification in complex mixtures. The results reveal that the neural network was successfully trained on metabolite identification from these 2D NMR spectra and achieved very good performance compared with other NMR-based metabolomic tools.

Natural Products with Potential to Treat RNA Virus Pathogens Including SARS-CoV-2.

Three families of RNA viruses, the Coronaviridae, Flaviviridae, and Filoviridae, collectively have great potential to cause epidemic disease in human populations. The current SARS-CoV-2 (Coronaviridae) responsible for the COVID-19 pandemic underscores the lack of effective medications currently available to treat these classes of viral pathogens. Similarly, the Flaviviridae, which includes such viruses as Dengue, West Nile, and Zika, and the Filoviridae, with the Ebola-type viruses, as examples, all lack effective therapeutics. In this review, we present fundamental information concerning the biology of these three virus families, including their genomic makeup, mode of infection of human cells, and key proteins that may offer targeted therapies. Further, we present the natural products and their derivatives that have documented activities to these viral and host proteins, offering hope for future mechanism-based antiviral therapeutics. By arranging these potential protein targets and their natural product inhibitors by target type across these three families of virus, new insights are developed, and crossover treatment strategies are suggested. Hence, natural products, as is the case for other therapeutic areas, continue to be a promising source of structurally diverse new anti-RNA virus therapeutics.

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

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

Portobelamides A and B and Caciqueamide, Cytotoxic Peptidic Natural Products from a Caldora sp. Marine Cyanobacterium.

Three new compounds, portobelamides A and B (1 and 2), 3-amino-2-methyl-7-octynoic acid (AMOYA) and hydroxyisovaleric acid (Hiva) containing cyclic depsipeptides, and one long chain lipopeptide caciqueamide (3), were isolated from a field-collection of a Caldora sp. marine cyanobacterium obtained from Panama as part of the Panama International Cooperative Biodiversity Group Program. Their planar structures were elucidated through analysis of 2D NMR and MS data, especially high resolution (HR) MS2/MS3 fragmentation methods. The absolute configurations of compounds 1 and 2 were deduced by traditional hydrolysis, derivative formation, and chromatographic analyses compared with standards. Portobelamide A (1) showed good cytotoxicity against H-460 human lung cancer cells (33% survival at 0.9 µM).

First Total Synthesis and Structure-Activity Relationship of Iheyamide A, an Antitrypanosomal Linear Peptide Isolated from a Dapis sp. Marine Cyanobacterium.

Iheyamide A (1) is an antitrypanosomal linear peptide isolated from a Dapis sp. marine cyanobacterium by our group in 2020, and based on structure-activity relationships of its natural analogues, the C-terminal pyrrolinone moiety has been identified as the phamacophore for its antiparasitic activity. Further, we isolated this pyrrolinone moiety by itself as a new natural product from the marine cyanobacterium and named it iheyanone (2). As expected, iheyanone (2) showed antitrypanosomal activity, but its potency was weaker than iheyamide A (1). To clarify more detailed structure-activity relationships, we completed a total synthesis of iheyamide A (1) along with iheyanone (2) and evaluated the antitrypanosomal activities of several synthetic intermediates. As a result, we found that the longer the peptide chain, the stronger the antitrypanosomal activity. As iheyamide A (1) showed selective toxicity against Trypanosoma brucei rhodesiense, these findings can provide design guidelines for antitrypanosomal drugs.

NPClassifier: A Deep Neural Network-Based Structural Classification Tool for Natural Products.

Computational approaches such as genome and metabolome mining are becoming essential to natural products (NPs) research. Consequently, a need exists for an automated structure-type classification system to handle the massive amounts of data appearing for NP structures. An ideal semantic ontology for the classification of NPs should go beyond the simple presence/absence of chemical substructures, but also include the taxonomy of the producing organism, the nature of the biosynthetic pathway, and/or their biological properties. Thus, a holistic and automatic NP classification framework could have considerable value to comprehensively navigate the relatedness of NPs, and especially so when analyzing large numbers of NPs. Here, we introduce NPClassifier, a deep-learning tool for the automated structural classification of NPs from their counted Morgan fingerprints. NPClassifier is expected to accelerate and enhance NP discovery by linking NP structures to their underlying properties.

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

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

Kalkitoxin Reduces Osteoclast Formation and Resorption and Protects against Inflammatory Bone Loss.

Osteoclasts, bone-specified multinucleated cells produced by monocyte/macrophage, are involved in numerous bone destructive diseases such as arthritis, osteoporosis, and inflammation-induced bone loss. The osteoclast differentiation mechanism suggests a possible strategy to treat bone diseases. In this regard, we recently examined the in vivo impact of kalkitoxin (KT), a marine product obtained from the marine cyanobacterium Moorena producens (previously Lyngbya majuscula), on the macrophage colony-stimulating factor (M-CSF) and on the receptor activator of nuclear factor κB ligand (RANKL)-stimulated in vitro osteoclastogenesis and inflammation-mediated bone loss. We have now examined the molecular mechanism of KT in greater detail. KT decreased RANKL-induced bone marrow-derived macrophages (BMMs) tartrate-resistant acid phosphatase (TRAP)-multinucleated cells at a late stage. Likewise, KT suppressed RANKL-induced pit area and actin ring formation in BMM cells. Additionally, KT inhibited several RANKL-induced genes such as cathepsin K, matrix metalloproteinase (MMP-9), TRAP, and dendritic cell-specific transmembrane protein (DC-STAMP). In line with these results, RANKL stimulated both genes and protein expression of c-Fos and nuclear factor of activated T cells (NFATc1), and this was also suppressed by KT. Moreover, KT markedly decreased RANKL-induced p-ERK1/2 and p-JNK pathways at different time points. As a result, KT prevented inflammatory bone loss in mice, such as bone mineral density (BMD) and osteoclast differentiation markers. These experiments demonstrated that KT markedly inhibited osteoclast formation and inflammatory bone loss through NFATc1 and mitogen-activated protein kinase (MAPK) signaling pathways. Therefore, KT may have potential as a treatment for destructive bone diseases.

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

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