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.

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

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

Lake Avernus Has Turned Red: Bioindicator Monitoring Unveils the Secrets of "Gates of Hades".

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.

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

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

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.

Discovery of pH-Selective Marine and Plant Natural Product Inhibitors of Cathepsin B Revealed by Screening at Acidic and Neutral pH Conditions.

Dysregulation of cathepsin B, which involves the translocation of the enzyme from acidic pH lysosomes to the neutral pH cytosol, followed by the initiation of cell death and inflammation, occurs in numerous brain disorders. The wide difference in the acidic pH (4.6) of lysosomes compared to the neutral pH (7.2) of the cytosol suggests that screening at different pH conditions may identify pH-selective modulators of cathepsin B. Therefore, a collection of pure marine and plant natural product (NP) compounds, with synthetic compounds, was screened at pH 4.6 and pH 7.2 in cathepsin B assays, which led to the identification of GER-12 (Crossbyanol B) and GER-24 ((7Z,9Z,12Z)-octadeca-7,9,12-trien-5-ynoic acid) marine NP inhibitors at acidic pH but not at neutral pH. GER-12 was effective for the reversible inhibition of cathepsin B, with an IC50 of 3 µM. GER-24 had an IC50 of 16 µM and was found to be an irreversible inhibitor. These results show that NP screening at distinct biological pH conditions can lead to the identification of pH-selective cathepsin B modulators. These findings suggest that screening efforts for molecular probes and drug discovery may consider the biological pH environment of the target in the disease process.

Discovery of Novel Tyrosinase Inhibitors From Marine Cyanobacteria.

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.

Secondary Metabolite Variation and Bioactivities of Two Marine Aspergillus Strains in Static Co-Culture Investigated by Molecular Network Analysis and Multiple Database Mining Based on LC-PDA-MS/MS.

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.

On the Hunt for New Toxin Families Produced by a Mediterranean Strain of the Benthic Dinoflagellate Ostreopsis cf. ovata.

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.

Structure and Candidate Biosynthetic Gene Cluster of a Manumycin-Type Metabolite from Salinispora pacifica.

A new manumycin-type natural product named pacificamide (1) and its candidate biosynthetic gene cluster (pac) were discovered from the marine actinobacterium Salinispora pacifica CNT-855. The structure of the compound was determined using NMR, electronic circular dichroism, and bioinformatic predictions. The pac gene cluster is unique to S. pacifica and found in only two of the 119 Salinispora genomes analyzed across nine species. Comparative analyses of biosynthetic gene clusters encoding the production of related manumycin-type compounds revealed genetic differences in accordance with the unique pacificamide structure. Further queries of manumycin-type gene clusters from public databases revealed their limited distribution across the phylum Actinobacteria and orphan diversity that suggests additional products remain to be discovered in this compound class. Production of the known metabolite triacsin D is also reported for the first time from the genus Salinispora. This study adds two classes of compounds to the natural product collective isolated from the genus Salinispora, which has proven to be a useful model for natural product research.

NPOmix: A machine learning classifier to connect mass spectrometry fragmentation data to biosynthetic gene clusters.

Microbial specialized metabolites are an important source of and inspiration for many pharmaceuticals, biotechnological products and play key roles in ecological processes. Untargeted metabolomics using liquid chromatography coupled with tandem mass spectrometry is an efficient technique to access metabolites from fractions and even environmental crude extracts. Nevertheless, metabolomics is limited in predicting structures or bioactivities for cryptic metabolites. Efficiently linking the biosynthetic potential inferred from (meta)genomics to the specialized metabolome would accelerate drug discovery programs by allowing metabolomics to make use of genetic predictions. Here, we present a k-nearest neighbor classifier to systematically connect mass spectrometry fragmentation spectra to their corresponding biosynthetic gene clusters (independent of their chemical class). Our new pattern-based genome mining pipeline links biosynthetic genes to metabolites that they encode for, as detected via mass spectrometry from bacterial cultures or environmental microbiomes. Using paired datasets that include validated genes-mass spectral links from the Paired Omics Data Platform, we demonstrate this approach by automatically linking 18 previously known mass spectra (17 for which the biosynthesis gene clusters can be found at the MIBiG database plus palmyramide A) to their corresponding previously experimentally validated biosynthetic genes (e.g., via nuclear magnetic resonance or genetic engineering). We illustrated a computational example of how to use our Natural Products Mixed Omics (NPOmix) tool for siderophore mining that can be reproduced by the users. We conclude that NPOmix minimizes the need for culturing (it worked well on microbiomes) and facilitates specialized metabolite prioritization based on integrative omics mining.

