Natural Products from Tongan Marine Organisms.

The islands of the South Pacific Ocean have been in the limelight for natural product biodiscovery, due to their unique and pristine tropical waters and environment. The Kingdom of Tonga is an archipelago in the central Indo-Pacific Ocean, consisting of 176 islands, 36 of which are inhabited, flourishing with a rich diversity of flora and fauna. Many unique natural products with interesting bioactivities have been reported from Indo-Pacific marine sponges and other invertebrate phyla; however, there have not been any reviews published to date specifically regarding natural products from Tongan marine organisms. This review covers both known and new/novel Marine Natural Products (MNPs) and their biological activities reported from organisms collected within Tongan territorial waters up to December 2020, and includes 109 MNPs in total, the majority from the phylum Porifera. The significant biological activity of these metabolites was dominated by cytotoxicity and, by reviewing these natural products, it is apparent that the bulk of the new and interesting biologically active compounds were from organisms collected from one particular island, emphasizing the geographic variability in the chemistry between these organisms collected at different locations.

Metagenomic Exploration of the Marine Sponge Mycale hentscheli Uncovers Multiple Polyketide-Producing Bacterial Symbionts.

Marine sponges have been a prolific source of unique bioactive compounds that are presumed to act as a deterrent to predation. Many of these compounds have potential therapeutic applications; however, the lack of efficient and sustainable synthetic routes frequently limits clinical development. Here, we describe a metagenomic investigation of Mycale hentscheli, a chemically gifted marine sponge that possesses multiple distinct chemotypes. We applied shotgun metagenomic sequencing, hybrid assembly of short- and long-read data, and metagenomic binning to obtain a comprehensive picture of the microbiome of five specimens, spanning three chemotypes. Our data revealed multiple producing species, each having relatively modest secondary metabolomes, that contribute collectively to the chemical arsenal of the holobiont. We assembled complete genomes for multiple new genera, including two species that produce the cytotoxic polyketides pateamine and mycalamide, as well as a third high-abundance symbiont harboring a proteusin-type biosynthetic pathway that appears to encode a new polytheonamide-like compound. We also identified an additional 188 biosynthetic gene clusters, including a pathway for biosynthesis of peloruside. These results suggest that multiple species cooperatively contribute to defensive symbiosis in M. hentscheli and reveal that the taxonomic diversity of secondary-metabolite-producing sponge symbionts is larger and richer than previously recognized.IMPORTANCEMycale hentscheli is a marine sponge that is rich in bioactive small molecules. Here, we use direct metagenomic sequencing to elucidate highly complete and contiguous genomes for the major symbiotic bacteria of this sponge. We identify complete biosynthetic pathways for the three potent cytotoxic polyketides which have previously been isolated from M. hentscheli Remarkably, and in contrast to previous studies of marine sponges, we attribute each of these metabolites to a different producing microbe. We also find that the microbiome of M. hentscheli is stably maintained among individuals, even over long periods of time. Collectively, our data suggest a cooperative mode of defensive symbiosis in which multiple symbiotic bacterial species cooperatively contribute to the defensive chemical arsenal of the holobiont.

Two new 4-methylidene containing steroids, craterol A and B, from the New Zealand two sponge association between Stelletta crater and Desmacella dendyi.

NMR-directed investigation of the two sponge association between Stelletta crater and Desmacella dendyi has resulted in the isolation of two new members of the rare 4-methylidene class of sterols. Craterol A (1) and B (2) represent the first examples of natural products reported from the species S. crater. The isolation of these compounds challenges the role of 4-methylidene sterols as chemotaxonomic markers for the sponge genus Theonella.

Halogenated Meroditerpenoids from a South Pacific Collection of the Red Alga Callophycus serratus.

A detailed examination of the red alga Callophycus serratus collected in Tonga led to the isolation of six new halogenated meroditerpenoids: callophycol C (1), callophycoic acid I (2), iodocallophycols E (3) and F (4), iodocallophycoic acid B (5), and callophycoic acid J (6). Of these, compounds 3-5 are new iodinated additions to the growing family of Callophycus meroditerpenoids. The relative configurations of compounds 1-6 were deduced by analyses of 1D NOE data and 1H-1H scalar coupling constants, and 3-6 are proposed to differ from the closely related compounds reported in the literature, iodocallophycoic acid A and iodocallophycols A-D. Iodocallophycol E (3) exhibited moderate cytotoxicity against the promyelocytic leukemia cell line HL-60 with an IC50 value of 6.0 µM.

