ciguatoxins and gambierol

Ciguatoxins (CTX) and brevetoxins (BTX) are polycyclic ethereal compounds biosynthesized by the worldwide distributed planktonic and epibenthic dinoflagellates of

Topics: Animals; Ciguatera Poisoning; Ciguatoxins; Dinoflagellida; Humans; Ligands; Marine Toxins; Oxocins; Potassium Channels, Voltage-Gated

Since the isolation of brevetoxin-B, a red tide toxin, many bioactive marine natural products featuring synthetically challenging trans-fused polycyclic ether ring systems have been reported. We have developed SmI(2)-induced cyclization of beta-alkoxyacrylate with aldehyde, affording 2,6-syn-2,3-trans-tetrahydropyran (THP) or 2,7-syn-2,3-trans-oxepane with complete stereoselection, as a key reaction of efficient iterative and bi-directional strategies for the construction of these polycyclic ethers. This reaction is also applicable to the synthesis of 3-, 5-, and 6-methyl-THPs and 3,5-dimethyl-THP. The synthesis of 2-methyl- and 2,6-dimethyl-THPs was accomplished by means of a unique methyl insertion. Recently, the SmI(2)-induced cyclization was extended to similar reactions using beta-alkoxyvinyl sulfone and sulfoxide. Reaction of (E)- and (Z)-beta-alkoxyvinyl sulfone-aldehyde afforded 2,6-syn-2,3-trans- and 2,6-syn-2,3-cis- THPs, respectively. Reaction of (E)-beta-alkoxyvinyl (R)- and (S)-sulfoxides gave 2,6-anti-2,3-cis- and 2,6-syn-2,3-trans-THPs, respectively. Reaction of (Z)-beta-alkoxyvinyl (R)-sulfoxides gave 2,6-syn-2,3-cis-THP and an olefinic product, while that of (Z)-beta-alkoxyvinyl (S)-sulfoxide afforded a mixture of many products. These SmI(2)-induced cyclizations have been applied to the total syntheses of various natural products, including brevetoxin-B, mucocin, pyranicin, and pyragonicin. Synthetic studies on gambierol and maitotoxin are also introduced.

Topics: Biological Products; Ciguatoxins; Cyclization; Ethers; Heterocyclic Compounds, 4 or More Rings; Iodides; Lactones; Marine Toxins; Oxocins; Polymers; Pyrans; Samarium

The unprecedented structure of the marine natural product brevetoxin B was elucidated by the research group of Nakanishi and Clardy in 1981. The ladderlike molecular architecture of this fused polyether molecule, its potent toxicity, and fascinating voltage-sensitive sodium channel based mechanism of action immediately captured the imagination of synthetic chemists. Synthetic endeavors resulted in numerous new methods and strategies for the construction of cyclic ethers, and culminated in several impressive total syntheses of this molecule and some of its equally challenging siblings. Of the marine polyethers, maitotoxin is not only the most complex and most toxic of the class, but is also the largest nonpolymeric natural product known to date. This Review begins with a brief history of the isolation of these biotoxins and highlights their biological properties and mechanism of action. Chemical syntheses are then described, with particular emphasis on new methods developed and applied to the total syntheses. The Review ends with a discussion of the, as yet unfinished, story of maitotoxin, and projects into the future of this area of research.

Topics: Animals; Ciguatoxins; Cyclization; Ethers; Marine Toxins; Molecular Structure; Oxocins

Marine polycyclic ether natural products continue to fascinate chemists and biologists due to their exceptionally large and complex molecular architectures and potent and diverse biological activities. Tremendous progress has been made over the past decade toward the total synthesis of marine polycyclic ether natural products. In this area, a convergent strategy for assembling small fragments into an entire molecule always plays a key role in successful total synthesis. This review describes our efforts to develop convergent strategies for the synthesis of polycyclic ethers and their application to the total synthesis of gambierol, gymnocin-A, and brevenal, and to the partial synthesis of the central part of ciguatoxins and the nonacyclic polyether skeleton of gambieric acids.

Topics: Ciguatoxins; Ethers, Cyclic; Marine Biology; Molecular Structure; Polycyclic Compounds; Thiopental

Gambierol inhibits voltage-gated K

Topics: Adenosine Triphosphate; Animals; Calcium; Catecholamines; Cells, Cultured; Chromaffin Cells; Ciguatoxins; Potassium; Rats

Marine ladder-shaped polyether toxins are implicated in neurological symptoms of fish-borne food poisonings. The toxin gambierol, produced by the marine dinoflagellate Gambierdiscus toxicus, belongs to the group of ladder-shaped polyether toxins and inhibits Kv3.1 channels with nanomolar affinity through a mechanism of gating modification. Binding determinants for gambierol localize at the lipid-exposed interface of the pore forming S5 and S6 segments, suggesting that gambierol binds outside of the permeation pathway. To explore a possible involvement of the voltage-sensing domain (VSD), we made different chimeric channels between Kv3.1 and Kv2.1, exchanging distinct parts of the gating machinery. Our results showed that neither the electro-mechanical coupling nor the S1-S3a region of the VSD affect gambierol sensitivity. In contrast, the S3b-S4 part of the VSD (paddle motif) decreased gambierol sensitivity in Kv3.1 more than 100-fold. Structure determination by homology modeling indicated that the position of the S3b-S4 paddle and its primary structure defines the shape and∖or the accessibility of the binding site for gambierol, explaining the observed differences in gambierol affinity between the channel chimeras. Furthermore, these findings explain the observed difference in gambierol affinity for the closed and open channel configurations of Kv3.1, opening new possibilities for exploring the VSDs as selectivity determinants in drug design.

Topics: Amino Acid Sequence; Animals; Binding Sites; Cell Line; Ciguatoxins; Dose-Response Relationship, Drug; Membrane Potentials; Mice; Models, Molecular; Mutant Chimeric Proteins; Patch-Clamp Techniques; Potassium Channel Blockers; Protein Conformation; Shab Potassium Channels; Shaw Potassium Channels

The marine polycyclic-ether toxin gambierol and 1-butanol (n-alkanol) inhibit Shaker-type Kv channels by interfering with the gating machinery. Competition experiments indicated that both compounds do not share an overlapping binding site but gambierol is able to affect 1-butanol affinity for Shaker through an allosteric effect. Furthermore, the Shaker-P475A mutant, which inverses 1-butanol effect, is inhibited by gambierol with nM affinity. Thus, gambierol and 1-butanol inhibit Shaker-type Kv channels via distinct parts of the gating machinery.

