Synthetic Pinnatoxins a and G Reversibly Block Mouse Skeletal Neuromuscular Transmission in Vivo and in Vitro

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Title Synthetic Pinnatoxins A and G Reversibly Block Mouse Skeletal Neuromuscular Transmission In Vivo and In Vitro.

Permalink https://escholarship.org/uc/item/4j20678f

Journal Marine drugs, 17(5)

ISSN 1660-3397

Authors Benoit, Evelyne Couesnon, Aurélie Lindovsky, Jiri et al.

Publication Date 2019-05-24

DOI 10.3390/md17050306

Peer reviewed

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Article Synthetic Pinnatoxins A and G Reversibly Block Mouse Skeletal Neuromuscular Transmission In Vivo and In Vitro

Evelyne Benoit 1,2 , Aurélie Couesnon 2 , Jiri Lindovsky 2, Bogdan I. Iorga 3 , 1,2 1 4 1,2, , Rómulo Aráoz , Denis Servent , Armen Zakarian and Jordi Molgó * † 1 Commissariat à l’Energie Atomique et aux énergies Alternatives (CEA), Institut des Sciences du Vivant Frédéric Joliot, Service d’Ingénierie Moléculaire des Protéines (SIMOPRO), CEA de Saclay, Université Paris-Saclay, F-91191 Gif-sur-Yvette, France; [email protected] (E.B.); [email protected] (R.A.); [email protected] (D.S.) 2 Centre National de la Recherche Scientifique (CNRS), Institut des Neurosciences Paris-Saclay (Neuro-PSI), UMR 9197 CNRS/Université Paris-Sud, F-91198 Gif-sur-Yvette, France; [email protected] (A.C.); [email protected] (J.L.) 3 Centre National de la Recherche Scientifique (CNRS), Institut de Chimie des Substances Naturelles, UPR 2301, Labex LERMIT, F-91198 Gif-sur-Yvette, France; [email protected] 4 Department of Chemistry and Biochemistry, University of California, Santa Barbara, CA 93106, USA; [email protected] * Correspondence: [email protected]; Tel.: +33-1-6908-5158 J.M. dedicates this article to honor Professor Stephen Thesleff from the University of Lund (Sweden) on the † occasion of his 95th birthday, for his human qualities and sincere friendship over the last 40 years.

Received: 7 May 2019; Accepted: 21 May 2019; Published: 24 May 2019

Abstract: Pinnatoxins (PnTXs) A-H constitute an emerging family belonging to the cyclic imine group of phycotoxins. Interest has been focused on these fast-acting and highly-potent toxins because they are widely found in contaminated shellfish. Despite their highly complex molecular structure, PnTXs have been chemically synthetized and demonstrated to act on various nicotinic acetylcholine receptor (nAChR) subtypes. In the present work, PnTX-A, PnTX-G and analogue, obtained by chemical synthesis with a high degree of purity (>98%), have been studied in vivo and in vitro on adult mouse and isolated nerve-muscle preparations expressing the mature muscle-type (α1)2β1δε nAChR. The results show that PnTX-A and G acted on the neuromuscular system of anesthetized mice and blocked the compound muscle action potential (CMAP) in a dose- and time-dependent manner, using a minimally invasive electrophysiological method. The CMAP block produced by both toxins in vivo was reversible within 6–8 h. PnTX-A and G, applied to isolated extensor digitorum longus nerve-muscle preparations, blocked reversibly isometric twitches evoked by nerve stimulation. The action of PnTX-A was reversed by 3,4-diaminopyridine. Both toxins exerted no direct action on muscle fibers, as revealed by direct muscle stimulation. PnTX-A and G blocked synaptic transmission at mouse neuromuscular junctions and PnTX-A amino ketone analogue (containing an open form of the imine ring) had no effect on neuromuscular transmission. These results indicate the importance of the cyclic imine for interacting with the adult mammalian muscle-type nAChR. Modeling and docking studies revealed molecular determinants responsible for the interaction of PnTXs with the muscle-type nAChR.