Zampanolides B-E from the Marine Sponge Cacospongia mycofijiensis: Potent Cytotoxic Macrolides with Microtubule-Stabilizing Activity.

Four new compounds (2-5) structurally related to the microtubule-stabilizing agent (-)-zampanolide (1) have been isolated from the Tongan marine sponge Cacospongia mycofijiensis. Three of these new structures, zampanolides B-D (2-4), exhibit nanomolar cytotoxicity toward the HL-60 cell line, are antimitotic, and induce in vitro tubulin polymerization at levels comparable to 1. Zampanolide E (5), saturated at C-8/C-9, was significantly less potent and does not stabilize purified tubulin, even at 10-fold higher concentrations. The structural differences across these compounds reveal a plasticity of the zampanolide pharmacophore. While unsaturation is required at Δ8, the configuration of this alkene and those of Δ4 and Δ4' have little effect on tubulin polymerization. The first natural co-occurrence of 1 and (-)-dactylolide (6) from the same sponge extract is also noted.

Peloruside E (22-Norpeloruside A), a Pelorusane Macrolide from the New Zealand Marine Sponge Mycale hentscheli, Retains Microtubule-Stabilizing Properties.

A new peloruside congener, peloruside E (5), has been isolated in sub-milligram quantities from a specimen of the New Zealand marine sponge Mycale hentscheli. The structure of 5 differs from the parent compound peloruside A (1) by replacement of the C-10 gem-dimethyl moiety with a monomethyl substituent and represents the first structural deviation in the pelorusane scaffold. Peloruside E (5) is potently antiproliferative (HL-60, IC50 90 nM, cf. 1, 19 nM) and polymerizes purified tubulin, albeit at a rate lower than that of 1.

Marine Invertebrate Natural Products that Target Microtubules.

Marine natural products as secondary metabolites are a potential major source of new drugs for treating disease. In some cases, cytotoxic marine metabolites target the microtubules of the eukaryote cytoskeleton for reasons that will be discussed. This review covers the microtubule-targeting agents reported from sponges, corals, tunicates, and molluscs and the evidence that many of these secondary metabolites are produced by bacterial symbionts. The review finishes by discussing the directions for future development and production of clinically relevant amounts of these natural products and their analogues through aquaculture, chemical synthesis, and biosynthesis by bacterial symbionts.

Hamigerans R and S: Nitrogenous Diterpenoids from the New Zealand Marine Sponge Hamigera tarangaensis.

Seven new members of the hamigeran family of diterpenoids have been isolated from the New Zealand marine sponge Hamigera tarangaensis. Among the new additions are hamigeran R (1), considered to be the first benzonitrile-based marine natural product, and hamigeran S (2), the first dimeric structure in the series. The formation of 1 and 2 is thought to occur via the reaction of hamigeran G with a nitrogen source, where the nitrile carbon of 1 is derived from the terpenoid skeleton.

Induction of accelerated senescence by the microtubule-stabilizing agent peloruside A.

Chemotherapeutic agents can induce accelerated senescence in tumor cells, an irreversible state of cell cycle arrest. Paclitaxel, a microtubule-stabilizing agent used to treat solid tumors of the breast, ovary, and lung and discodermolide, another stabilizing agent from a marine sponge, induce senescence in cultured cancer cells. The aim of this study was to determine if the microtubule-stabilizing agent peloruside A, a polyketide natural product from a marine sponge, can induce accelerated senescence in a breast cancer cell line MCF7. Doxorubicin, a DNA-damaging agent, paclitaxel, and discodermolide were used as positive controls. Senescence-associated-ß-galactosidase activity was increased by peloruside A, similar to paclitaxel, discodermolde, and doxorubicin, with a potency heirarchy of doxorubicin > paclitaxel > discodermolide > peloruside, based on IC25 concentrations that inhibit proliferation. Clonogenic survival was significantly decreased by peloruside A, similar to doxorubicin and the two other microtubule-stabilizing agents. The tumor suppressor protein p53 increased after treatment, whereas pRb decreased in response to all four compounds. It was concluded that in addition to apoptosis, peloruside A causes accelerated senescence in a subpopulation of MCF7 cells that contributes to its potential anticancer activity in a breast cancer cell line.

