Review Cite This: J. Nat. Prod. 2018, 81, 691−702
pubs.acs.org/jnp
Marine Invertebrate Natural Products that Target Microtubules John H. Miller,*,† Jessica J. Field,† Arun Kanakkanthara,‡ Jeremy G. Owen,† A. Jonathan Singh,§ and Peter T. Northcote§ †
School of Biological Sciences and Centre for Biodiscovery and §The Ferrier Research Institute, Victoria University of Wellington, Wellington, New Zealand ‡ Department of Oncology and Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Rochester, Minnesota, United States ABSTRACT: 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.
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Corals are marine invertebrates in the phylum Cnidaria, class Anthozoa. Corals are sessile and typically live in compact colonies of thousands of genetically identical polyps. Corals, like sponges, are a rich source of bioactive molecules. Tunicates, also known as sea squirts, are in the phylum Chordata, subphylum Urochordata, class Ascidiacea. They are generally solitary, sessile marine organisms representing the most primitive chordates. Both corals and tunicates harbor symbionts, similar to sponges. Molluscs (phylum Mollusca) are a large and diverse group of soft-bodied, often shelled, marine organisms that include snails, chitons, bivalves (clams and mussels), nudibranchs, cephalopods, and sea slugs (sea hare Aplysia).
hemical compounds derived from living organisms, such as terrestrial plants, microorganisms, and marine organisms, serve as important sources of medicinal products for the treatment of human cancers1−3 and other diseases. Marine organisms in particular are a rich and diverse source of unique natural products with bioactivities ranging from growth inhibition to induction of apoptosis.4,5 Natural products have been shaped by evolution to generate a battery of defenses against other predatory or competing organisms or parasites. A major step in the evolution of the production of compounds by marine organisms has been the acquisition of symbiotic bacteria that act as factories for synthesis of these unique compounds. Sponges have been especially prolific as a source of bioactive compounds that display activity against cytoskeletal proteins and enzymes in higher metazoans. Marine natural products (MNPs) have been isolated mostly from sponges (47%), then corals (34%), then smaller amounts from echinoderms, molluscs, and tunicates (18%).6 Sponges are thought to be the oldest, simplest multicellular animals, lacking true tissues but having different cell types for different functions.7 Marine sponges are classified based on their skeletal components (or lack thereof), which are made up of either collagen fibers and filaments (organic skeleton) or separate or fused spicules of calcium carbonate or silicon dioxide. Sponges have a symbiotic relationship with bacteria. Lectins, sugar binding proteins important in cellular recognition, are involved in allowing bacteria to coexist with sponge tissue because lectins contain binding sites for particular symbionts.8,9 Approximately 40−60% of the total sponge mass consists of bacteria.10,11 These bacteria are considered the source of many of the secondary metabolites found in sponges;12 however, the relationship in terms of benefit between the two remains poorly understood. © 2018 American Chemical Society and American Society of Pharmacognosy
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MNPS THAT TARGET MICROTUBULES Many excellent reviews already exist on natural products from the marine environment; therefore, this review is focused on MNPs that target the eukaryote cytoskeleton, specifically the microtubules. See Liu et al. 201413 for a relatively recent review that includes five selected microtubule targeting agents (MTAs) isolated from marine invertebrates and two from cyanobacteria. This compares with the present review that includes a total of 20 confirmed MTAs from marine invertebrates reported in the literature that includes the five also reviewed by Liu et al.13 The advent of MTAs for anticancer treatment began in the 1950s and 1960s with microtubule-destabilizing agents isolated from terrestrial plants, including colchicine (1) from the plant Colchicum autumnale, already used medically by the turn of the Special Issue: Special Issue in Honor of Susan Horwitz Received: November 14, 2017 Published: February 12, 2018 691
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19th century, and the Vinca alkaloids (e.g., vincristine, 2), which were isolated from leaves of the Madagascar periwinkle, Catharanthus roseus, in the 1950s.14 In 1979, another plant compound, paclitaxel (3), isolated from the bark and needles of the Pacific yew tree, was the first natural product to be shown to stabilize microtubules by Susan Horwitz at the Albert Einstein College of Medicine in New York.15−17 For a current review of the history of paclitaxel’s development, see Yang and Horwitz, 2017.18 The mechanism of how 3 kills cancer cells is described by Weaver et al.19 In 1987, the epothilones (e.g., epothilone B, 4) were discovered and isolated from the cellulose-degrading soil myxobacterium Sorangium cellulosum20 and led to the development of ixabepilone (5).21 Since then, many other new MTAs have been identified, particularly from the marine environment.