Keywords: pinnatoxins; marine phycotoxins; mouse neuromuscular system; compound muscle action potential; synaptic potentials; emerging toxins; cyclic imines

Mar. Drugs 2019, 17, 306; doi:10.3390/md17050306 www.mdpi.com/journal/marinedrugs Mar. Drugs 2019, 17, 306 2 of 17

1. Introduction Pinnatoxins (PnTXs) A–D were originally identified in the digestive glands of the bivalve mollusks Pinna attenuata and Pinna muricata following food poisoning outbreaks that were linked to these shellfish in China and Japan [1–4]. However, it is still unclear whether PnTXs were the cause of these poisoning events. Later, three new PnTX-A analogues, the PnTX-E, F, and G, were isolated and structurally characterized from extracts of Pacific oysters (Crassostrea gigas) and razorfish (Pinna bicolor) from South Australia [5,6]. Analysis of surface sediments in New Zealand and South Australia, where shellfish were reported positive for PnTXs,Mar. Drugs led 2019, to 17, x the FOR PEER discovery REVIEW of a peridinoid dinoflagellate producing2 ofPnTX-E 17 and F in

New Zealand,1. PnTX-E, Introduction F, and G in South Australia [7–9] and PnTX-G in Japan [10]. Morphological and phylogenetic similaritiesPinnatoxins (PnTXs) were A–D observed were originally between identified these in the dinoflagellates digestive glands of and the abivalve new dinoflagellate named Vulcanodiniummollusks Pinna rugosum attenuatadiscovered and Pinna muricata in water following samples food poisoning of Mediterranean outbreaks that were lagoonslinked to in the French these shellfish in China and Japan [1–4]. However, it is still unclear whether PnTXs were the cause of coast [11]. Contaminationthese poisoning of events. mussels Later, three and new clams PnTX-A by an PnTXs,alogues, the and PnTX-E, the link F, and to G, thewereV. isolated rugosum and dinoflagellate was first reportedstructurally in France characterized in 2011 from [12 extracts], but retro-analysisof Pacific oysters (Crassostrea of contaminated gigas) and razorfish shellfish (Pinna samples revealed high levels of PnTX-Gbicolor) from since South Australia 2006 [13 [5,6].]. Likewise, PnTXs have been found in other European waters Analysis of surface sediments in New Zealand and South Australia, where shellfish were and seafood sincereported 2010 positive [14– for18 PnTXs,], and led in to Canada the discovery as wellof a peridinoid [19]. There dinoflagellate is also producing evidence PnTX-E that the harmful V. rugosum dinoflagellateand F in New can Zealand, be transported PnTX-E, F, and in G ballast in South tanks Australia of [7–9] shipping and PnTX-G vessels in Japan [20], [10]. which is of global Morphological and phylogenetic similarities were observed between these dinoflagellates and a new concern. Also, newdinoflagellate strains named of the VulcanodiniumV. rugosum rugosumdinoflagellate, discovered in water isolated samplesfrom of Mediterranean the South lagoons China Sea [21] and the Arabian Gulfin the [22 French], were coast reported [11]. Contamination to produce of mussels only and PnTX-H clams by PnTXs, and and portimine the link to the [21 V., 23rugosum], as determined by dinoflagellate was first reported in France in 2011 [12], but retro-analysis of contaminated shellfish liquid chromatography-tandemsamples revealed high levels mass of PnTX-G spectrometry since 2006 [13]. (LC-MS Likewise,/MS). PnTXs have been found in other PnTXs belongEuropean to awaters heterogeneous and seafood since and 2010 growing[14–18], and groupin Canada of as macrocyclic well [19]. There is compounds also evidence called “cyclic imines toxins”that that the include harmful V. therugosum prorocentrolides, dinoflagellate can be transported spiro-prorocentrimine, in ballast tanks of shipping gymnodimines, vessels [20], spirolides, which is of global concern. Also, new strains of the V. rugosum dinoflagellate, isolated from the South pteriatoxins, andChina portimines Sea [21] and ([ the24 –Arabian26] for Gulf reviews, [22], were and reported [27, 28to produce] for recently only PnTX-H described and portimine cyclic imine toxins). Up until now, eight[21,23], PnTXsas determined (A–H) by liquid have chromatogr been reported.aphy-tandem Theirmass spectrometry chemical (LC-MS/MS). structure contains a common PnTXs belong to a heterogeneous and growing group of macrocyclic compounds called “cyclic scaffold characterizedimines toxins” by that a dimethyl include the prorocentrolides, substituted spiro-prorocentrimine, 7-membered cyclic gymnodimines, imine as spirolides, part of a spiroimine ring system, apteriatoxins, 6,5,6-spiroketal and portimines ring ([24–26] system, for reviews, and a and bridged [27,28] for ketal recently which described is typicalcyclic imine of this family of toxins). Up until now, eight PnTXs (A–H) have been reported. Their chemical structure contains a toxins [24–26],common as exemplified scaffold characterized for PnTX-A by a dimethyl and G subs in Figuretituted 7-membered1. It has cyclic been imine proposed as part of that a PnTX-F and G are the precursorsspiroimine of ring all system, PnTXs, a 6,5,6-spiroketal as well as ring of system, the structurally and a bridged ketal related which pteriatoxins,is typical of this via metabolic and hydrolyticfamily transformations of toxins [24–26], inas shellfishexemplified [for6]. PnTX-A Interestingly, and G in Figure in contrast 1. It has been to proposed other cyclic that imine toxins, PnTX-F and G are the precursors of all PnTXs, as well as of the structurally related pteriatoxins, via PnTXs exhibit anmetabolic outstanding and hydrolytic chemical transformations stability in shellfish at acid [6]. pH Interestingly, (pH 1.5 in and contrast pH to 4.0) other [6 cyclic,29]. imine toxins, PnTXs exhibit an outstanding chemical stability at acid pH (pH 1.5 and pH 4.0) [6,29].