Topics: 1-Butanol; Binding Sites; Ciguatoxins; Ion Channel Gating; Potassium Channel Blockers; Shaker Superfamily of Potassium Channels

Gambierol is a marine polycyclic ether toxin, produced along with ciguatoxin congeners by the dinoflagellate Gambierdiscus toxicus. We have recently reported that two truncated skeletal analogs of gambierol comprising the EFGH- and BCDEFGH-rings of the parent compound showed similar potency to gambierol on voltage-gated potassium channels (Kv) inhibition in neurons. Gambierol and its truncated analogs share the main crucial elements for biological activity, which are the C28=C29 double bond within the H-ring and the unsaturated side chain. Since Kv channels are critical for the regulation of calcium signaling, proliferation, secretion and migration in human T lymphocytes, we evaluated the activity of both the tetracyclic and heptacyclic analogs of gambierol on potassium currents in resting T lymphocyte and their effects on interleukin-2 (IL-2) release and gene expression in activated T lymphocytes. The results presented in this work clearly demonstrate that both truncated analogs of gambierol inhibit Kv channels present in resting T lymphocytes (Kv1.3) and prevented lymphocyte activation by concanavalin A. The main effects of the heptacyclic and tetracyclic analogs of gambierol in human T cells are: (1) inhibition of potassium channels in resting and concanavalin-activated T cells in the nanomolar range, (2) inhibition of IL-2 release from concanavalin-activated T cells and (3) negatively affect the expression of genes involved in cell proliferation and immune response observed in concanavalin-activated lymphocytes. These results together with the lack of toxicity in this cellular model, indicates that both analogs of gambierol have additional potential for the development of therapeutic tools in autoimmune diseases.

Topics: Cell Proliferation; Cells, Cultured; Ciguatoxins; Dose-Response Relationship, Drug; Gene Expression Regulation; Humans; Interleukin-2; Lymphocyte Activation; Macrolides; Membrane Potentials; Potassium; Potassium Channel Blockers; Potassium Channels, Voltage-Gated; T-Lymphocytes; Transcription, Genetic

Gambierol and its heptacyclic and tetracyclic analogs were tested for inhibitory activity against the human voltage-gated potassium channel Kv1.2 (hKv1.2), which was stably expressed in Chinese hamster ovary (CHO) cells. Gambierol, the heptacyclic analog, and the tetracyclic analog inhibited the potassium current evoked by a step pulse from -80mV to 40mV. The IC50 values for the three compounds were 0.75±0.15nM, 7.6±1.2nM, and 28±4.0nM (the mean±SEM, n=3), respectively. The cytotoxic activity was examined in order to assess a relationship between cytotoxicity and inhibition of the hKv1.2. The IC50 values for gambierol, the heptacyclic analog, and the tetracyclic analog in the wild-type CHO cells were 95±7.1μM, 6.5±0.8μM (the mean±SEM, n=3), and >100μM (n=3), respectively, whereas those in the CHO cells stably expressing hKv1.2 were 78±5.8μM, 6.0±1.0μM (the mean±SEM, n=3), and >100μM (n=3). These results suggested that cytotoxicity is not triggered by inhibition of the human Kv1.2. The electrophysiological recording at the resting potential in the presence of gambierol, the heptacyclic analog, and the tetracyclic analog revealed the dose-dependent leak current, which was largest when the heptacyclic analog was administered to the cells. We thus propose that the leak current induced by these compounds might cause a fatal effect on the cultured cells.

Topics: Animals; Cell Proliferation; CHO Cells; Ciguatoxins; Cricetinae; Cricetulus; HEK293 Cells; Humans; Kv1.2 Potassium Channel; Membrane Potentials; Protein Binding; Recombinant Proteins

Gambierol is a marine polycyclic ether toxin produced by the marine dinoflagellate Gambierdiscus toxicus and is a member of the ciguatoxin toxin family. Gambierol has been demonstrated to be either a low-efficacy partial agonist/antagonist of voltage-gated sodium channels or a potent blocker of voltage-gated potassium channels (Kvs). Here we examined the influence of gambierol on intact cerebrocortical neurons. We found that gambierol produced both a concentration-dependent augmentation of spontaneous Ca(2+) oscillations, and an inhibition of Kv channel function with similar potencies. In addition, an array of selective as well as universal Kv channel inhibitors mimicked gambierol in augmenting spontaneous Ca(2+) oscillations in cerebrocortical neurons. These data are consistent with a gambierol blockade of Kv channels underlying the observed increase in spontaneous Ca(2+) oscillation frequency. We also found that gambierol produced a robust stimulation of phosphorylation of extracellular signal-regulated kinases 1/2 (ERK1/2). Gambierol-stimulated ERK1/2 activation was dependent on both inotropic [N-methyl-d-aspartate (NMDA)] and type I metabotropic glutamate receptors (mGluRs) inasmuch as MK-801 [NMDA receptor inhibitor; (5S,10R)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate], S-(4)-CGP [S-(4)-carboxyphenylglycine], and MTEP [type I mGluR inhibitors; 3-((2-methyl-4-thiazolyl)ethynyl) pyridine] attenuated the response. In addition, 2-aminoethoxydiphenylborane, an inositol 1,4,5-trisphosphate receptor inhibitor, and U73122 (1-[6-[[(17b)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione), a phospholipase C inhibitor, both suppressed gambierol-induced ERK1/2 activation, further confirming the role of type I mGluR-mediated signaling in the observed ERK1/2 activation. Finally, we found that gambierol produced a concentration-dependent stimulation of neurite outgrowth that was mimicked by 4-aminopyridine, a universal potassium channel inhibitor. Considered together, these data demonstrate that gambierol alters both Ca(2+) signaling and neurite outgrowth in cerebrocortical neurons as a consequence of blockade of Kv channels.