of critical macromolecules and organelles, cell motility, and maintenance of cell shape and polarity to adapt to diverse extracellular environments;25,26 During cell division, microtubules form the mitotic spindle apparatus that is essential for chromosome segregation during mitosis.24 Owing to the central role of microtubules in the key cellular processes of cell proliferation and survival, compounds that target microtubules and disrupt their functions have proven to be one of the most successful classes of anticancer drugs available in the clinic to date.27 Two mechanisms of action have been described for the anticancer effects of microtubule-targeting agents. One well-described mechanism is that MTAs cause cancer cell death by blocking mitosis, which is dependent on an intense regulation of microtubule polymerization/depolymerization dynamics.23,27 MTAs exert distinct effects on microtubule dynamics based on their concentration. At higher concentrations, MTAs either prevent microtubule polymerization (compounds in this class are known as microtubule-destabilizing agents and include 1 and 2), causing decreased microtubule polymer mass or promote microtubule polymerization (compounds in this class are known as microtubule-stabilizing agents and include 3−5), causing increased polymer mass.27 At lower concentrations, MTAs inhibit microtubule dynamics without markedly affecting the polymer mass. At the cellular level, both microtubule-stabilizing and -destabilizing agents inhibit microtubule dynamics and lead to cellcycle arrest in mitosis through sustained activation of the spindle assembly checkpoint that ultimately triggers cell death via apoptosis.27 Another mechanism that was recognized more recently is that MTAs induce cell death by perturbing interphase microtubule functions.28−32 Microtubules are involved in numerous essential activities of the cells both during mitosis (mitotic spindles and asters) and during interphase (cell trafficking of organelles, cell shape, cell movement). The interphase microtubules act as tracks for intracellular transport of diverse proteins and nucleic acid cargoes that are crucial for cell survival.33 MTAs have been shown to disrupt interphase microtubule dynamics and trafficking of critical proteins that favor cancer cell growth.28−30 For example, taxanes antagonized androgen receptor signaling that drives prostate cancer by preventing dynein-mediated trafficking of androgen receptor to the nucleus along interphase microtubules.34,35 Moreover, paclitaxel (3) decreased the velocity of endocytic trafficking of epidermal growth factor receptor and shuttled it to lysosomes situated in the periphery in lung carcinoma cells.36 Recently, Poruchynsky et al.37 showed that MTA treatment increases cytoplasmic retention of key DNA repair proteins (ATM, ATR, DNA-PK, Rad50, Mre11, p95/NBS1, p53, and 53BP1) by preventing their nuclear transport via interphase microtubules in multiple cancer cell types. The increased cytoplasmic sequestration of the DNA repair proteins by 2 and 3 induced prolonged DNA damage in the cells.38 By showing an interphase mechanism of action of MTAs, the study by Poruchynsky et al.37 also explains why combinations of MTAs and DNA damaging agents are successful in cancer therapy.
Microtubules are one of the three main structural components of the eukaryotic cytoskeleton along with actin microfilaments and intermediate filaments. Heterodimers of α- and β-tubulin polymerize head-to-tail to form a protofilament, 13 of which bind together laterally to form a microtubule with a diameter of 25 nm (Figure 1).22 Microtubules are dynamic structures that grow and
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Figure 1. Structure and dynamics of microtubules.