Figure 1. ChemicalFigure structures1. Chemical structures of PnTX-A, of PnTX-A, PnTX-G PnTX-G and and PnTX-A PnTX-A amino aminoketone analogue ketone (PnTX-AK). analogue (PnTX-AK).

In mouse bioassays, PnTx-E, F, and G were shown to produce rapid lethality by respiratory In mousedepression bioassays, upon PnTx-E, intraperitoneal F, and administration, G were shownwith both toneurological produce symptoms rapid and lethality skeletal by respiratory depression uponmuscle intraperitoneal flaccid paralysis administration,[6,23,30]. Among the withcyclic imine both phycotoxins neurological purified, symptoms PnTX-E, F, and and G skeletal muscle flaccid paralysiswere [6 ,the23 ones,30]. that Among exhibited thethe highest cyclic acute imine oral mouse phycotoxins toxicity [30]. purified, PnTX-E, F, and G were the PnTXs, like other cyclic imine toxins, are known to be potent antagonists of both Torpedo muscle- ones that exhibitedtype (α the12β1γδ highest) and neuronal acute α7, oral α4β2 mouse and α3β toxicity2 nicotinic acetylcholine [30]. receptors (nAChRs) [31–33]. PnTXs, likeStudies other on cyclic isolated imine rat phrenic-hemidiaphragm toxins, are known preparations to be potent showed antagonists that crude extract of bothcontainingTorpedo a muscle-type mixture of PnTX-E and PnTX-F, as well as purified PnTX-F [34] and purified PnTX-E, PnTX-F, and (α12β1γδ) and neuronal α7, α4β2 and α3β2 nicotinic acetylcholine receptors (nAChRs) [31–33]. Studies on isolated rat phrenic-hemidiaphragm preparations showed that crude extract containing a mixture of PnTX-E and PnTX-F, as well as purified PnTX-F [34] and purified PnTX-E, PnTX-F, and PnTX-G [35] produced concentration-dependent decreases in nerve-evoked muscle twitches with a rank order of Mar. Drugs 2019, 17, 306 3 of 17 potency of PnTX-F > PnTX-G > PnTX-E, incomplete washout profiles for PnTX-F and PnTX-G, and the inability toMar. be Drugs reversed 2019, 17, x byFOR the PEER anticholinesterase REVIEW inhibitor neostigmine [34,35]. 3 of 17 To the best of our knowledge, neither PnTX-A nor PnTX-G, obtained by chemical synthesis and having anPnTX-G established [35] produced degree concentration-dependent of purity (>98%), have decre beenases studied in nerve-evokedin vivo muscleand in twitches vitro on with adult a mouse and isolatedrank nerve-muscleorder of potency preparationsof PnTX-F > PnTX-G expressing > PnTX-E, the incomplete mature washout muscle profilesα1 β1 forδε PnTX-FnAChR. and Therefore, PnTX-G, and the inability to be reversed by the anticholinesterase inhibitor neostigmine2 [34,35]. aims of the currentTo the best study of our knowledge, were (i) to neither study PnTX-A the localnor PnTX-G, action obtained of synthetic by chemical PnTX-A synthesis and and G on the neuromuscularhaving an system establishe ofd anesthetized degree of purity mice (>98%),in vivohave ,been using studied a minimally in vivo and invasive in vitro on electrophysiological adult mouse method,and (ii) isolated to study nerve-musclein vitro preparatiothe actionsns expressing of PnTX-A the mature and G,muscle as wellα12β1δε as nAChR. PnTX-A Therefore, amino ketone derivativeaims (PnTX-AK, of the current containing study were an (i) opento study form the local of the action imine of synthetic ring), onPnTX-A isometric and G twitchon the tension neuromuscular system of anesthetized mice in vivo, using a minimally invasive electrophysiological and on cholinergic transmission at single neuromuscular junctions, (iii) to determine the eventual method, (ii) to study in vitro the actions of PnTX-A and G, as well as PnTX-A amino ketone derivative efficacy of(PnTX-AK, 3,4-diaminopyridine containing an open for form reversing of the imine the neuromuscular ring), on isometric blockade twitch tension produced and on cholinergic by PnTX-A and G, and (iv) totransmission model the at molecular single neuromuscular interactions junctions, between (iii) PnTX-A to determine and Gthe and eventual the α 1efficacy2β1δε ofnAChR. 3,4- diaminopyridine for reversing the neuromuscular blockade produced by PnTX-A and G, and (iv) to 2. Resultsmodel the molecular interactions between PnTX-A and G and the α12β1δε nAChR.