Zampanolide, a Microtubule-Stabilizing Agent, Is Active in Resistant Cancer Cells and Inhibits Cell Migration.

Zampanolide, first discovered in a sponge extract in 1996 and later identified as a microtubule-stabilizing agent in 2009, is a covalent binding secondary metabolite with potent, low nanomolar activity in mammalian cells. Zampanolide was not susceptible to single amino acid mutations at the taxoid site of ß-tubulin in human ovarian cancer 1A9 cells, despite evidence that it selectively binds to the taxoid site. As expected, it did not synergize with other taxoid site microtubule-stabilizing agents (paclitaxel, ixabepilone, discodermolide), but surprisingly also did not synergize in 1A9 cells with laulimalide/peloruside binding site agents either. Efforts to generate a zampanolide-resistant cell line were unsuccessful. Using a standard wound scratch assay in cell culture, it was an effective inhibitor of migration of human umbilical vein endothelial cells (HUVEC) and fibroblast cells (D551). These properties of covalent binding, the ability to inhibit cell growth in paclitaxel and epothilone resistant cells, and the ability to inhibit cell migration suggest that it would be of interest to investigate zampanolide in preclinical animal models to determine if it is effective in vivo at preventing tumor growth and metastasis.

Peloruside A, a microtubule-stabilizing agent, induces aneuploidy in ovarian cancer cells.

To ensure proper chromosome segregation, mitosis is tightly regulated by the spindle assembly checkpoint (SAC). Low concentrations of microtubule-stabilizing agents can induce aneuploid populations of cells in the absence of G2/M block, suggesting pertubation of the spindle checkpoint. We investigated the effects of peloruside A, a microtubule-stabilizing agent, on expression levels of several key cell cycle proteins, MAD2, BUBR1, p55CDC and cyclin B1. Synchronized 1A9 ovarian carcinoma cells were allowed to progress through the cell cycle in the presence or absence of peloruside A. Co-immunoprecipitation and Western blotting were used to probe the cell cycle kinetics of MAD2 and BUBR1 dissociation from p55CDC. Using confocal microscopy, we investigated whether premature dissociation of MAD2 and BUBR1 at low (40 nM) but not high (100 nM) concentrations of peloruside A was caused by defects in the attachment of chromosomes to the mitotic spindle. An increased frequency of polar chromosomes was observed at low concentrations of peloruside A, suggesting that an increased frequency of pseudo-metaphase cells, which are not detected by the spindle assembly checkpoint, may be underlying the induction of aneuploidy.

Peloruside A: a lead non-taxoid-site microtubule-stabilizing agent with potential activity against cancer, neurodegeneration, and autoimmune disease.

Covering: 2000 up to 2016Peloruside A, a macrocyclic secondary metabolite from a New Zealand marine sponge, Mycale hentscheli, has shown potent antiproliferative activity in cultured cancer cells as well as inhibitory effects on tumor growth in mouse models. The compound also has promising effects against cell models of neurodegenerative and autoimmune diseases. In mechanistic studies, peloruside A shares with paclitaxel (Taxol®) the ability to stabilize microtubules by binding to ß-tubulin. Peloruside A, however, occupies a unique external site on ß-tubulin that does not overlap the classical taxoid site that is located on the inside of the microtubule. As such, peloruside A has been of central importance in defining a new microtubule-stabilizer binding site localized on the exterior surface of the microtubule that has led to increased interest in the design of an upscaled total synthesis of the natural product and its analogues. Here, we review advances in the biochemical and biological validation of peloruside A as an attractive therapeutic candidate for the treatment of cancer, neurodegeneration, and autoimmune disease.

Polyhalogenated Indoles from the Red Alga Rhodophyllis membranacea: The First Isolation of Bromo-Chloro-Iodo Secondary Metabolites.

An unusual tetrahalogenated indole with the exceptionally rare inclusion of the three halogens bromine, chlorine, and iodine was found using mass spectrometry within a fraction of a semipurified extract obtained from the red alga Rhodophyllis membranacea. We report herein the isolation and structure elucidation, using a combination of NMR spectroscopy and mass spectrometry, of 11 new tetrahalogenated indoles (1-11), including four bromochloroiodoindoles (5-7, 10). Several were evaluated for cytotoxic and antifungal activities against the HL-60 promyelocytic cell line and Saccharomyces cerevisiae, respectively.