WHY DO MARINE ORGANISMS PRODUCE MTAS? Microtubules are the target of many natural products produced by marine organisms;39 therefore, an important question that should be asked is why tubulin is such a good target that so many MTAs have evolved in marine organisms? It is known that most marine natural products that show bioactivity against other organisms’ proteins are secondary metabolites, produced not to support
shorten in length as a function of modulation by GTP/GDP transitions and binding of microtubule-associated proteins such as MAPs, stathman, and motor proteins.23 Microtubules are critical in many different cellular processes, especially throughout the cell cycle.24 In interphase cells, microtubules are organized into a radial array from a central microtubule-organizing center (or centrosome) and play important roles in intracellular trafficking 692
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tubulin and sponge tubulin based on a database search in Uniprot (Table 1, Figure 2). Complete FASTA sequences were downloaded from UniProt, and Geneious Software was used to align the sequences and develop sequence identity. The four sponge species were chosen because complete β-tubulin sequences were available for those sponges, whereas only fragments of β-tubulin sequences were available for other sponges. Consensus sequences were determined by the software from the most frequent residues between the β-tubulin chain sequences being compared. Human tubulin β chain: gene = TUBB, protein ID = P07437. Amphimedon queenslandica tubulin β chain: protein ID = A0A1X7VM44. Sycon raphanus tubulin β chain: gene = tubb, protein ID = I2FLM0. Geodia cydonium tubulin β chain: gene = tub, protein ID = O96950. Suberites domuncula tubulin β chain: gene = tub, protein ID = Q70SJ8. Schütze et al.45 cloned and sequenced sponge β-tubulin from the marine sponge Geodia cydonium. Comparing the β-tubulin sequence of G. cydonium to human tubulin indicated 91% identity (41 differences out of 449 amino acids). Twelve of the differences were clustered at the C-terminus, where most of the variation is found between human tubulin isotypes as well. Resistance to microtubule-stabilizing agents by cells can arise from mutations in the binding sites to taxoid drugs or peloruside A and laulimalide (see MTAs from Marine Invertebrates);46,47 however, there is no obvious lack of sequence identity in the binding sites on sponge tubulin, making this an unlikely explanation for the protection of the sponge from the toxins contained in its tissues. It would be an interesting experiment to clone the gene for sponge tubulin from M. hentscheli, express and purify the protein in a bacterium, and test directly whether a marine invertebrate-derived MTA can stabilize polymerization of sponge tubulin in vitro.
primary metabolism of the marine species but having other secondary functions.40 These secondary metabolites are often toxins that provide a chemical defense against environmental stress factors, including predation, overgrowth by fouling organisms, or competition for space.41 This is suggested by the observation that the most toxic secondary metabolites are found in organisms that grow in habitats where intense competition for space and feeding pressure occurs, such as in coral reefs. Marine sponges, corals, and tunicates are typically found in coral reefs and are a food source for other marine organisms. The microtubule is a highly vulnerable target in eukaryotes because there is no redundancy in microtubule structure and function in cells. Although there are a number of isotypes of β-tubulin, most MTAs interact to some degree with all the isotypes.18,27,42 This lack of rescue by back-up tubulins may contribute to the explanation of why so many MNPs have been identified that are MTAs. It is also possible that the predominance of cytotoxicity assays for bioassay-guided isolation of MNPs contributes a bias toward discovering MTAs. It is unclear why actin microfilaments have not been pursued more in drug discovery programs, as they also have no back-up in the cells. Numerous actin-targeting MNPs have been described, leaving the potential for further development of actin inhibitors as a future anticancer approach.43
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SPONGE β-TUBULIN SEQUENCE SIMILARITY TO HUMAN β-TUBULIN Considering the high concentrations of toxic secondary metabolites that may be contained in marine invertebrates, it would be interesting to compare the sequences of invertebrate β-tubulin with higher vertebrate or mammalian β-tubulin to determine if the MTAs are likely to interact with the tubulin. Sponges are the most common ancestor of all multicellular organisms, containing the earliest proteins in animal lineages. Sponges have orthologue genes encoding proteins that are found throughout eukaryotes. Interestingly, sponge proteins share a higher degree of homology with proteins found in mammals than with those found in lower eukaryotes such as Caenorhabditis elegans;44 thus, many sponge proteins are extremely similar to those found in humans. In the context of β1-tubulin, there is 90−98% similarity between human