Topics: Animals; Calcium; Calcium Signaling; Cells, Cultured; Cerebral Cortex; Ciguatoxins; Dose-Response Relationship, Drug; Mice; Neurons; Potassium Channel Blockers; Potassium Channels, Voltage-Gated

Voltage-gated potassium (Kv) and sodium (Nav) channels are key determinants of cellular excitability and serve as targets of neurotoxins. Most marine ciguatoxins potentiate Nav channels and cause ciguatera seafood poisoning. Several ciguatoxins have also been shown to affect Kv channels, and we showed previously that the ladder-shaped polyether toxin gambierol is a potent Kv channel inhibitor. Most likely, gambierol acts via a lipid-exposed binding site, located outside the K(+) permeation pathway. However, the mechanism by which gambierol inhibits Kv channels remained unknown. Using gating and ionic current analysis to investigate how gambierol affected S6 gate opening and voltage-sensing domain (VSD) movements, we show that the resting (closed) channel conformation forms the high-affinity state for gambierol. The voltage dependence of activation was shifted by >120 mV in the depolarizing direction, precluding channel opening in the physiological voltage range. The (early) transitions between the resting and the open state were monitored with gating currents, and provided evidence that strong depolarizations allowed VSD movement up to the activated-not-open state. However, for transition to the fully open (ion-conducting) state, the toxin first needed to dissociate. These dissociation kinetics were markedly accelerated in the activated-not-open state, presumably because this state displayed a much lower affinity for gambierol. A tetrameric concatemer with only one high-affinity binding site still displayed high toxin sensitivity, suggesting that interaction with a single binding site prevented the concerted step required for channel opening. We propose a mechanism whereby gambierol anchors the channel's gating machinery in the resting state, requiring more work from the VSD to open the channel. This mechanism is quite different from the action of classical gating modifier peptides (e.g., hanatoxin). Therefore, polyether toxins open new opportunities in structure-function relationship studies in Kv channels and in drug design to modulate channel function.

Topics: Action Potentials; Animals; Binding Sites; Cell Line; Ciguatoxins; Fibroblasts; Ion Channel Gating; Kinetics; Membrane Potentials; Mice; Permeability; Potassium Channels, Voltage-Gated; Shaw Potassium Channels; Structure-Activity Relationship

Gambierol is a potent neurotoxin that belongs to the family of marine polycyclic ether natural products and primarily targets voltage-gated potassium channels (K(v) channels) in excitable membranes. Previous work in the chemistry of marine polycyclic ethers has suggested the critical importance of the full length of polycyclic ether skeleton for potent biological activity. Although we have previously investigated structure-activity relationships (SARs) of the peripheral functionalities of gambierol, it remained unclear whether the whole polycyclic ether skeleton is needed for its cellular activity. In this work, we designed and synthesized two truncated skeletal analogues of gambierol comprising the EFGH- and BCDEFGH-rings of the parent compound, both of which surprisingly showed similar potency to gambierol on voltage-gated potassium channels (K(v)) inhibition. Moreover, we examined the effect of these compounds in an in vitro model of Alzheimer's disease (AD) obtained from triple transgenic (3xTg-AD) mice, which expresses amyloid beta (Aβ) accumulation and tau hyperphosphorylation. In vitro preincubation of the cells with the compounds resulted in significant inhibition of K(+) currents, a reduction in the extra- and intracellular levels of Aβ, and a decrease in the levels of hyperphosphorylated tau. In addition, pretreatment with these compounds reduced the steady-state level of the N-methyl-D-aspartate (NMDA) receptor subunit 2A without affecting the 2B subunit. The involvement of glutamate receptors was further suggested by the blockage of the effect of gambierol on tau hyperphosphorylation by glutamate receptor antagonists. The present study constitutes the first discovery of skeletally simplified, designed polycyclic ethers with potent cellular activity and demonstrates the utility of gambierol and its synthetic analogues as chemical probes for understanding the function of K(v) channels as well as the molecular mechanism of Aβ metabolism modulated by NMDA receptors.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Cell Survival; Cells, Cultured; Cerebral Cortex; Ciguatoxins; Humans; Mice; Mice, Transgenic; Neurons; Potassium Channels, Voltage-Gated; Receptors, N-Methyl-D-Aspartate; tau Proteins

The polycyclic ether class of marine natural products has attracted the attention of researchers due to their complex and large chemical structures and diverse biological activities. Gambierol is a marine polycyclic ether toxin, first isolated along with ciguatoxin congeners from the dinoflagellate Gambierdiscus toxicus. The parent compound gambierol and the analogues evaluated in this work share the main crucial elements for biological activity, previously described to be the C28=C29 double bond within the H ring and the unsaturated side chain [Fuwa, H., Kainuma, N., Tachibana, K., Tsukano, C., Satake, M., and Sasaki, M. (2004) Diverted total synthesis and biological evaluation of gambierol analogues: Elucidation of crucial structural elements for potent toxicity. Chem. Eur. J. 10, 4894-4909]. With the aim to gain a deeper understanding of the cellular mechanisms involved in the biological activity of these compounds, we compared its activity in primary cultured neurons. The three compounds inhibited voltage-gated potassium channels (Kv) in a concentration-dependent manner and with similar potency, caused a small inhibition of voltage-gated sodium channels (Nav), and evoked cytosolic calcium oscillations. Moreover, the three compounds elicited a "loss of function" effect on Kv channels at concentrations of 0.1 nM. Additionally, both the tetracyclic and the heptacyclic derivatives of gambierol elicited synchronous calcium oscillations similar to those previously described for gambierol in cultured cerebellar neurons. Neither gambierol nor its tetracyclic derivative elicited cell toxicity, while the heptacyclic analogue caused a time-dependent decrease in cell viability. Neither the tetracyclic nor the heptacyclic analogues of gambierol exhibited lethality in mice after ip injection of 50 or 80 μg/kg of each compound. Altogether, the results presented in this work support an identical mechanism of action for gambierol and its tetracyclic and heptacyclic analogues and indicate a "loss of function" effect on potassium channels even after administration of the three compounds at subnanomolar concentrations. In addition, because gambierol is known to stabilize the closed state of Kv3 channels, the results presented in this paper may have implications for understanding of channel functions and for future development of therapies against ciguatera poisoning and potassium channel-related diseases.