Peloruside A Inhibits Growth of Human Lung and Breast Tumor Xenografts in an Athymic nu/nu Mouse Model.

Peloruside A is a microtubule-stabilizing agent isolated from a New Zealand marine sponge. Peloruside prevents growth of a panel of cancer cell lines at low nanomolar concentrations, including cell lines that are resistant to paclitaxel. Three xenograft studies in athymic nu/nu mice were performed to assess the efficacy of peloruside compared with standard anticancer agents such as paclitaxel, docetaxel, and doxorubicin. The first study examined the effect of 5 and 10 mg/kg peloruside (QD×5) on the growth of H460 non-small cell lung cancer xenografts. Peloruside caused tumor growth inhibition (%TGI) of 84% and 95%, respectively, whereas standard treatments with paclitaxel (8 mg/kg, QD×5) and docetaxel (6.3 mg/kg, Q2D×3) were much less effective (%TGI of 50% and 18%, respectively). In a second xenograft study using A549 lung cancer cells and varied schedules of dosing, activity of peloruside was again superior compared with the taxanes with inhibitions ranging from 51% to 74%, compared with 44% and 50% for the two taxanes. A third xenograft study in a P-glycoprotein-overexpressing NCI/ADR-RES breast tumor model showed that peloruside was better tolerated than either doxorubicin or paclitaxel. We conclude that peloruside is highly effective in preventing the growth of lung and P-glycoprotein-overexpressing breast tumors in vivo and that further therapeutic development is warranted. Mol Cancer Ther; 14(8); 1816-23. ©2015 AACR.

ßI-tubulin mutations in the laulimalide/peloruside binding site mediate drug sensitivity by altering drug-tubulin interactions and microtubule stability.

Peloruside A (PLA) and laulimalide (LAU) are potent microtubule-stabilizing natural products that are effective against a broad spectrum of cancer cells. The interactions of PLA and LAU with tubulin have attracted a great deal of attention, mainly because they bind to ß-tubulin at a site that is different from the classical taxoid site. Multiple ßI-tubulin amino acid residues have been predicted by computer modelling studies and more recently by protein crystallography to participate in the binding of PLA and LAU to tubulin. The relevance of these residues in determining cellular sensitivity to the compounds, however, remains largely uncertain. To determine the role of four binding site residues, Q291, D295, V333, and N337 on PLA and LAU activity, we introduced single mutations to these sites by site-directed mutagenesis and transfected each mutant tubulin separately into HEK and/or HeLa cells. We found that a Q291M ßI-tubulin mutation increased sensitivity of the cells to PLA, but not to LAU, paclitaxel (PTX), or vinblastine (VBL). In contrast, V333W and N337L mutations led to less stable microtubules, with the V333W causing resistance to PLA and PTX, but not LAU, and the N337L causing resistance to PLA, LAU, and PTX. Moreover, cells expressing either W333 or L337 were hypersensitive to the microtubule-destabilizing agent, VBL. The D295I mutation conferred resistance to both PLA and LAU without affecting microtubule stability or sensitivity to PTX or ixabepilone (IXB). This study identifies the first mammalian ßI-tubulin mutation that specifically increases sensitivity to PLA, and reports mutations at PLA and LAU binding site residues that can either reduce microtubule stability or impair drug-tubulin binding, conferring resistance to these microtubule-stabilizing agents. This information provides insights on ß-tubulin residues important for maintaining microtubule structural integrity and for sensitivity to microtubule-targeting agents, and suggests novel directions for rational structure-based design of new and more potent agents for cancer treatment that target the LAU/PLA site.

Inhibition of human vascular endothelial cell migration and capillary-like tube formation by the microtubule-stabilizing agent peloruside A.