2.1. Eﬀects2. ofResults PnTX-A and G on the Excitability Properties of Mouse Neuromuscular System In Vivo The e2.1.ﬀects Effects of of intramuscular PnTX-A and G on injections the Excitability of PnTX-A Properties and of Mouse G were Neurom studieduscular on System the multimodal in Vivo excitability properties ofThe the effects mouse of neuromuscular intramuscular injections system of in PnTX-A vivo (Figure and G 2were). studied on the multimodal excitability properties of the mouse neuromuscular system in vivo (Figure 2).

Figure 2.FigureEﬀects 2. ofEffects local of injections local injections of PnTX-A of PnTX-A and and G on G theon the multimodal multimodal excitability excitability properties properties of of mouse neuromuscularmouse neuromuscular system in vivo system.(a) Tracesin vivo. of(a) CMAPTraces of recorded CMAP recorded from thefrom tail the muscle tail muscle following following increasing intensitiesincreasing of caudal intensities motor of nerve caudal stimulation motor nerve stimul (scheme),ation (scheme), before (control) before (control) and after and after injection injection of PnTX-A

(5.44 nmol/kg of mouse, upper traces) or PnTX-G (3.20 nmol/kg of mouse, lower traces). (b) On-line recordings of the eﬀects of PnTX-A (5.44 nmol/kg of mouse), PnTX-G (3.20 nmol/kg of mouse) and/or methanol (1%) injections on the CMAP maximal amplitude registered continuously over time. Values are expressed relatively to those before injections. The arrow indicates the time of injections. Mar. Drugs 2019, 17, x FOR PEER REVIEW 4 of 17

of PnTX-A (5.44 nmol/kg of mouse, upper traces) or PnTX-G (3.20 nmol/kg of mouse, lower traces). (b) On-line recordings of the effects of PnTX-A (5.44 nmol/kg of mouse), PnTX-G (3.20 nmol/kg of mouse) and/or methanol (1%) injections on the CMAP maximal amplitude registered continuously over time. Values are expressed relatively to those before injections. The arrow indicates the time of Mar. Drugs 2019, 17, 306 4 of 17 injections.