Topics: Animals; Calcium; Cell Survival; Cells, Cultured; Cerebellum; Ciguatoxins; Dinoflagellida; Electrophysiological Phenomena; Mice; Neurons; Potassium; Potassium Channels, Voltage-Gated; Sodium; Structure-Activity Relationship

Many microalgae produce compounds that exhibit potent biological activities. Ingestion of marine organisms contaminated with those toxins results in seafood poisonings. In many cases, the lack of toxic material turns out to be an obstacle to make the toxicological investigations needed. In this study, we evaluate the cytotoxicity of several marine toxins on neuroblastoma cells, focusing on gambierol and its effect on cytosolic calcium levels. In addition, we compared the effects of this toxin with ciguatoxin, brevetoxin, and gymnocin-A, with which gambierol shares a similar ladder-like backbone, as well as with polycavernoside A analogue 5, a glycosidic macrolide toxin. For this purpose, different fluorescent dyes were used: Fura-2 to monitor variations in cytosolic calcium levels, Alamar Blue to detect cytotoxicity, and Oregon Green 514 Phalloidin to quantify and visualize modifications in the actin cytoskeleton. Data showed that, while gambierol and ciguatoxin were successful in producing a calcium influx in neuroblastoma cells, gymnocin-A was unable to modify this parameter. Nevertheless, none of the toxins induced morphological changes or alterations in the actin assembly. Although polycavernoside A analogue 5 evoked a sharp reduction of the cellular metabolism of neuroblastoma cells, gambierol scarcely reduced it, and ciguatoxin, brevetoxin, and gymnocin-A failed to produce any signs of cytotoxicity. According to this, sharing a similar polycyclic ether backbone is not enough to produce the same effects on neuroblastoma cells; therefore, more studies should be carried out with these toxins, whose effects may be being underestimated.

Topics: Actins; Calcium; Cell Line, Tumor; Ciguatoxins; Cytosol; Dinoflagellida; Humans; Marine Toxins

Gambierol is a marine polyether ladder toxin derived from the dinoflagellate Gambierdiscus toxicus. To date, gambierol has been reported to act either as a partial agonist or as an antagonist of sodium channels or as a blocker of voltage-dependent potassium channels. In this work, we examined the cellular effect of gambierol on cytosolic calcium concentration, membrane potential and sodium and potassium membrane currents in primary cultures of cerebellar granule cells. We found that at concentrations ranging from 0.1 to 30 microM, gambierol-evoked [Ca(2+)]c oscillations that were dependent on the presence of extracellular calcium, irreversible and highly synchronous. Gambierol-evoked [Ca(2+)]c oscillations were completely eliminated by the NMDA receptor antagonist APV and by riluzole and delayed by CNQX. In addition, the K(+) channel blocker 4-aminopyridine (4-AP)-evoked cytosolic calcium oscillations in this neuronal system that were blocked by APV and delayed in the presence of CNQX. Electrophysiological recordings indicated that gambierol caused membrane potential oscillations, decreased inward sodium current amplitude and decreased also outward IA and IK current amplitude. The results presented here point to a common mechanism of action for gambierol and 4-AP and indicate that gambierol-induced oscillations in cerebellar neurons are most likely secondary to a blocking action of the toxin on voltage-dependent potassium channels and hyperpolarization of sodium current activation.

Topics: Animals; Calcium; Cells, Cultured; Cerebellum; Ciguatoxins; Cytoplasmic Granules; Glutamic Acid; Mice; Sodium Channels

Gambierol was isolated as a neurotoxin from the cultured cells of the ciguatera causative dinoflagellate Gambierdiscus toxicus and classified as a member of the polycyclic ether family of marine toxins. The structure consists of a ladder-shaped trans-fused octacyclic ring system that includes 18 stereogenic centers, two 1,3-diaxial dimethyl-substituted tetrahydropyranyl rings, and a partially conjugated triene side chain. The total synthesis of gambierol has been achieved by utilizing an oxiranyl anion strategy in an iterative manner. Synthetic highlights of this route include direct carbon-carbon formation on epoxides, sulfonyl-assisted 6-endo cyclization, and an expansion reaction of the tetrahydropyranyl rings to oxepanes to forge the polycyclic architecture of the target molecule.

Topics: Anions; Ciguatoxins; Cyclization; Dinoflagellida; Epoxy Compounds; Ethylene Oxide; Marine Toxins; Molecular Structure; Neurotoxins; Polycyclic Compounds; Structure-Activity Relationship

Gambierol is a complex marine toxin first isolated with ciguatoxins from cell cultures of the toxic dinoflagellate Gambierdiscus toxicus. Despite the chemical complexity of the polycyclic ether toxin, the total successful synthesis of gambierol has been achieved by different chemical strategies. In the present work the effects of synthetic gambierol on mouse and frog skeletal neuromuscular preparations and Xenopus skeletal myocytes have been studied. Gambierol (0.1-5 muM) significantly increased isometric twitch tension in neuromuscular preparations stimulated through the motor nerve. Less twitch augmentation was observed in directly stimulated muscles when comparing twitch tension-time integrals obtained by nerve stimulation. Also, gambierol induced small spontaneous muscle contraction originating from presynaptic activity that was completely inhibited by d-tubocurarine. Gambierol slowed the rate of muscle action potential repolarization, triggered spontaneous and/or repetitive action potentials, and neither affected action potential amplitude nor overshoot in skeletal muscle fibers. These results suggest that gambierol through an action on voltage-gated K(+) channels prolongs the duration of action potentials, enhances the extent and time course of Ca(2+) release from the sarcoplasmic reticulum, and increases twitch tension generation. Further evidence is provided that gambierol at sub-micromolar concentrations blocks a fast inactivating outward K(+) current that is responsible for action potential prolongation in Xenopus skeletal myocytes.

