Search

The marine environment is known to be occupied by microorganisms. The potential toxicity of some of these marine microorganisms, that are capable of producing unknown biotoxins, has always been underestimated. Indeed, these biotoxins may be a threat to human health through the consumption of contaminated seafood and fish. For more than ten years, recurrent but atypical toxicity has been detected in mussels from Bizerte lagoon (North of Tunisia) during routine tests. In this study, we have isolated and characterized a new proteinaceous marine biotoxin, named Mussel Toxic Peptide (MTP). Using HPLC, electrophoresis and LC/MS studies, we showed that MTP has a protein characteristic UV-spectrum, can be visualized by protein specific reagents such as Coomassie, and has a molecular mass of 6.4 kDa. Patch-clamp experiments performed on cultured N18 neuroblastoma cells revealed that MTP (0.9-18 µM) markedly inhibited voltage-gated Na current, but was about 23 times less active in blocking voltage-gated K current at equimolar concentrations. To the best of our knowledge, this is the first time that a proteinaceous marine biotoxin with relatively high molecular mass is isolated and involved in the contamination of mussels harvested from shellfish farming areas.

Spirolides are a large family of lipophilic marine toxins produced by dinoflagellates that have been detected in contaminated shellfish. Among them, 13,19-didesmethyl and 13-desmethyl spirolide C phycotoxins are widely distributed and their mode of action needs to be clearly defined. In order to further characterize the pharmacological profiles of these phycotoxins on various nicotinic acetylcholine receptor (nAChR) subtypes and to examine whether they act on muscarinic receptors (mAChRs), functional electrophysiological studies and competition binding experiments have been performed. While 13-desmethyl spirolide C interacted efficiently with sub-nanomolar affinities and low selectivity with muscular and neuronal nAChRs, 13,19-didesmethyl spirolide C was more selective of muscular and homopentameric α7 receptors and recognized only weakly neuronal heteropentameric receptors, especially the α4β2 subtype. Thus, the presence of an additional methyl group on the tetrahydropyran ring significantly modified the pharmacological profile of 13-desmethyl spirolide C by notably increasing its affinity on certain neuronal nAChRs. Structural explanations of this selectivity difference are proposed, based on molecular docking experiments modeling different spirolide-receptor complexes. In addition, the 2 spirolides interacted only with low micromolar affinities with the 5 mAChRs, highlighting that the toxicity of the spirolide C analogs is mainly due to their high inhibition potency on various peripheral and central nAChRs and not to their low ability to interact with mAChR subtypes., (© The Author 2015. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Pinnatoxins are macrocyclic imine phycotoxins associated with algal blooms and shellfish toxicity. Functional analysis of pinnatoxin A and pinnatoxin G by binding and voltage-clamp electrophysiology on membrane-embedded neuronal α7, α4β2, α3β2, and muscle-type α12βγδ nicotinic acetylcholine receptors (nAChRs) reveals high-affinity binding and potent antagonism for the α7 and α12βγδ subtypes. The toxins also bind to the nAChR surrogate, acetylcholine-binding protein (AChBP), with low Kd values reflecting slow dissociation. Crystal structures of pinnatoxin-AChBP complexes (1.9-2.2 Å resolution) show the multiple anchoring points of the hydrophobic portion, the cyclic imine, and the substituted bis-spiroketal and cyclohexene ring systems of the pinnatoxins that dictate tight binding between the opposing loops C and F at the receptor subunit interface, as observed for the 13-desmethyl-spirolide C and gymnodimine A congeners. Uniquely, however, the bulky bridged EF-ketal ring specific to the pinnatoxins extends radially from the interfacial-binding pocket to interact with the sequence-variable loop F and govern nAChR subtype selectivity and central neurotoxicity., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

From a small group of exotic compounds isolated only two decades ago, Cyclic Imine (CI) toxins have become a major class of marine toxins with global distribution. Their distinct chemical structure, biological mechanism of action, and intricate chemistry ensures that CI toxins will continue to be the subject of fascinating fundamental studies in the broad fields of chemistry, chemical biology, and toxicology. The worldwide occurrence of potent CI toxins in marine environments, their accumulation in shellfish, and chemical stability are important considerations in assessing risk factors for human health. This review article aims to provide an account of chemistry, biology, and toxicology of CI toxins from their discovery to the present day.

