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.

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.

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.

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.

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.

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 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.

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.

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.

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.

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.

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).

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.

Microtubule-stabilizing agents delay the onset of EAE through inhibition of migration.

We have shown previously that microtubule-stabilizing agents (MSA), a class of anti-proliferative compounds, can delay disease onset and reduce cumulative disease in an experimental model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE). To explore how MSA could alter EAE disease processes, we compared the effect of administering MSA before or after peak antigen-specific proliferation and found that treatment before proliferation completely inhibited antigen-specific responses in the spleen; whereas administration of an MSA such as paclitaxel or docetaxel after peak proliferation did not. Despite the presence of antigen-specific responses in mice treated at the later time point, both treatment periods resulted in similar protection against EAE, suggesting that the protective effect of MSA in EAE could not be solely attributed to anti-proliferative activity. Instead, using in vivo migration assays, it was shown that MSA inhibit immune cell infiltration into the central nervous system (CNS). Furthermore, we found that the efficacy of an MSA could be enhanced by administering low doses of two different MSA together, such as peloruside A and ixabepilone, indicating that these MSA synergize in vivo to suppress disease. Taken together, these data suggest that MSA can suppress EAE by at least two distinct mechanisms of action--prevention of proliferation and inhibition of migration into the CNS. Finally, we have shown that a combination treatment with synergizing MSA may provide enhanced protection at lower therapeutic doses.

Structurally diverse hamigerans from the New Zealand marine sponge Hamigera tarangaensis: NMR-directed isolation, structure elucidation and antifungal activity.

The NMR-directed investigation of the New Zealand marine sponge Hamigera tarangaensis has afforded ten new compounds of the hamigeran family, and a new 13-epi-verrucosane congener. Notably, hamigeran F (6) possesses an unusual carbon­carbon bond between C-12 and C-13, creating an unprecedented skeleton within this class. In particular, the structural features of 6, hamigeran H (10) and hamigeran J (12) imply a diterpenoid origin, which has allowed the putative biogenesis of three hamigeran carbon skeletons to be proposed based on geranyl geranyl pyrophosphate. All new hamigerans exhibited micromolar activity towards the HL-60 promyelocytic leukaemic cell line, and hamigeran G also selectively displayed antifungal activity in the budding yeast Saccharomyces cerevisiae. Homozygous deletion profiling (HOP) analysis suggests Golgi apparatus function as a potential target of this unusual class of sponge-derived terpenoids.

The cellular target specificity of pateamine A.

The natural product pateamine A (pateamine) from the sponge Mycale hentscheli is active against a wide range of dividing cells and has been shown to inhibit the functions of the eukaryotic initiation factor 4A (eIF4A). We have identified that pateamine is additionally able to modulate the formation of actin filaments and microtubules in vitro but at higher concentrations than required for inhibition of eIF4A. Cell cycle analysis confirmed that actin and tubulin are not major mediators of the cellular activity of pateamine. The range of targets identified demonstrates the value of multiple approaches to determining the mode of action of biologically active compounds.

Hallmarks of molecular action of microtubule stabilizing agents: effects of epothilone B, ixabepilone, peloruside A, and laulimalide on microtubule conformation.

Microtubule stabilizing agents (MSAs) comprise a class of drugs that bind to microtubule (MT) polymers and stabilize them against disassembly. Several of these agents are currently in clinical use as anticancer drugs, whereas others are in various stages of development. Nonetheless, there is insufficient knowledge about the molecular modes of their action. Recent studies from our laboratory utilizing hydrogen-deuterium exchange in combination with mass spectrometry (MS) provide new information on the conformational effects of Taxol and discodermolide on microtubules isolated from chicken erythrocytes (CET). We report here a comprehensive analysis of the effects of epothilone B, ixabepilone (IXEMPRA(TM)), laulimalide, and peloruside A on CET conformation. The results of our comparative hydrogen-deuterium exchange MS studies indicate that all MSAs have significant conformational effects on the C-terminal H12 helix of α-tubulin, which is a likely molecular mechanism for the previously observed modulations of MT interactions with microtubule-associated and motor proteins. More importantly, the major mode of MT stabilization by MSAs is the tightening of the longitudinal interactions between two adjacent αß-tubulin heterodimers at the interdimer interface. In contrast to previous observations reported with bovine brain tubulin, the lateral interactions between the adjacent protofilaments in CET are particularly strongly stabilized by peloruside A and laulimalide, drugs that bind outside the taxane site. This not only highlights the significance of tubulin isotype composition in modulating drug effects on MT conformation and stability but also provides a potential explanation for the synergy observed when combinations of taxane and alternative site binding drugs are used.