Topics: Action Potentials; Animals; Ciguatoxins; Female; Mice; Muscle Contraction; Muscle, Skeletal; Patch-Clamp Techniques; Potassium Channel Blockers; Tubocurarine; Xenopus

We have used proteomic analyses to probe the responses induced by a pair of marine biotoxins, okadaic acid (OA) and gambierol (GB), added alone or in combination to a cultured cell line and carried out a preliminary investigation into the possible interactions between toxins possessing two different molecular mechanisms of action at a cellular level. When MCF-7 cells were treated with OA, we found that cellular levels of 30 proteins were significantly affected, including several isoforms of nonphosphorylated and phosphorylated hsp 27, as well as enzymes involved in the maintenance of nucleoside triphosphate pools and the control of redox states of the cell. When we repeated our analysis using GB, nine proteins were significantly affected, including some isoforms of nonphosphorylated hsp 27, as well as semenogelin-1, myosin-7, and the ATP synthase subunit delta. The combined addition of OA and GB to MCF-7 cells, in turn, affected 14 proteins, including some isoforms of nonphosphorylated and phosphorylated hsp 27, as well as myosin-7, the ATP synthase subunit delta, and enzymes involved in the control of redox states of the cell. If components affected by either OA or GB, as well as by the combined treatment, were classified according to the detected changes, two sets of data were obtained, including the components whose levels were found affected by the combined treatment, regardless of the effect observed after addition of only one agent, and those that had been found affected in cells that had been challenged with only one toxin but not when cells had been subjected to the combined treatment. Multiple patterns of responses to the toxin mixture were recorded in the two sets, consisting of both independent and interacting actions, among which we detected synergistic, similar, and antagonistic effects.

Topics: Amino Acid Sequence; Cell Line, Tumor; Ciguatoxins; Humans; Intracellular Signaling Peptides and Proteins; Marine Toxins; Molecular Sequence Data; Okadaic Acid; Protein Isoforms; Protein Serine-Threonine Kinases; Proteomics

Gambierol is a marine polycyclic ether toxin belonging to the group of ciguatera toxins. It does not activate voltage-gated sodium channels (VGSCs) but inhibits Kv1 potassium channels by an unknown mechanism. While testing whether Kv2, Kv3, and Kv4 channels also serve as targets, we found that Kv3.1 was inhibited with an IC(50) of 1.2 +/- 0.2 nM, whereas Kv2 and Kv4 channels were insensitive to 1 microM gambierol. Onset of block was similar from either side of the membrane, and gambierol did not compete with internal cavity blockers. The inhibition did not require channel opening and could not be reversed by strong depolarization. Using chimeric Kv3.1-Kv2.1 constructs, the toxin sensitivity was traced to S6, in which T427 was identified as a key determinant. In Kv3.1 homology models, T427 and other molecular determinants (L348, F351) reside in a space between S5 and S6 outside the permeation pathway. In conclusion, we propose that gambierol acts as a gating modifier that binds to the lipid-exposed surface of the pore domain, thereby stabilizing the closed state. This site may be the topological equivalent of the neurotoxin site 5 of VGSCs. Further elucidation of this previously undescribed binding site may explain why most ciguatoxins activate VGSCs, whereas others inhibit voltage-dependent potassium (Kv) channels. This previously undescribed Kv neurotoxin site may have wide implications not only for our understanding of channel function at the molecular level but for future development of drugs to alleviate ciguatera poisoning or to modulate electrical excitability in general.

Topics: Amino Acid Sequence; Binding Sites; Ciguatoxins; Marine Toxins; Models, Molecular; Molecular Sequence Data; Potassium Channels, Voltage-Gated; Sequence Homology, Amino Acid

The total synthesis of gambierol has been achieved utilizing an oxiranyl anion strategy in an iterative manner. Synthetic highlights of this route include direct carbon-carbon formation on epoxides, sulfonyl-assisted 6-endo cyclization, and expansion reaction of tetrahydropyranyl rings to oxepanes to forge the polycyclic architecture of the target molecule.

Topics: Ciguatoxins; Cyclization; Molecular Conformation; Stereoisomerism

In this study, we pharmacologically characterized gambierol, a marine polycyclic ether toxin which is produced by the dinoflagellate Gambierdiscus toxicus. Besides several other polycyclic ether toxins like ciguatoxins, this scarcely studied toxin is one of the compounds that may be responsible for ciguatera fish poisoning (CFP). Unfortunately, the biological target(s) that underlies CFP is still partly unknown. Today, ciguatoxins are described to specifically activate voltage-gated sodium channels by interacting with their receptor site 5. But some dispute about the role of gambierol in the CFP story shows up: some describe voltage-gated sodium channels as the target, while others pinpoint voltage-gated potassium channels as targets. Since gambierol was never tested on isolated ion channels before, it was subjected in this work to extensive screening on a panel of 17 ion channels: nine cloned voltage-gated ion channels (mammalian Na(v)1.1-Na(v)1.8 and insect Para) and eight cloned voltage-gated potassium channels (mammalian K(v)1.1-K(v)1.6, hERG and insect ShakerIR) expressed in Xenopus laevis oocytes using two-electrode voltage-clamp technique. All tested sodium channel subtypes are insensitive to gambierol concentrations up to 10 microM. In contrast, K(v)1.2 is the most sensitive voltage-gated potassium channel subtype with almost full block (>97%) and an half maximal inhibitory concentration (IC(50)) of 34.5 nM. To the best of our knowledge, this is the first study where the selectivity of gambierol is tested on isolated voltage-gated ion channels. Therefore, these results lead to a better understanding of gambierol and its possible role in CFP and they may also be useful in the development of more effective treatments.