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MTAS FROM MARINE INVERTEBRATES In total, this review includes 14 MNPs from sponges, two from corals, three from tunicates, and one from a mollusc (Table 2). These MNPs consist mostly of polyketide macrolides and lactones, peptides, alkaloids, and terpenes. Many congeners and synthetic analogues of these MNPs have also appeared in the literature with structural differences that may improve (or at least impart similar) activity and increased selectivity toward their biological targets. In the summary table (Table 2), the MNPs are separated by the animal type from which they were isolated. In the detailed descriptions of MTAs that follow, the main sections are based on whether the MTAs are stabilizers or destabilizers. Microtubule-Stabilizing Agents. Sarcodictyins and Eleutherobin. The cembranoid-derived diterpenoids sarcodictyins A (6), B (7), and eleutherobin (8) were isolated from marine soft corals of the genus Sarcodictyon and Eleutherobia, respectively.48−50
Table 1. Percent Sequence Homology between Four Sponge β1-Tubulins and Human Tubulin human A. queenslandica S. raphanus G. cydonium S. domuncula human A. queenslandica S. raphanus G. cydonium S. domuncula
96.4 96.4 94.8 90.8 97.1
96.0 91.1 98.4
94.8 96.0 90.0 96.0
90.8 91.1 90.0
97.1 98.4 96.0 91.3
91.3
Figure 2. Identity between human β-tubulin and four sponge tubulins. 693
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Table 2. Microtubule-Targeting Agents from Marine Invertebrates compound
chemical type
Laulimalide (9) Discodermolide (10) Dictyostatin-1 (11) Zampanolide (12) Peloruside A (13) Ceratamines A, B (14, 15) Halichondrin B (17) Spongistatin-1 (26) Hemiasterlin (27) Arenastatin A (30) Jaspolide B (32) PM050489/PM060184 (33, 34) CALe (35)c Leiodermatolide (36)
polyketide macrolide polyhydroxylated lactone polyketide macrolide polyketide macrolide polyketide macrolide heterocyclic alkaloid polyether macrolide polyether macrolide linear tripeptide depsipeptide triterpenoid polyketide diterpenoid polyketide macrolide
Sarcodictyins A, B (6, 7) Eleutherobin (8)
diterpenoid diterpenoid
Rigidin (23) Diazonamide A (25) Vitilevuamide (31)
heterocyclic alkaloid halogenated cyclic peptide bicyclic peptide
Dolastatins 10, 15 (19, 20)
peptide/depsipeptide
organism Sponges (Porifera) Cacospongia mycofijiensis Discodermia dissoluta Spongia sp. Cacospongia mycofijiensis Mycale hentscheli Pseudoceratina sp. Halichondria okadai Spongia sp. Hemiasterella minor Dysidea arenaria Jaspis sp. Lithoplocamia lithistoides Axinella sp. Leiodermatium sp. Corals (Cnidaria) Sarcodictyon roseum Eleutherobia sp. Tunicates (Chordata) Eudistoma cf. rigida Diazona angulata Didemnum cuculiferum Sea Hare (Mollusca) Dolabella auricularia
microtubule mode of actiona
binding site on β-tubulinb
S S S S S D D D D D D D protection MT dynamics
Lau/Pel taxoid taxoid taxoid Lau/Pel unknown vinca vinca vinca vinca unknown vinca colchicine unknown
S S
taxoid taxoid
D D D
colchicine vinca vinca
D
vinca
S = stabilization; D = destabilization. bBinding sites on β-tubulin (Dumontet and Jordan 2010) are as follows: taxoid site on the inside of microtubules that binds paclitaxel (3), docetaxel, and epothilones in addition to the compounds listed; Lau/Pel site on the outside of microtubules that binds laulimalide (9) and peloruside A (13); vinca site at the interdimer interface that binds vinblastine, vincristine (2), vinorelbine, and others in addition to the compounds listed; and colchicine site also located between heterodimers that binds colchicine (1), colcemid, podophyllotoxin, 2-methoxyestradiol, combretastatin, noscapine, maytansine, and others. cCALe (35) binds the colchicine site and prevents or slows colcemid from exerting its effect of destabilization or depolymerization of the microtubules. a
activity and was associated with severe toxicity and mortality.61 However, several analogues of laulimalide (9) and its natural congener, isolaulimalide, offer potential for further development as an anticancer drug.63,64
The compounds differ from each other through the oxidation state and glycosylation at one of the diterpene methyl groups. Eleutherobin and sarcodictyins A and B are paclitaxel-like microtubulestabilizing agents that bind to the taxoid site on β-tubulin48,51,52 with 8 in particular noted to be a substrate for the P-glycoprotein (P-gp) multidrug-resistant efflux pump.51 SAR studies of eleutherobin analogues showed necessity for the N(1)-methylurocanate and sugar moieties for activity.53 Despite these findings, development of these compounds seems to have stagnated.
