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.

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.

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.

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.

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.

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.

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.

Cytotoxic Microcolin Lipopeptides from the Marine Cyanobacterium Moorea producens.

Nine new linear lipopeptides, microcolins E-M (1-9), together with four known related compounds, microcolins A-D (10-13), were isolated from the marine cyanobacterium Moorea producens using bioassay-guided and LC-MS/MS molecular networking approaches. Catalytic hydrogenation of microcolins A-D (10-13) yielded two known compounds, 3,4-dihydromicrocolins A and B (14, 15), and two new derivatives, 3,4-dihydromicrocolins C and D (16, 17), respectively. The structures of these new compounds were determined by a combination of spectroscopic and advanced Marfey's analysis. Structurally unusual amino acid units, 4-methyl-2-(methylamino)pent-3-enoic (Mpe) acid and 2-amino-4-methylhexanoic acid (N-Me-homoisoleucine), in compounds 1-3 and 8, respectively, are rare residues in naturally occurring peptides. These metabolites showed significant cytotoxic activity against H-460 human lung cancer cells with IC50 values ranging from 6 nM to 5.0 µM. The variations in structure and attendant biological activities provided fresh insights concerning structure-activity relationships for the microcolin class of lipopeptides.

Exploration of the carmaphycins as payloads in antibody drug conjugate anticancer agents.

Antibody-drug conjugates (ADCs) represent a new dimension of anticancer chemotherapeutics, with warheads to date generally involving either antitubulin or DNA-directed agents to achieve low-to sub-nanomolar potency. However, other potent cytotoxins working by different pharmacological mechanisms are under investigation, such as α,ß-epoxyketone based proteasome inhibitors. These proteasome active agents are an emerging class of anticancer drug that possesses ultra-potent cytotoxicity to some cancer cell lines. The carmaphycins are representatives of this latter class that we isolated and characterized from a marine cyanobacterium, and these as well as several synthetic analogues exhibit this level of potency. In the current work, we investigated the use of these highly potent cytotoxic compounds as warheads in the design of novel ADCs. We designed and synthesized a library of carmaphycin B analogues that contain amine handles, enabling their attachment to an antibody linker. The basicity of these incorporated amine handles was shown to strongly affect their cytotoxic properties. Linear amines resulted in the greatest reduction in cytotoxicity whereas less basic aromatic amines retained potent activity as demonstrated by a 4-sulfonylaniline derivative. These investigations resulted in identifying the P2 residue in the carmaphycins as the most suitable site for linker attachment point, and hence, we synthesized a highly potent analogue of carmaphycin B that contained a 4-sulfonylaniline handle as an attachment point for the linker antibody.

Samholides, Swinholide-Related Metabolites from a Marine Cyanobacterium cf. Phormidium sp.

Cancer cell cytotoxicity was used to guide the isolation of nine new swinholide-related compounds, named samholides A-I (1-9), from an American Samoan marine cyanobacterium cf. Phormidium sp. Their structures were determined by extensive analysis of 1D and 2D NMR spectroscopic data. The new compounds share an unusual 20-demethyl 44-membered lactone ring composed of two monomers, and they demonstrate structural diversity arising from geometric isomerization of double bonds, sugar units with unique glyceryl moieties and varied methylation patterns. All of the new samholides were potently active against the H-460 human lung cancer cell line with IC50 values ranging from 170 to 910 nM. The isolation of these new swinholide-related compounds from a marine cyanobacterium reinvigorates questions concerning the evolution and biosynthetic origin of these natural products.

Discovery and Synthesis of Caracolamide A, an Ion Channel Modulating Dichlorovinylidene Containing Phenethylamide from a Panamanian Marine Cyanobacterium cf. Symploca Species.

A recent untargeted metabolomics investigation into the chemical profile of 10 organic extracts from cf. Symploca spp. revealed several interesting chemical leads for further natural product drug discovery. Subsequent target-directed isolation efforts with one of these, a Panamanian marine cyanobacterium cf. Symploca sp., yielded a phenethylamide metabolite that terminates in a relatively rare gem-dichlorovinylidene moiety, caracolamide A (1), along with a known isotactic polymethoxy-1-alkene (2). Detailed NMR and HRESIMS analyses were used to determine the structures of these molecules, and compound 1 was confirmed by a three-step synthesis. Pure compound 1 was shown to have in vitro calcium influx and calcium channel oscillation modulatory activity when tested as low as 10 pM using cultured murine cortical neurons, but was not cytotoxic to NCI-H460 human non-small-cell lung cancer cells in vitro (IC50 > 10 µM).

Dudawalamides A-D, Antiparasitic Cyclic Depsipeptides from the Marine Cyanobacterium Moorea producens.

A family of 2,2-dimethyl-3-hydroxy-7-octynoic acid (Dhoya)-containing cyclic depsipeptides, named dudawalamides A-D (1-4), was isolated from a Papua New Guinean field collection of the cyanobacterium Moorea producens using bioassay-guided and spectroscopic approaches. The planar structures of dudawalamides A-D were determined by a combination of 1D and 2D NMR experiments and MS analysis, whereas the absolute configurations were determined by X-ray crystallography, modified Marfey's analysis, chiral-phase GCMS, and chiral-phase HPLC. Dudawalamides A-D possess a broad spectrum of antiparasitic activity with minimal mammalian cell cytotoxicity. Comparative analysis of the Dhoya-containing class of lipopeptides reveals intriguing structure-activity relationship features of these NRPS-PKS-derived metabolites and their derivatives.

Basis for selectivity of cationic antimicrobial peptides for bacterial versus mammalian membranes.

Novel cationic antimicrobial peptides typified by structures such as KKKKKKAAXAAWAAXAA-NH2, where X = Phe/Trp, and several of their analogues display high activity against a variety of bacteria but exhibit no hemolytic activity even at high dose levels in mammalian erythrocytes. To elucidate their mechanism of action and source of selectivity for bacterial membranes, phospholipid mixtures mimicking the compositions of natural bacterial membranes (containing anionic lipids) and mammalian membranes (containing zwitterionic lipids + cholesterol) were challenged with the peptides. We found that peptides readily inserted into bacterial lipid mixtures, although no insertion was detected in model "mammalian" membranes. The depth of peptide insertion into model bacterial membranes was estimated by Trp fluorescence quenching using doxyl groups variably positioned along the phospholipid acyl chains. Peptide antimicrobial activity generally increased with increasing depth of peptide insertion. The overall results, in conjunction with molecular modeling, support an initial electrostatic interaction step in which bacterial membranes attract and bind peptide dimers onto the bacterial surface, followed by the "sinking" of the hydrophobic core segment to a peptide sequence-dependent depth of approximately 2.5-8 A into the membrane, largely parallel to the membrane surface. Antimicrobial activity was likely enhanced by the fact that the peptide sequences contain AXXXA sequence motifs, which promote their dimerization, and possibly higher oligomerization, as assessed by SDS-polyacrylamide gel analysis and fluorescence resonance energy transfer experiments. The high selectivity of these peptides for nonmammalian membranes, combined with their activity toward a wide spectrum of Gram-negative and Gram-positive bacteria and yeast, while retaining water solubility, represent significant advantages of this class of peptides.