Topics: Animals; Ciguatera Poisoning; Ciguatoxins; Dinoflagellida; Dose-Response Relationship, Drug; Oocytes; Potassium Channel Blockers; Potassium Channels, Voltage-Gated; Sodium Channels; Xenopus laevis

The marine toxin gambierol, a polyether ladder toxin derived from the marine dinoflagellate Gambierdiscus toxicus, was evaluated for interaction with voltage-gated sodium channels (VGSCs) in cerebellar granule neuron (CGN) cultures. At concentrations ranging from 10 nM to 10 microM, gambierol alone had no effect on the intracellular Ca2+ concentration [Ca2+]i of exposed CGN cultures. Furthermore, there was no evidence of neurotoxicity in CGN cultures exposed for 2 h to gambierol (1 nM-10 microM). However, gambierol was a potent inhibitor (IC50 = 189 nM) of the elevation of [Ca2+]i that accompanies exposure of CGN cultures to the VGSC activator brevetoxin-2 (PbTx-2). To further explore the potential interaction of gambierol with VGSCs, the influence of gambierol on PbTx-2-induced neurotoxicity was assessed. Gambierol reduced the PbTx-2-induced efflux of lactate dehydrogenase in exposed CGN cultures in a concentration-dependent manner (IC50 = 471 nM). It is noteworthy that the potencies of gambierol as an inhibitor of both PbTx-2-induced Ca2+ influx and cytotoxicity were coincident. Finally, the inhibitory effects of gambierol on PbTx-2-induced elevation of [Ca2+]i were compared with those of brevenal, a natural inhibitor of the toxic effects of brevetoxin isolated from cultures of Karina brevis. Like gambierol, brevenal inhibited PbTx-2-induced elevation of [Ca2+]i in a concentration-dependent manner (IC50 = 108.6 nM). These results provide evidence for gambierol acting as a functional antagonist of neurotoxin site 5 on neuronal VGSCs.

Topics: Animals; Animals, Newborn; Binding Sites; Calcium; Cell Survival; Cells, Cultured; Cerebellum; Ciguatoxins; Ethers, Cyclic; Marine Toxins; Molecular Structure; Neurons; Oxocins; Polycyclic Compounds; Rats; Rats, Sprague-Dawley; Sodium Channel Blockers; Sodium Channels; Synaptosomes

Ciguatera fish poisoning and neurotoxic shellfish poisoning are distinct clinical entities characterized by gastrointestinal and neurological disturbances, following the consumption of certain reef fish and shellfish containing toxic polyether compounds sporadically present in certain toxic marine dinoflagellates. The biotransformation and bioaccumulation of gambierol and brevetoxin, and their congeners, are believed to be involved in the pathogenesis of these "food-chain diseases", for which no effective treatments are available. Here, we describe for the first time the potent effect of gambierol and brevetoxin on TRPV1 channels, a key player in thermal and pain sensation. Our findings may lead to promising new therapeutic interventions.

Topics: Animals; Ciguatera Poisoning; Ciguatoxins; Ethers, Cyclic; Humans; Marine Toxins; Oxocins; Patch-Clamp Techniques; Polycyclic Compounds; Shellfish; TRPV Cation Channels; Xenopus laevis

Gambierol, a representative of the marine ladder toxin family, consists of eight ether rings, 18 stereocenters, and two challenging pyranyl rings having methyl groups that are in a 1,3-diaxial orientation to one another. Herein we describe the generation of gambierol's A-C and F-H ring systems and demonstrate the versatility of the glycosyl anhydride, enol ether-olefin RCM strategy to fused polycyclic ethers. This work has both enabled us to generate sufficient quantities of the gambierol precursors and has enabled us to better understand the chemical transformations that were key to these efforts. Fundamental work included efforts to C-glycosides and C-ketosides, Claisen rearrangements, and enol ether-olefin RCM reactions.

Topics: Ciguatera Poisoning; Ciguatoxins; Cyclization; Epoxy Compounds; Ethers; Ethers, Cyclic; Glycosides; Indicators and Reagents; Magnetic Resonance Spectroscopy; Marine Toxins; Polycyclic Compounds

The preceding manuscript detailed our synthesis of the gambierol A-C and F-H ring precursors. Reported herein is a description of the coupling of the two precursors and the conversion of the coupled material into gambierol. Coupling of the subunits involved ester formation, enol ether RCM, and mixed thioketal formation and reduction. By employing this strategy we were able to bring highly advanced subunits into the coupling and, as a result, we were able to minimize the number of post-coupling transformations required to complete gambierol. At the completion of the synthesis, we generated 7.5 mg (1.5 % overall yield) of (-)-gambierol in 44 steps (longest linear sequence).

Topics: Catalysis; Ciguatoxins; Cyclization; Esters; Ethers, Cyclic; Indicators and Reagents; Polycyclic Compounds

The combination of a trimethylsilyl group, a Brønsted base, a fluoride source, and a hydroxylic solvent enables the first construction of the tetrad of tetrahydropyran rings found in the majority of the ladder polyether natural products by way of a cascade of epoxide-opening events that emulates the final step of Nakanishi's proposed biosynthetic pathway. The trimethylsilyl group disappears during the course of the cascade, and thus these are the first epoxide ring-opening cascades that afford ladder polyether subunits containing no directing groups at the end of the cascade.

Topics: Ciguatoxins; Epoxy Compounds; Ethers, Cyclic; Furans; Molecular Conformation; Mollusk Venoms; Oxocins; Polycyclic Compounds; Polymers

Gambierol is a polycyclic ether toxin with the same biogenetic origin as ciguatoxins. Gambierol has been associated with neurological symptoms in humans even though its mechanism of action has not been fully characterized.. We studied the effect of gambierol in human neuroblastoma cells by using bis-oxonol to measure membrane potential and FURA-2 to monitor intracellular calcium.. We found that this toxin: i) produced a membrane depolarization, ii) potentiated the effect of veratridine on membrane potential iii) decreased ciguatoxin-induced depolarization and iv) increased cytosolic calcium in neuroblastoma cells.. These results indicate that gambierol modulate ion fluxes by acting as a partial agonist of sodium channels.

Topics: Calcium; Cell Polarity; Ciguatoxins; Ethers, Cyclic; Humans; Membrane Potentials; Neuroblastoma; Neurons; Polycyclic Compounds; Saxitoxin; Sodium Channels; Sodium-Calcium Exchanger; Tumor Cells, Cultured; Veratridine

The marine dinoflagellate Gambierdiscus toxicus produces highly lipophilic, polycyclic ether toxins that cause a seafood poisoning called ciguatera. Ciguatoxins (CTXs) and gambierol represent the two major causative agents of ciguatera intoxication, which include taste alterations (dysgeusiae). However, information on the mode of action of ciguatera toxins in taste cells is scarce. Here, we have studied the effect of synthetic CTX3C (a CTX congener) on mouse taste cells. By using the patch-clamp technique to monitor membrane ion currents, we found that CTX3C markedly affected the operation of voltage-gated Na(+) channels but was ineffective on voltage-gated K(+) channels. This result was the exact opposite of what we obtained earlier with gambierol, which inhibits K(+) channels but not Na(+) channels. Thus, CTXs and gambierol affect with high potency the operation of separate classes of voltage-gated ion channels in taste cells. Our data suggest that taste disturbances reported in ciguatera poisoning might be due to the ability of ciguatera toxins to interfere with ion channels in taste buds.