Discodermolide. In 1990, the Gunasekera laboratory at the Harbor Branch Oceanographic Institute reported the structure of discodermolide (10), a polyhydroxylated lactone, from the Caribbean marine sponge Discodermia dissoluta, collected at a depth of 33 m.65 Discodermolide stabilizes microtubules by binding to the taxoid site,66,67 but it also synergizes with paclitaxel.68 Studies from the Horwitz and Steinmetz laboratories demonstrated that paclitaxel interacts mainly with the M-loop of β-tubulin, whereas 10 has a binding orientation away from this loop and toward the N-terminal H1−S2 loop.69−71 Discodermolide stabilizes microtubules potentially through tubulin interdimer contacts on the αtubulin side and to a lesser extent on interprotofilament contacts between adjacent β-tubulin subunits. Discodermolide (10) was obtained by Novartis in 1998 and in 2004 reported Phase I clinical trials in patients with solid tumors, but the trial was discontinued due to severe lung toxicities.72 Work continues on total synthetic approaches and semisynthetic analogues73−75 as well as its mechanism of stabilization.71
Laulimalide. In 1988, the Crews (University of California, Santa Cruz) and the Moore and Scheuer (University of Hawaii, Manoa) laboratories independently reported the isolation of laulimalide (fijianolide B, 9), a 20-membered polyketide macrolide from marine sponges collected in Vanuatu (Spongia mycof ijiensis) and Indonesia (Hyattella sp.), respectively.54,55 Laulimalide is cytotoxic at nanomolar concentrations against a number of cancer cell lines and was reported to be the first microtubule-stabilizing agent that bound to a nontaxoid site on β-tubulin.56−58 Laulimalide is active against paclitaxel- and epothilone-resistant ovarian cancer cell lines that have mutations in the drug binding site on β-tubulin and also bypasses P-gp-mediated multidrug resistance by cancer cells. Laulimalide binds to an exterior site on the microtubule that is different from the taxoid site.59,60 Unfortunately, in one of two preclinical in vivo studies,61,62 9 had only marginal antitumor 694
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Peloruside A. Peloruside A (13) is a 16-membered polyketide macrolide that was first reported in 2000 from the New Zealand marine sponge Mycale hentscheli.87 Peloruside A is a well-studied nontaxoid site microtubule-stabilizing agent that shares a similar or overlapping binding site with laulimalide (9) on β-tubulin.57,88−90 Like laulimalide, it is not a substrate for the P-gp drug efflux pump and is active against paclitaxel- and epothilone-resistant cancer cells that have mutations at the taxane site on tubulin.89 Moreover, 13 demonstrated potent antitumor activity in mice bearing various types of human tumor xenografts.91 Peloruside A exhibited improved activity over taxoid site MTAs. This in vivo study supports the continued clinical development of 13 as an anticancer drug. The binding sites of 13 on β-tubulin and the details of its natural congeners and synthetic/semisynthetic analogues have been covered in recent in-depth reviews by Kanakkanthara et al.92 and Brackovic et al.93
Dictyostatin-1. The 22-membered macrolide dictyostatin-1 (11) was first reported by the Pettit laboratory (Arizona State University) in 1994 from a Spongia sp. marine sponge collected from the Maldives.76 Despite its potent cytotoxicity toward murine leukemic P388 cells and others, the microtubule-stabilizing activity of 11 was only reported in 2003 following reisolation from a Jamaican sponge of the family Corallistidae (order Lithistida).77 Dictyostatin-1 binds to the taxoid site on β-tubulin and inhibits the growth of a number of cancer cells, including those that overexpress Pgp.77 Unfortunately, 11 did not progress into in vivo preclinical testing or clinical trials because of its complex structure and difficult total synthesis; however, improvements in simplifying its total synthesis and the development of synthetic and semisynthetic analogues78−80 may increase the likelihood that dictyostatin-1 will be advanced for future clinical development.
