Mechanism of action of pinnatoxins E, F and G

Abstract

Pinnatoxins E, F and G are the three most recently discovered isomers of the pinnatoxin family. Pinnatoxins belong to the cyclic imine group of toxins that causes a fast-acting, “all or nothing” toxic response in rodent bioassays. Symptoms of toxicity and eventual death of animals via respiratory depression has been shown to be a result of nicotinic receptor antagonism by other members of this toxin group. However, very little information is available on the mechanism of action of the pinnatoxins. Therefore, the aim of this thesis was to characterise the mechanism of action and relative in vitro toxicity of pinnatoxins E, F and G. An in vitro phrenic nerve-hemidiaphragm preparation revealed that all three isomers produced a dose-dependent reduction in nerve-evoked twitch responses, with a rank order of potency F > G > E. Stimulation of the muscle preparations with a depolarising agent showed that there was no direct myotoxic effect of these toxins, indicating a site of action at the neuromuscular synapse. Intracellular recordings of spontaneous and evoked electrical events in paralysed muscle preparations revealed that pinnatoxins F and G reduced the amplitude of miniature and evoked endplate potentials, without affecting the resting membrane potential of the muscle fibres or the frequency of spontaneous potentials. This profile of effects is indicative of antagonism of post-synaptic muscle-type nicotinic receptors. In order to visualise pinnatoxin binding to these receptors, an aminated derivative of pinnatoxin F was conjugated to a red fluorophore. The resulting fluorophore-conjugated pinnatoxin retained neuromuscular blocking activity and in vivo toxicity, albeit at a level lower than native pinnatoxin F. Fluorescent pinnatoxin signals co-localised with motor-nerve fluorescent signals in muscle sections from transgenic mice that express a yellow fluorescent protein in motor nerve terminals. These signals did not overlap, indicating that the fluorescent pinnatoxin was binding to a site adjacent to the nerve terminals. An inhibition of fluorescent labeling in the presence of the nicotinic antagonist α-bungarotoxin confirmed that this binding site was post-synaptic nicotinic receptors. With the nicotinic receptor antagonist properties of the pinnatoxins proven, radioligand binding assays were then employed to study the receptor affinity of the pinnatoxins for both muscle-type and α7 and α4β2 neuronal nicotinic receptors. Binding studies revealed that all 3 isomers bound with high (picomolar) affinity to muscle-type and α7 receptors, and with a slightly lower (nanomolar) affinity to α4β2 receptors. The neuronal subtypes had an order of potency of F > G > E, with muscle-type receptors showing similar affinity for all three isomers. Molecular modeling studies revealed potential ligand-receptor interactions that could be responsible for the different binding affinities seen for different subtypes of nicotinic receptors. Multiple interactions were seen between the pinnatoxin conformers and the muscle-type and α7 receptor models, with fewer interactions in α4β2 complexes. In conclusion, this thesis has unequivocally shown that pinnatoxins E, F and G are potent nicotinic receptor antagonists, and that this mechanism of action is almost certainly responsible for the symptoms of toxicity and cause of death in cases of pinnatoxin intoxication.