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 α1 2 βγδ and neuronal α3β2 and α4β2 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 (≈2.4Å) show toxins neatly imbedded within the nest of aromatic 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 bisspiroacetal or gymnodimine tetrahydrofuran and their common cyclohexene-butyrolactone further anchor the toxins in apical and membrane directions, along the subunit interface. In contrast, the sequence-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.acetylcholine binding protein | marine phycotoxins | nicotinic acetylcholine receptor | pharmacological and structural analyses | seafood poisoning

Gymnodimines (GYMs) are phycotoxins exhibiting unusual structural features including a spirocyclic imine ring system and a trisubstituted tetrahydrofuran embedded within a 16‐membered macrocycle. The toxic potential and the mechanism of action of GYM‐A, highly purified from contaminated clams, have been assessed. GYM‐A in isolated mouse phrenic hemidiaphragm preparations produced a concentration‐ and time‐dependent block of twitch responses evoked by nerve stimulation, without affecting directly elicited muscle twitches, suggesting that it may block the muscle nicotinic acetylcholine (ACh) receptor (nAChR). This was confirmed by the blockade of miniature endplate potentials and the recording of subthreshold endplate potentials in GYM‐A paralyzed frog and mouse isolated neuromuscular preparations. Patch‐clamp recordings in Xenopus skeletal myocytes revealed that nicotinic currents evoked by constant iontophoretical ACh pulses were blocked by GYM‐A in a reversible manner. GYM‐A also blocked, in a voltage‐independent manner, homomeric human α7 nAChR expressed in Xenopus oocytes. Competition‐binding assays confirmed that GYM‐A is a powerful ligand interacting with muscle‐type nAChR, heteropentameric α3β2, α4β2, and chimeric α7‐5HT3 neuronal nAChRs. Our data show for the first time that GYM‐A broadly targets nAChRs with high affinity explaining the basis of its neurotoxicity, and also pave the way for designing specific tests for accurate GYM‐A detection in shellfish samples.

The distribution and function of sympathetic innervation in skeletal muscle have largely remained elusive. Here we demonstrate that sympathetic neurons make close contact with neuromuscular junctions and form a network in skeletal muscle that may functionally couple different targets including blood vessels, motor neurons, and muscle fibers. Direct stimulation of sympathetic neurons led to activation of muscle postsynaptic β2-adrenoreceptor (ADRB2), cAMP production, and import of the transcriptional coactivator peroxisome proliferator-activated receptor γ-coactivator 1α (PPARGC1A) into myonuclei. Electrophysiological and morphological deficits of neuromuscular junctions upon sympathectomy and in myasthenic mice were rescued by sympathicomimetic treatment. In conclusion, this study identifies the neuromuscular junction as a target of the sympathetic nervous system and shows that sympathetic input is crucial for synapse maintenance and function

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.

The effects of 0.15-250 microM riluzole, a novel psychotropic agent with anticonvulsant properties, were studied on voltage-clamped nodes of Ranvier of isolated nerve fibres of the frog. When added to the external solution, the drug rapidly and reversibly inhibited both K and Na currents with an apparent dissociation constant of 0.09 mM. The riluzole-induced decrease of these currents was not "use-dependent". At concentrations up to 100 microM, the drug had no noticeable effect on the time course of Na current inactivation nor on the shape and the position along voltage axis of the Na conductance/voltage relationship. On the other hand, it induced substantial shifts towards negative voltages of the steady-state Na inactivation/voltage curve. From these results, according to the modulated-receptor model, an apparent dissociation constant of 0.29 microM could be calculated for riluzole-induced blockage of inactivated Na channels. The recovery from Na current inactivation was also affected by the drug. It is concluded that riluzole is a highly specific blocker of inactivated Na channels, which is more than 300 times more effective on these channels than on K or resting Na channels.

Background. The majority of patients receiving the platinum-based chemotherapy drug oxaliplatin develop peripheral neurotoxicity. Because this neurotoxicity involves ROS production, we investigated the efficacy of mangafodipir, a molecule that has antioxidant properties and is approved for use as an MRI contrast enhancer.Methods. The effects of mangafodipir were examined in mice following treatment with oxaliplatin. Neurotoxicity, axon myelination, and advanced oxidized protein products (AOPPs) were monitored. In addition, we enrolled 23 cancer patients with grade ≥2 oxaliplatin-induced neuropathy in a phase II study, with 22 patients receiving i.v. mangafodipir following oxaliplatin. Neuropathic effects were monitored for up to 8 cycles of oxaliplatin and mangafodipir. Funding.

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