Ceratamines. Ceratamines A (14) and B (15) were reported in 2003 by the Roberge and Andersen laboratories (University of British Columbia, Canada) from a Pseudoceratina sp. marine sponge collected in Papua New Guinea.94 The authors employed a cellbased screen to detect antimitotic behavior from crude marine invertebrate extracts. Antimitotic activity was reported in the low micromolar range. These heterocyclic alkaloids are relatively simple in structure, having been proposed to be derived from tyrosinylhistidine, and because of this, make for ideal candidates for further development. Roberge and Andersen took advantage of the SAR potential with a subsequent study showing replacement of bromine in 14 with methyl groups producing a compound (16) with enhanced antimitotic activity.95 The ceratamines stabilize microtubules by binding to a nontaxoid site and lead to an unusual, new mitotic arrest phenotype.96
Zampanolide. Tanaka and Higa (University of the Ryukyus, Japan) first disclosed the structure of zampanolide (12) in 1996 from the Okinawan marine sponge Fasciospongia rimosa81 and reported potent cytotoxicity (IC50 1−5 ng/mL) against the P388, A549, HT29, and MEL28 cell lines. In 2009, the Northcote and Miller laboratories reported the isolation of 12 following an NMRdirected investigation of the Tongan marine sponge Cacospongia mycof ijiensis and consequentially discovered its antimitotic and microtubule-stabilizing activity.82 Zampanolide binds to the taxoid site on tubulin and forms a covalent bond to the luminal site of the microtubule.83,84 This mode of action may also explain how zampanolide overcomes the P-gp-mediated resistance seen in some cancer cells.82 Covalent binding of a drug prevents its efflux from the cell by drug efflux pumps, thereby increasing its effective concentration in the cell and prolonging its duration of action.85 Zampanolide is also cytotoxic in cells that are resistant to taxoid site drugs or peloruside/laulimalide site drugs.86 In addition, 12 inhibits cell migration, which is associated with tumor metastasis.86 Further studies are needed, however, to determine its clinical utility. A 20-membered macrolide, 12, possesses relatively low oxygenation and few stereogenic centers, features normally associated with polyketide-derived macrolides. These qualities make 12 an attractive target for structure−activity relationship (SAR) studies.
Microtubule-Destabilizing Agents. Halichondrin B. Halichondrin B (17) is a potent polyether macrolide first reported in 1986 from the marine sponge Halichondria okadai by Hirata and Uemura.97 It occupies the vinca site on tubulin, prevents microtubule polymerization, and has shown remarkable antitumor activity both in vitro and in vivo.98,99 Early work in association with David Newman of the Natural Products Branch of the National Cancer Institute in Frederick, MD, USA used aquaculture to obtain 300 mg of 17 from more than a ton of the sponge Lissodendoryx sp. grown on 40 m deep lines off the New Zealand coast.100 Although the limited availability of halichondrin B became a barrier to its further development, the establishment of a synthetic approach despite the 32 stereocenters of the parent compound generated structurally simplified synthetic analogues that have similar anticancer activity as the parent compound.101−103 695
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One of the impressive analogues was eribulin (18).104 The mesylate salt of 18 was approved in 2010 by the FDA for the treatment of patients with metastatic breast cancer who had initially received at least two chemotherapeutic regimens for the treatment of metastatic disease.104 Eribulin mesylate is the only agent that is known so far to prolong overall survival of patients with pretreated metastatic breast cancer when administered as a monotherapy.104
0.4−0.5 nM in selected cancer cell lines. Five years later, dolastain 15 (20) (IC50 3−5 nM) was reported from the same organism.106 Both compounds caused microtubule depolymerzation in vitro with IC50 values of 1.2 μM for 19 and 23 μM for 20, possibly binding to the vinca site on tubulin.106 In 2001, 19 was identified as a constituent of the cyanobacterium Caldora penicillata.107,108 The activity profiles of these particular compounds initiated detailed studies into their development into clinically relevant drug candidates; however, trials were discontinued due to toxicity. Monomethyl auristatin E (21), an analogue of 19, was developed into the antibody−drug conjugate brentuximab vedotin (Adcetris, 22), which gained FDA approval for the treatment of Hodgkin lymphoma.109 Rigidin. Rigidin (23) is a pyrrole alkaloid isolated from the tunicate Eudistoma cf. rigida.110 Although 23 was shown to have no direct interaction with tubulin, the synthetic preparation of analogues featuring the modification of its 7-deazaxanthine scaffold to a 7-deazahypoxanthine variant afforded compounds (e.g., alkyne 24) with substantial antiproliferative and antimicrotubule activity.111,112 Using 3H-colchicine competition experiments, the colchicine site was determined to be the likely binding site of the 7-deazahypoxanthine analogues.111,112