Gymnodimine-A and 13-desmethyl spirolide C are marine toxins belonging to the cyclic imine group produced by Karenia selliformis and Alexandrium ostenfeldii/peruvianum dinoflagellates, respectively. The aim of this work was to analyze the pharmacological properties of both toxins (at sub-lethal doses) on neuromuscular excitability, when injected locally to isoflurane-anesthetized mice, using a multimodal minimally-invasive in vivo electrophysiological approach. The main effect of both toxins was a marked reversible time- and dose-dependent decrease of the compound muscle action potential recorded from the tail muscle in response to caudal motor nerve stimulation. The dose-response curves of gymnodimine-A and 13-desmethyl spirolide C effect on the maximal amplitude of compound muscle action potential revealed 50% inhibitory doses of 51 ng/mouse (i.e. 1.6 μg/kg or 3.3 nmol/kg mouse) and 0.18 ng/mouse (i.e. 6 ng/kg or 0.01 nmol/kg mouse), respectively. The blocking effect occurred without significant modification of motor nerve excitability parameters. It is concluded that the inhibition of the mouse compound muscle action potential, induced by gymnodimine-A and 13-desmethyl spirolide C, results from an action of these toxins at the level of the skeletal neuromuscular junction, since both cyclic imine toxins are known to interact and block muscle-type nicotinic acetylcholine receptors. In the present in vivo study, 13-desmethyl spirolide C was about 300 fold more active than gymnodimine-A on equimolar basis. The present in vivo approach, associated to recent progress in chemical synthesis of cyclic imine toxins, paves the way for more detailed structure-activity studies to obtain new and more potent synthetic analogs., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Severe toxicity was detected in mussels from Bizerte Lagoon (Northern Tunisia) using routine mouse bioassays for detecting diarrheic and paralytic toxins not associated to classical phytoplankton blooming. The atypical toxicity was characterized by rapid mouse death. The aim of the present work was to understand the basis of such toxicity. Bioassay-guided chromatographic separation and mass spectrometry were used to detect and characterize the fraction responsible for mussels' toxicity. Only a C17-sphinganine analog mycotoxin (C17-SAMT), with a molecular mass of 287.289 Da, was found in contaminated shellfish. The doses of C17-SAMT that were lethal to 50% of mice were 750 and 150 μg/kg following intraperitoneal and intracerebroventricular injections, respectively, and 900 μg/kg following oral administration. The macroscopic general aspect of cultures and the morphological characteristics of the strains isolated from mussels revealed that the toxicity episodes were associated to the presence of marine microfungi (Fusarium sp., Aspergillus sp. and Trichoderma sp.) in contaminated samples. The major in vivo effect of C17-SAMT on the mouse neuromuscular system was a dose- and time-dependent decrease of compound muscle action potential amplitude and an increased excitability threshold. In vitro, C17-SAMT caused a dose- and time-dependent block of directly- and indirectly-elicited isometric contraction of isolated mouse hemidiaphragms.

Introduction: Nicotinic acetylcholine receptors (nAChRs) have been extensively studied because of their importance in physiological processes and their involvement in a number of muscle and neuronal human pathologies. However, the role of specific subtypes remains poorly understood due to the lack of selective nAChR probes. During the last decade, drug design strategies have been powered by a wide variety of natural compounds with diverse chemical structures, and by the structural characterization of several nAChRs structural homologs., Areas Covered: In this review, the authors present a short overview of nAChRs, and some natural sources of bioactive molecules targeting these receptors. The authors provide an emphasis on α-conotoxins from Conus venoms, which provide the most diverse selective antagonists of nAChRs known to date, as well briefly discussing macrocyclic imine toxins. The authors, furthermore, review valuable radioactive and non-radioactive methods used for discovering novel ligands targeting nAChRs and highlight high-throughput developments in receptor-binding and electrophysiological assays. Finally, the authors review the molecular modeling approaches used in the last few years with an aim to provide an overview of their potential to identify and optimize selective nAChR ligands., Expert Opinion: Recent years have provided new valuable techniques for the detection and identification of new nAChRs ligands, along with an increasing use of different molecular modeling tools. This furthering of knowledge has had an impact on the design and discovery of more potent and selective nAChRs ligands. There is still however a lack of high-resolution structural information that will require new developments.