On-line recordingsrecordings were were initiated initiated approximately approximately 10 min 10 beforemin abefore given a injection given ofinjection PBS containing of PBS methanolcontaining (0.1–1%) methanol and (0.1–1%) various dosesand various of either doses PnTX-A of (fromeither0.54 PnTX-A to 5.44 (from nmol /0.54kg of to mouse) 5.44 nmol/kg or PnTX-G of (1.60mouse) and or 3.20 PnTX-G nmol /(1.60kg of and mouse), 3.20 nmol/kg to observe of themouse) alterations, to observe occurring the alterations on some selected occurring excitability on some parameters,selected excitability such as theparameters, CMAP amplitude such as the and CMAP excitability amplitude threshold, and registeredexcitability continuously threshold, registered over time. Thecontinuously major effect over of thetime. two The toxins major was effect a decrease of the oftw CMAPo toxins amplitude, was a decrease as exemplified of CMAP in Figureamplitude,2a,b for as 5.44exemplified nmol of in PnTX-A Figure per 2a kgand of b mouse for 5.44 and nmol 3.20 of nmol PnTX-A of PnTX-G per kg per of mouse kg of mouse. and 3.20 This nmol effect of occurredPnTX-G atper various kg of mouse. times, dependingThis effect occurred on the dose at various of toxin time injecteds, depending [PnTX-A: on from the 43.3 dose of0.2 toxin min injected (n = 3 mice) [PnTX- for ± 0.54A: from nmol 43.3/kg ± of 0.2 mouse min (n to = 26.1 3 mice)7.7 for min 0.54 (n =nmol/kg4 mice) of for mouse 5.44 nmol to 26.1/kg ± of 7.7 mouse, min (n and = 4 PnTX-G:mice) for from 5.44 ± 24.4nmol/kg9.7 ofmin mouse, (n = 4and mice) PnTX-G: for 1.60 from nmol 24.4/kg of± 9.7 mouse min to(n 15.5= 4 mice)7.0 minfor 1.60 (n = nmol/kg4 mice) forof mouse 3.20 nmol to /15.5kg of ± ± ± mouse].7.0 min (n The = 4 toxin-inducedmice) for 3.20 nmol/kg decrease of of mouse]. CMAP amplitude The toxin-induced was completely decrease reversed of CMAP within amplitude 6-8 h afterwas PnTX-Acompletely (5.44 reversed nmol/kg within of mouse) 6-8 h or after PnTX-G PnTX-A (3.20 (5. nmol44 nmol/kg/kg of mouse) of mouse) injection, or PnTX-G leading (3.20 to a 93.7nmol/kg8.0% of ± (nmouse)= 8 mice) injection, recovery leading compared to a 93.7 to control ± 8.0% conditions, (n = 8 mice) i.e., recovery CMAP amplitude compared before to control injection conditions, (Figure2 i.e.a). Finally,CMAP amplitude the CMAP before amplitude injection remained (Figure stable 2a). Finally, before toxinthe CMAP injections, amplitude or before remained and after stable injection before (5toxinµL) injections, of PBS added or before only and with after 1% methanolinjection (5 (Figure µL) of2 PBSb). Underadded theseonly with latter 1% conditions, methanol the(Figure CMAP 2b). maximalUnder these amplitude latter conditions, was not significantly the CMAP a ffmaximalected (P =amplitude0.332), i.e., was 95.0 not5.6% significantly within 57.7 affected13.3 ( minP = ± ± (0.332),n = 3 mice), i.e. 95.0 compared ± 5.6% towithin control 57.7 conditions. ± 13.3 min This (n indicates= 3 mice), that compared injections to of control the vehicle conditions. associated This to theindicates highest that doses injections of toxin of studied the vehicle had no associated significant etoff ectthe on highest the CMAP doses amplitude. of toxin studied had no significantThe dose-response effect on the CMAP curves amplitude. of PnTX-A and G effects on the CMAP maximal amplitude were establishedThe dose-response and analyzed curves using individual of PnTX-A values and insteadG effects of meanon the values, CMAP for maximal a better comparison amplitude ofwere the eestablishedfficacy between and analyzed the two toxins. using individual Results reveal values that instead the doses of mean necessary values, to inhibit for a better 50% ofcomparison the response of the efficacy between the two toxins. Results reveal that the doses necessary to inhibit 50% of the (ID50) was 3.1 0.2 (n = 18 mice) and 2.8 0.1 (n = 8 mice) nmol/kg of mouse, respectively (Figure3). response (ID50±) was 3.1 ± 0.2 (n = 18 mice)± and 2.8 ± 0.1 (n = 8 mice) nmol/kg of mouse, respectively These two ID50 values were not significantly different (P = 0.123). The comparison of PnTX-A and (Figure 3). These two ID50 values were not significantly different (P = 0.123). The comparison of PnTX- G ID50 values to those of some other cyclic imine toxins previously studied, i.e., gymnodimine-A (GYM-A)A and G ID [3650], values 13-desmethyl to those spirolideof some other C (13-SPX-C) cyclic imine [36] andtoxins 20-methyl previously spirolide studied, G (20-meSPX-G)i.e. gymnodimine- [37], showsA (GYM-A) that PnTX-A [36], 13-desmethyl and G were spirolide as potent C as (13-SPX- GYM-A,C) but [36] at and least 20-methyl 300-fold less spirolide efficient G than(20-meSPX-G) 13-SPX-C and[37], 20-meSPX-G,shows that PnTX-A to inhibit and the G CMAPwere as maximal potent as amplitude GYM-A, but (Table at 1least). 300-fold less efficient than 13- SPX-C and 20-meSPX-G, to inhibit the CMAP maximal amplitude (Table 1).