Dolastatins. The Pettit laboratory reported the constitutional structure of dolastatin 10 (19) in 1987 in which 28.7 mg was isolated from 1000 kg of the sea hare Dolabella auricularia105 and identified it as a heavily modified pentapeptide with an IC50 of
Diazonamide A. Lindquist and Fenical from the Scripps Institute of Oceanography in La Jolla, CA, USA reported on the isolation of a halogenated cyclic peptide, diazonamide A (25),
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from the colonial ascidian Diazona chinensis that binds the vinca site on β-tubulin.113 Diazonamide A caused unusual effects on microtubules compared to other vinca site MTAs like vincristine (2), slowing microtubule growth but increasing the rescue of depolymerizing microtubules by straightening or reducing the curvature of the microtubules.114
both in vitro and in vivo, was derived by modifying the Nterminus portion of the parent molecule.124,125 Compound 29 has undergone a phase I trial for patients with advanced solid tumors.124 Arenastatin A. The depsipeptide arenastatin A (cryptophycin24, 30) was reported in 1994 by the Kitagawa laboratory (Osaka University, Japan) from the Okinawan marine sponge Dysidea arenaria.126 The compound showed potent cytotoxicity against the KB cell line (IC50 8 pM) and is structurally similar to cryptophycin-1 (isolated from a cyanobacterium), which was reported to inhibit microtubule assembly and bind to the vinca site.127,128 The microtubule-inhibiting activities of 30 (IC50 2.3 μM) and synthetic analogues were reported in 1996.129 Further studies showed arenastatin A to be a competitive binder with vinblastine and rhizoxin but not colchicine.130 However, the authors of this work noted that 30 is chemically unstable, which may explain the reason behind its restricted development.
Spongistatin-1. Spongistatin-1 (26) is a macrocyclic lactone polyether that was initially isolated from a Spongia sp. marine sponge.115,116 Spongistatin-1 is highly cytotoxic at picomolar concentrations in the NCI-60 panel of human cancer cell lines.115 It also retained its remarkable anticancer activity in vivo, exhibiting a potent effect against various tumor xenografts without major associated toxicity.117 Spongistatin-1 occupies the vinca site on tubulin and inhibits microtubule polymerization.115,116 It also has an antimetastatic effect and shows caspase-independent pro-apoptotic activity.117,118 As with most sponge-derived compounds, the limited supply of 26 has so far prevented its clinical development, but several approaches are in place to resolve this issue.119,120
Vitilevuamide. The bicyclic 13 amino acid peptide vitilevuamide (31) was isolated from two ascidians Didemnum cuculiferum and Polysyncraton lithostrotum and showed destabilizing activity in the nanomolar range.131 Competition experiments suggested noncompetitive inhibition of vinblastine but not colchicine binding, suggesting an interaction with the vinca binding site on tubulin.
Hemiasterlin. Hemiasterlin (27) is a potent cytotoxic tripeptide that was isolated from the marine sponge Hemiasterella minor.121 Its excellent anticancer cell activity is based on microtubule depolymerization via binding to the vinca domain on tubulin. A synthetic analogue of hemiasterlin,122 HTI-286 (SPA110, 28), with an IC50 of 2.5 nM in a panel of 18 cancer cell lines, was the first analogue of hemiasterlin to enter Phase I clinical trials in 2002 and moved into Phase II trials from which it was later withdrawn.123 Another analogue, E7974 (29), which is also effective against a wide variety of human cancer cell types
Jaspolide B. The isomalabaricane triterpenoid jaspolide B (32) was reported among several related compounds in 2006 from a marine sponge of the genus Jaspis, collected from the South China Sea.132 Compound 32 showed micromolar growth inhibition against Bel-7402 and HepG2 cells, caused apoptosis in
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demonstrated that 36 disrupted microtubule dynamics through a unique mechanism.140
the G1 phase of the cell cycle, and showed dose-dependent microtubule disassembly.133 In the same year, Li et al. reported G2/M arrest and induction of apoptosis by 32 in HL-60 cells.134 To date, no binding site has been established for this compound. PM050489 and PM060184. In 2013, researchers from PharmaMar (Madrid, Spain) reported the isolation of PM050489 (33) and PM060184 (34), two polyketide-derived metabolites from a Madagascar collection of the marine sponge Lithoplocamia lithistoides.135 Both compounds exhibited antimitotic behavior and showed growth inhibition at subnanomolar concentrations against a variety of human tumor cell lines. The supply issue was circumvented in this case through a multigram total synthesis of both compounds reported in the same article,135 allowing for sufficient material to continue development. In the same year, Pera et al. established the mechanism of action and showed binding within the vinca domain.136