Spirolide and gymnodimine macrocyclic imine phycotoxins belong to an emerging class of chemical agents associated with marine algal blooms and shellfish toxicity. Analysis of 13-desmethyl spirolide C and gymnodimine A by binding and voltage-clamp recordings on muscle-type alpha1(2)betagammadelta and neuronal alpha3beta2 and alpha4beta2 nicotinic acetylcholine receptors reveals subnanomolar affinities, potent antagonism, and limited subtype selectivity. Their binding to acetylcholine-binding proteins (AChBP), as soluble receptor surrogates, exhibits picomolar affinities governed by diffusion-limited association and slow dissociation, accounting for apparent irreversibility. Crystal structures of the phycotoxins bound to Aplysia-AChBP ( approximately 2.4A) show toxins neatly imbedded within the nest of ar-omatic side chains contributed by loops C and F on opposing faces of the subunit interface, and which in physiological conditions accommodates acetylcholine. The structures also point to three major features: (i) the sequence-conserved loop C envelops the bound toxins to maximize surface complementarity; (ii) hydrogen bonding of the protonated imine nitrogen in the toxins with the carbonyl oxygen of loop C Trp147 tethers the toxin core centered within the pocket; and (iii) the spirolide bis-spiroacetal or gymnodimine tetrahydrofuran and their common cyclohexene-butyrolactone further anchor the toxins in apical and membrane directions, along the subunit interface. In contrast, the se-quence-variable loop F only sparingly contributes contact points to preserve the broad receptor subtype recognition unique to phycotoxins compared with other nicotinic antagonists. These data offer unique means for detecting spiroimine toxins in shellfish and identify distinctive ligands, functional determinants and binding regions for the design of new drugs able to target several receptor subtypes with high affinity.

The effects of 31 plant extracts, which most are traditionally used to treat ciguatera fish poisoning in the Pacific area, were studied on the cytotoxicity of mouse neuroblastoma cells produced by ouabain, veratridine and/or brevetoxin-3 or Pacific ciguatoxin-1. The cell viability was determined using a quantitative colorimetric method. A marked cytotoxicity of seven of the 31 plant extracts studied, was observed. Despite this, these plant extracts were suspected to contain active compound(s) against the cytotoxicity produced by brevetoxin (2 extracts), brevetoxin, ouabain and/or veratridine (3 extracts), or only against that of ouabain and/or veratridine (2 extracts). Among the 24 plant extracts that exhibited by themselves no cytotoxicity, 22 were active against the effect of brevetoxin or against that of both veratridine and brevetoxin. Similar results were obtained when the seven most active plant extracts were reassayed using ciguatoxin instead of brevetoxin. In conclusion, the present work reports the first activity assessment of some plant extracts, achieved in vitro on a quite large scale. The fact that 27 plant extracts were found to exert, in vitro, a protective effect against the action of ciguatoxin and/or brevetoxin, paves the way for finding new active compounds to treat ciguatera fish poisoning, provided these compounds also reverse the effects of sodium channel activators.

The French Society of Toxinology (SFET) organized its 27th annual meeting on 9–10 December 2021 as a virtual meeting (e-RT27). The central theme of this meeting was "Toxins: Mr Hyde or Dr Jekyll?", emphasizing the latest findings on plant, fungal, algal, animal and bacterial toxins during 10 lectures, 15 oral communications (shorter lectures) and 20 posters shared by ca. 80 participants. The abstracts of lectures and posters, as well as the winners of the best oral communication and poster awards, are presented in this report. [ABSTRACT FROM AUTHOR]

Pinnatoxins belong to an emerging class of potent marine toxins of the cyclic imine group. Detailed studies of their biological effects have been impeded by unavailability of the complex natural product from natural sources. This work describes the development of a robust, scalable synthetic sequence relying on a convergent strategy that delivered a sufficient amount of the toxin for detailed biological studies and its commercialization for use by other research groups and regulatory agencies. A central transformation in the synthesis is the highly diastereoselective Ireland-Claisen rearrangement of a complex α,α-disubstituted allylic ester based on a unique mode for stereoselective enolization through a chirality match between the substrate and the lithium amide base. With synthetic pinnatoxin A, a detailed study has been performed that provides conclusive evidence for its mode of action as a potent inhibitor of nicotinic acetylcholine receptors selective for the human neuronal α7 subtype. The comprehensive electrophysiological, biochemical, and computational studies support the view that the spiroimine subunit of pinnatoxins is critical for blocking nicotinic acetylcholine receptor subtypes, as evidenced by analyzing the effect of a synthetic analogue of pinnatoxin A containing an open form of the imine ring. Our studies have paved the way for the production of certified standards to be used for mass-spectrometric determination of these toxins in marine matrices and for the development of tests to detect these toxins in contaminated shellfish. [ABSTRACT FROM AUTHOR]

Abstract: 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 μM) 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 Ca2+ 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. [ABSTRACT FROM AUTHOR]