Topics: Animals; Ciguatoxins; Ethers, Cyclic; Ion Channel Gating; Mice; Organ Culture Techniques; Patch-Clamp Techniques; Polycyclic Compounds; Potassium Channels, Voltage-Gated; Sodium Channels; Taste; Taste Buds

This communication describes the total synthesis of the marine polyether toxin, gambierol. This work couples our iterative C-glycoside/enol ether-olefin metathesis strategy to the subunits with a unique olefin metathesis/carbonyl olefination reaction to bring the subunits together.

Topics: Animals; Ciguatoxins; Dinoflagellida; Ethers, Cyclic; Glycosides; Polycyclic Compounds

Ciguatera is a food poisoning caused by toxins of Gambierdiscus toxicus, a marine dinoflagellate. The neurological features of this intoxication include sensory abnormalities, such as paraesthesia, heightened nociperception, and also taste alterations. Here, we have evaluated the effect of gambierol, one of the possible ciguatera toxins, on the voltage-gated ion currents in taste cells. Taste cells are excitable cells endowed with voltage-gated Na+, K+, and Cl- currents (I(Na), I(K), and I(Cl), respectively). By applying the patch-clamp technique to single cells in isolated taste buds obtained from the mouse vallate papilla, we have recorded such currents and determined the effect of bath-applied gambierol. We found that this toxin markedly inhibited I(K) in the nanomolar range (IC50 of 1.8 nM), whereas it showed no significant effect on I(Na) or I(Cl) even at high concentration (1 microM). The block of I(K) was irreversible even after a 50-min wash. In addition to affecting the current amplitude, we found that gambierol significantly altered both the activation and inactivation processes of I(K). In conclusion, unlike other toxins involved in ciguatera, such as ciguatoxins, which affect the functioning of voltage-gated sodium channels, the preferred molecular target of gambierol is the voltage-gated potassium channel, at least in taste cells. Voltage-gated potassium currents play an important role in the generation of the firing pattern during chemotransduction. Thus, gambierol may alter action potential discharge in taste cells and this could be associated with the taste alterations reported in the clinical literature.

Topics: Action Potentials; Animals; Cells, Cultured; Ciguatoxins; Ethers, Cyclic; Mice; Patch-Clamp Techniques; Polycyclic Compounds; Potassium Channels, Voltage-Gated; Sodium Channels; Taste Buds

A fully functionalized ABCD ring moiety of gambierol, a marine polycyclic ether toxin, was synthesized by the use of the oxiranyl anion strategy and reductive cycloetherification of a beta,delta-dihydroxy ketone.

Topics: Animals; Ciguatoxins; Dinoflagellida; Ethers, Cyclic; Marine Toxins; Molecular Structure; Polycyclic Compounds; Stereoisomerism

Gambierol is a polycyclic ether toxin, which has been isolated from the marine dinoflagellate Gambierdiscus toxicus. A series of gambierol analogues have been prepared from an advanced intermediate of our total synthesis of gambierol and investigated for their toxicity against mice, thus providing the first systematic structure-activity relationships (SAR) of this polycyclic ether class of marine toxin. The SAR studies described herein clearly indicate that 1) the C28=C29 double bond within the H ring and the unsaturated side chain are the crucial structural elements required for exerting potent biological activity and 2) the C1 and C6 hydroxy groups, the C30 methyl group, and the C37=C38 double bond have little influence on the degree of neurotoxicity against mice.

Topics: Animals; Ciguatoxins; Ethers, Cyclic; Male; Mice; Mice, Inbred Strains; Models, Molecular; Polycyclic Compounds; Structure-Activity Relationship

The convergent total synthesis of gambierol (1) is described. The octacyclic ether framework of 1 was constructed via the intramolecular allylation of alpha-chloroacetoxy ether followed by ring-closing metathesis. A modified Stille coupling was successfully applied to the synthesis of the triene side chain.

Topics: Animals; Ciguatoxins; Dinoflagellida; Ethers, Cyclic; Marine Toxins; Neurotoxins; Polycyclic Compounds

[structure: see text] This manuscript describes our synthesis of the F-H subunit of gambierol. In addition to the synthesis of the tricycle, of note is an interesting protecting group influence on the generation of a C(23) C-glycoside as well as the use of ring-closing metathesis to generate a tetrasubstituted enol ether.

Topics: Animals; Ciguatoxins; Cyclization; Dinoflagellida; Ethers, Cyclic; Glycosides; Indicators and Reagents; Oxidation-Reduction; Polycyclic Compounds

Brevetoxins (BTXs) and ciguatoxins (CTXs) bind to site 5 of the voltage-gated sodium channel of excitable membranes. In the present study, we performed a competitive inhibition assay with other structurally distinct naturally occurring polyethers using isotope-labeled dihydro BTX-B ([3H]PbTx-3), which showed, for the first time, that gambierol and gambieric acid-A inhibit the binding of [3H]PbTx-3 while yessotoxins are inactive in this assay. The inhibition assay also suggested that there is a significant relationship between the size of the polycyclic region and inhibitory activity. Interestingly, the acute mouse toxicities of the compounds do not correspond directly to their inhibitory activities. These observations will serve as a guide for designing artificial polyethers with desired activity.

Topics: Animals; Binding, Competitive; Brain; Ciguatoxins; Drug Interactions; Ethers, Cyclic; Marine Toxins; Molecular Structure; Mollusk Venoms; Oxocins; Polycyclic Compounds; Protein Binding; Rats; Sodium Channels; Synaptosomes

Gambierol is a polycyclic ether toxin, isolated as a toxic constituent from the marine dinoflagellate Gambierdiscus toxicus. We describe here the synthesis and biological evaluation of structural analogues of gambierol. The present preliminary structure-activity relationship studies clearly indicate that the H ring functionality and the unsaturated side chain of gambierol are crucial for its potent toxicity.