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.

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.

Applying a Chemogeographic Strategy for Natural Product Discovery from the Marine Cyanobacterium Moorena bouillonii.

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.

An anti-inflammatory isoflavone from soybean inoculated with a marine fungus Aspergillus terreus C23-3.

A new isoflavone derivative compound 1 (psoralenone) was isolated from soybean inoculated with a marine fungus Aspergillus terreus C23-3, together with seven known compounds including isoflavones 2-6, butyrolactone I (7) and blumenol A (8). Their structures were elucidated by MS, NMR, and ECD. Psoralenone displayed moderate in vitro anti-inflammatory activity in the LPS-induced RAW264.7 cell model. Compound 2 (genistein) showed moderate acetylcholinesterase (AChE) inhibitory activity whereas compounds 2, 5 (biochanin A), 6 (psoralenol), and 7 exhibited potent larvicidal activity against brine shrimp. Compounds 3 (daidzein), 4 (4'-hydroxy-6,7-dimethoxyisoflavone), and 5-7 showed broad-spectrum anti-microbial activity, and compound 7 also showed moderate 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity.

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.

Tutuilamides A-C: Vinyl-Chloride-Containing Cyclodepsipeptides from Marine Cyanobacteria with Potent Elastase Inhibitory Properties.

Marine cyanobacteria (blue-green algae) have been shown to possess an enormous capacity to produce structurally diverse natural products that exhibit a broad spectrum of potent biological activities, including cytotoxic, antifungal, antiparasitic, antiviral, and antibacterial activities. Using mass-spectrometry-guided fractionation together with molecular networking, cyanobacterial field collections from American Samoa and Palmyra Atoll yielded three new cyclic peptides, tutuilamides A-C. Their structures were established by spectroscopic techniques including 1D and 2D NMR, HR-MS, and chemical derivatization. Structure elucidation was facilitated by employing advanced NMR techniques including nonuniform sampling in combination with the 1,1-ADEQUATE experiment. These cyclic peptides are characterized by the presence of several unusual residues including 3-amino-6-hydroxy-2-piperidone and 2-amino-2-butenoic acid, together with a novel vinyl chloride-containing residue. Tutuilamides A-C show potent elastase inhibitory activity together with moderate potency in H-460 lung cancer cell cytotoxicity assays. The binding mode to elastase was analyzed by X-ray crystallography revealing a reversible binding mode similar to the natural product lyngbyastatin 7. The presence of an additional hydrogen bond with the amino acid backbone of the flexible side chain of tutuilamide A, compared to lyngbyastatin 7, facilitates its stabilization in the elastase binding pocket and possibly explains its enhanced inhibitory potency.

Pagoamide A, a Cyclic Depsipeptide Isolated from a Cultured Marine Chlorophyte, Derbesia sp., Using MS/MS-Based Molecular Networking.

A thiazole-containing cyclic depsipeptide with 11 amino acid residues, named pagoamide A (1), was isolated from laboratory cultures of a marine Chlorophyte, Derbesia sp. This green algal sample was collected from America Samoa, and pagoamide A was isolated using guidance by MS/MS-based molecular networking. Cultures were grown in a light- and temperature-controlled environment and harvested after several months of growth. The planar structure of pagoamide A (1) was characterized by detailed 1D and 2D NMR experiments along with MS and UV analysis. The absolute configurations of its amino acid residues were determined by advanced Marfey's analysis following chemical hydrolysis and hydrazinolysis reactions. Two of the residues in pagoamide A (1), phenylalanine and serine, each occurred twice in the molecule, once in the d- and once in the l-configuration. The biosynthetic origin of pagoamide A (1) was considered in light of other natural products investigations with coenocytic green algae.

MetaMiner: A Scalable Peptidogenomics Approach for Discovery of Ribosomal Peptide Natural Products with Blind Modifications from Microbial Communities.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are an important class of natural products that contain antibiotics and a variety of other bioactive compounds. The existing methods for discovery of RiPPs by combining genome mining and computational mass spectrometry are limited to discovering specific classes of RiPPs from small datasets, and these methods fail to handle unknown post-translational modifications. Here, we present MetaMiner, a software tool for addressing these challenges that is compatible with large-scale screening platforms for natural product discovery. After searching millions of spectra in the Global Natural Products Social (GNPS) molecular networking infrastructure against just eight genomic and metagenomic datasets, MetaMiner discovered 31 known and seven unknown RiPPs from diverse microbial communities, including human microbiome and lichen microbiome, and microorganisms isolated from the International Space Station.