Peloruside A is a microtubule-stabilizing agent that is currently under investigation as a potential anticancer agent. Peloruside A binds to a site on ß-tubulin that is distinct to that of the taxanes (paclitaxel and docetaxel) and the epothilones. An attractive clinical quality of microtubule-stabilizing agents is their ability to target multiple mechanisms of tumour growth. In addition to inducing tumour cell apoptosis by arresting cells in mitosis, microtubule-stabilizing agents also inhibit angiogenesis, a process needed by tumor cells for growth and metastasis. In this study, the effects of peloruside A on endothelial cell processes important for angiogenesis were assessed in comparison to docetaxel. Both peloruside A and docetaxel potently inhibited the proliferation of human umbilical vein endothelial cells, with IC50 values of 1.4 and 1.7 nM, respectively. Peloruside also potently blocked endothelial cell migration during wound closure and the three-dimensional organization of the endothelial cells into capillary-like tubes. In the wound scratch assay, peloruside A inhibited wound recovery with an IC50 of 6.3 nM after 18 h. Docetaxel was approximately 3-fold more potent than peloruside A. The number of capillary-like tubes that formed after 16 h culture in Matrigel™ was also inhibited in a dose-dependent manner with an IC50 of 4.5 nM. Docetaxel was about 2-fold more potent than peloruside A in preventing tube formation. This inhibition of endothelial cell function occurred at relatively non-cytotoxic concentrations over the 16-18 h incubations for both stabilizing agents, suggesting that anti-angiogenic effects are likely to occur before therapeutically relevant doses begin to inhibit tumor growth or adverse side effects develop.

The Nitrogenous Hamigerans: Unusual Amino Acid-Derivatized Aromatic Diterpenoid Metabolites from the New Zealand Marine Sponge Hamigera tarangaensis.

The NMR-directed isolation and structure elucidation of nine new nitrogenous hamigeran diterpenoids from the New Zealand marine sponge Hamigera tarangaensis are described. Featured in this set are the oxazole-containing hamigeran M (4) and eight compounds (5a-6a and 7a-8c) related to the constitutional structure of hamigeran D (1). Moderate cytotoxicity in the low-micromolar range against the HL-60 promyeloid leukemic cell line is reported for seven of the new compounds. The structural nature of these compounds suggests that their adducts are derived from an amino acid source and has allowed for revision of the configuration about C-18 of the archetypal compound, hamigeran D, from 1a to 1b. The existence of three constitutionally identical forms of hamigeran Q (8a-8c) requires the involvement of an allo-isoleucine stereoisomer and suggests the intriguing possibility of partial prokaryotic biogenesis of these unusual secondary metabolites.

The Oxeatamides: nitrogenous spongian diterpenes from the New Zealand marine sponge Darwinella oxeata.

As part of our ongoing search for novel and bioactive compounds from New Zealand marine organisms, we investigated the extracts of the sponge Darwinella oxeata. NMR-guided fractionation led to the isolation of nine new nitrogenous spongian diterpenes, oxeatamide A (1), iso-oxeatamide A (2), oxeatamides B-G (3-8), and oxeatamide A 23-methyl ester (9), as well as two known compounds, membranolides C and D (10, 11).

13C NMR analysis of 3,6-dihydro-2H-pyrans: assignment of remote stereochemistry using axial shielding effects.

The rational analysis of (13)C NMR axial shielding effects has enabled the assignment of remote relative stereochemistry in 3,6-oxygen-substituted 3,6-dihydro-2H-pyrans. Comparison of the (13)C NMR shifts of equivalent centers in cis- and trans-substituted 3,6-dihydro-2H-pyrans allows the relative configuration at the C3 and C6 positions to be defined in diastereoisomeric mixtures. Density functional calculations were used to validate this method and assess the conformational bias present in the ring system. Ultimately, the coupling of computational chemistry with this (13)C NMR-based method provided a reliable and convenient method for stereochemical assignment of a single diastereomer. This approach provides a facile and complementary alternative to the practices previously employed for determining the relative configuration in 3,6-dihydro-2H-pyrans.

Structural basis of microtubule stabilization by laulimalide and peloruside A.

Laulimalide and peloruside A are microtubule-stabilizing agents (MSAs), the mechanism of action on microtubules of which is poorly defined. Here, using X-ray crystallography it is shown that laulimalide and peloruside A bind to a unique non-taxane site on ß-tubulin and use their respective macrolide core structures to interact with a second tubulin dimer across protofilaments. At the same time, they allosterically stabilize the taxane-site M-loop that establishes lateral tubulin contacts in microtubules. Structures of ternary complexes of tubulin with laulimalide/peloruside A and epothilone A are also solved, and a crosstalk between the laulimalide/peloruside and taxane sites via the M-loop of ß-tubulin is found. Together, the data define the mechanism of action of laulimalide and peloruside A on tubulin and microtubules. The data further provide a structural framework for understanding the synergy observed between two classes of MSAs in tubulin assembly and the inhibition of cancer cell growth.