Topics: Animals; Ciguatoxins; Dinoflagellida; Ethers, Cyclic; Indicators and Reagents; Lethal Dose 50; Mice; Polycyclic Compounds; Structure-Activity Relationship

The convergent total syntheses of gambierol (1) and 16-epi-gambierol (2) have been achieved. The ABC and FGH ring segments 4 and 5 were prepared from known compounds 6 and 13, respectively, by linear manners. The fragments prepared were connected by our own synthetic strategy including the intramolecular allylation of alpha-acetoxy ether followed by ring-closing metathesis to furnish the octacyclic ether 3. The diiodoalkene 45, prepared from 3, was converted to the Z-iodoalkene 50 via a novel and stereoselective hydrogenolysis followed by deprotection. Construction of the triene side chain was performed by the modified Stille coupling of 50 with the Z-vinylic stannane 41 to afford 1. The similar transformations were carried out on the epimeric octacycle 34 to give 2, which showed no toxicity against mice at the concentration of 14 mg/kg.

Topics: Animals; Ciguatoxins; Ethers, Cyclic; Isomerism; Lethal Dose 50; Mice; Models, Molecular; Nuclear Magnetic Resonance, Biomolecular; Polycyclic Compounds

Gambierol was isolated from Gambierdiscus toxicus, which causes ciguatera fish poisoning. The acute toxicological effects induced in mice by synthesized gambierol were studied. The lethal doses were about 80 microg/kg by i.p. and i.v., and 150 microg/kg by p.o. The main injury by this toxin was observed in the lung, and secondary in the heart, resulting in systemic congestion. Another toxic effect was seen in the stomach, inducing hypersecretion and ulceration. With survival from the severe stage during the initial 3 h, recovery was favorable, especially after 4 days. Additional effects were not evident during 1-week post-administration observation.

Topics: Administration, Oral; Animals; Ciguatera Poisoning; Ciguatoxins; Dinoflagellida; Disease Models, Animal; Ethers, Cyclic; Injections, Intraperitoneal; Injections, Intravenous; Lethal Dose 50; Lung; Male; Mice; Mice, Inbred ICR; Myocardium; Polycyclic Compounds; Stomach

[structure: see text] The first total synthesis of gambierol, a marine polycyclic ether toxin, has been achieved. The synthesis features the Pd(PPh3)4/CuCl/LiCl-promoted Stille coupling for the stereoselective construction of the sensitive triene side chain that includes a conjugated (Z,Z)-diene moiety.

Topics: Animals; Ciguatoxins; Dinoflagellida; Ethers, Cyclic; Marine Toxins; Palladium; Polycyclic Compounds; Polyenes; Stereoisomerism; Vinyl Compounds

The first total synthesis of (-)-gambierol (1), a marine polycyclic ether toxin, has been achieved. Key features of the successful synthesis include (1) a convergent union of the ABC and EFGH ring fragments (5 and 6, respectively) via our developed B-alkyl Suzuki-Miyaura cross-coupling strategy leading to the octacyclic polyether core 4 and (2) a late-stage introduction of the sensitive triene side chain by use of Pd(PPh(3))(4)/CuCl/LiCl-promoted Stille coupling. The ABC ring fragment 5 was synthesized in a linear manner (B --> AB --> ABC), wherein the A ring was formed by intramolecular hetero-Michael reaction and the C ring was constructed via 6-endo cyclization of hydroxy epoxide 7. An improved synthetic entry to the EFGH ring fragment 6 is also described, in which SmI(2)-induced reductive cyclization methodology was applied to the stereoselective construction of the F and H rings, leading to 6 with remarkable overall efficiency. Stereoselective hydroboration of 5 and subsequent Suzuki-Miyaura coupling with 6 provided endocyclic enol ether 45 in high yield, which was then converted to octacyclic polyether core 4. Careful choice of the global deprotection stage was a key element for the successful total synthesis. Functionalization of the H ring and global desilylation gave (Z)-vinyl bromide 2. Finally, cross-coupling of 2 with (Z)-vinyl stannane 3 under Corey's Pd(PPh(3))(4)/CuCl/LiCl-promoted Stille conditions completed the total synthesis of (-)-gambierol (1).

Topics: Ciguatoxins; Ethers, Cyclic; Marine Toxins; Nuclear Magnetic Resonance, Biomolecular; Polycyclic Compounds; Stereoisomerism

The Lewis acid mediated reaction of alpha-acetoxy ethers 15-22 gave the corresponding cyclized products 23, 25, 27, 29, 31, 32, 34, and 36 in good yields with high stereoselectivities. Those cyclized products were subjected to ring-closing metathesis to afford the polycyclic ethers 38-42, 44, and 45 in good yields. The usefulness of the present methodology was demonstrated by the convergent synthesis of the CDEF ring system of brevetoxin B (1) and the CDEFG ring system of gambierol (2).

Topics: Allyl Compounds; Ciguatoxins; Ethers, Cyclic; Polycyclic Compounds

[structure: see text]. The stereoselective syntheses of the C'D'E'F'-ring system of maitotoxin and the FG-ring system of gambierol were accomplished. The key steps involve 6-endo-cyclization of methylepoxide, SmI2-induced reductive cyclization, 6-endo-cyclization of vinylepoxide, and formation of the lactone ring.

Topics: Ciguatoxins; Ethers, Cyclic; Marine Toxins; Molecular Structure; Oxocins; Polycyclic Compounds; Stereoisomerism

[reaction: see text]. This Letter describes our use of C-glycosides to synthesize the A-D ring system of the marine ladder toxin gambierol in 20 steps.

Topics: Animals; Ciguatoxins; Dinoflagellida; Ethers; Ethers, Cyclic; Glycosides; Marine Biology; Molecular Conformation; Polycyclic Compounds

[structure: see text]. A convergent synthetic route to the octacyclic polyether core of gambierol, a marine polycyclic ether toxin, has been developed. The synthesis involves construction of two fragments representing the ABC and EFGH ring systems followed by their coupling via a B-alkyl Suzuki reaction.

Topics: Animals; Ciguatoxins; Ethers, Cyclic; Eukaryota; Indicators and Reagents; Marine Toxins; Polycyclic Compounds