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DEVELOPMENT OF MARINE-DERIVED MTAS Supply of the natural product is a well-documented hurdle in the development of these potent marine toxins into drug candidates. The ecological ramifications and concerns over overprospecting are well-justified. However, recent advances in isolation techniques, structure elucidation, synthetic methodology, and sequencing technologies all lend themselves to overcoming the supply issue. The production of sufficient quantities of MNPs for preclinical and clinical trials is limited by the difficulty in collecting the organisms and small amounts obtained after extraction, purification, and identification. For upscaling to greater amounts (gram rather than milligram amounts), there are a number of options available, including (a) collecting more material from the marine environment for extraction (often not possible or practical), (b) aquaculture, (c) synthesis from an abundant precursor, (d) total synthesis (upscaled for large amounts), (e) design and synthesis of a simplified analogue, and f) biosynthesis by bacterial symbionts resident in the marine organisms’ tissues either via isolation and cultivation of the producing strain or heterologous expression of the biosynthetic gene cluster (BGC). As we have considerable experience working with peloruside A (13) in some of these upscaling methods, the following sections will concentrate on this compound where possible. Aquaculture. The collection and growth of marine organisms in a controlled aquaculture setting is an approach that has been tried, but there are difficulties with this controlled production method.11 Some of the problems are that marine organisms require special conditions that may not be attainable except in the ocean. This complicates the logistics of the process. For example, the environment of the marine organisms affects growth and survival, including variations in depth, light, temperature, nutrients, and surface availability for grazing or attachment. Stress to a marine organism may also influence the production of metabolites.11 Studies by Page et al. with a sponge141,142 indicated that there was significant spatial and temporal variability in the amount of a particular MNP being produced by the species being studied. This investigation involved ecological studies on the New Zealand marine sponge Mycale hentscheli, which produces three highly toxic compounds: mycalamide A, pateamine, and peloruside A (13).87 The levels of 13, for example, in different sponge populations (chemotypes) changed considerably depending on the time of year and location. Depth and the site of collection were other important factors. Peloruside A obtained from different populations did not show consistent patterns; however, in a particular sponge population that produced 13, the production was maintained for generations. The spatial and temporal patchiness of the production was probably related to the specific symbiotic bacteria found in the sponge that are believed to produce polyketide 13. As sponge mass is believed to consist of up to 40−60% bacteria,10,11 it is not surprising that sponges may contain as much as 100 or 200 μg of purified 13 per gram (wet weight) of sponge material. Page et al.142 went on to apply aquaculture techniques over a seven-year period. The aquaculture was not viable in aquaria but needed to be set up off-shore in a New Zealand coastal region where peloruside-containing sponges were found (Capsize Point in Pelorus Sound in the Marlborough Sounds). The sponges were grown on lines in an area where a mussel farming venture was underway. Growth was very good, reaching 2437% increases
Other MTAs from Marine Invertebrates. Other MNPs with less defined microtubule-targeting actions have been extracted that bind either to the colchicine or vinca site or an unknown site on β-tubulin. CALe. CALe (N-formyl-7-amino-11-cycloamphilectene, 35), a diterpenoid isolated from an Axinella sp. marine sponge collected in Vanuatu was reported by Gomez-Paloma and co-workers in 2002.137 The structure of 35 was determined by spectroscopic and crystallographic methods and is unusual in that, apart from an N-formyl attachment, it is relatively devoid of functionalization. Using a chemoproteomics approach, Casapullo and coworkers identified α,β-tubulin as one of the targets of CALe.138 Compound 35 showed a protective effect on microtubule integrity against the colchicine analogue colcemid when HeLa cells were treated with the compound.137
Leiodermatolide. Leiodermatolide (36) is a polyketidederived, 16-membered macrolide, isolated from a deep-water marine sponge Leiodermatium sp. collected from Florida, USA.139 The Paterson and Wright laboratories reported the structure of 36 in 2011, following structure elucidation using NMR spectroscopic and computational techniques.139 Leiodermatolide exhibited potent antimitotic activity (