100 PnTX-A PnTX-G

25 Relative CMAPRelative (%) amplitude 0 0.1 1.0 10.0 Dose of PnTX (nmol/kg of mouse) Figure 3.3. Dose-response curves of the effects eﬀects of PnTX-APnTX-A and G determineddetermined from CMAPCMAP maximalmaximal amplitude values recorded from the mouse tail muscle inin vivovivo.. Each value is expressedexpressed relativelyrelatively toto that obtained before injections. The curves were calculated calculated from typical sigmoid non-linear regression through datadata pointspoints (r(r22 = 0.9550.955 and and 0.875 0.875 for for PnTX-A PnTX-A and G, resp respectively).ectively). The dose required to block

50% of thethe CMAPCMAP maximalmaximal amplitudeamplitude (ID(ID50)) and and nH were, respectively, 3.13.1 ± 0.2 nmolnmol/kg/kg of mouse 50 ± and 1.6 0.2 (n = 18 mice) for PnTX-A, and 2.8 0.1 nmol/kg of mouse and 2.7 0.3 (n = 8 mice) ± ± ± for PnTX-G. Mar. Drugs 2019, 17, 306 5 of 17

The five different excitability tests (stimulus-response, strength-duration and current-threshold relationships, as well as threshold electrotonus and recovery cycle), performed together before and approximately 45 min after injections of PBS containing methanol (1%) and either PnTX-A (5.44 nmol/kg of mouse) or PnTX-G (3.20 nmol/kg of mouse) did not reveal further effects of the two toxins since no apparent modification of excitability waveforms was detected (data not shown). This was confirmed by analyzing the parameters determined from excitability tests, as exemplified in Table2 for PnTX-A, with the exception of a significant lower “TEd (undershoot)” after PnTX-A-injection. Although this result may be due to alterations in slow voltage-gated K+ channels, it was not confirmed by analyzing the other parameters (such as “TEd (40–60 ms)”, “TEd (90–100 ms)” and “TEd (accommodation)”) related to this type of ion channel.

Table 1. ID50 values (in nmol/kg of mouse) of some cyclic imine toxins, determined from CMAP maximal amplitude recorded from mouse tail muscle in vivo.

ID50 PnTX-A PnTX-G GYM-A 13-SPX-C 20-meSPX-G Mean 3.080 2.830 3.474 0.009 0.002 S.D. 0.203 0.090 0.166 0.001 0.000 n (mice) 18 8 15 19 26 Reference Present study Present study [36][36][37]

Table 2. Excitability parameters (means S.D.) from tail muscle recordings of mice in vivo, before ± (control, n = 15 mice) and approximately 45 min after injection of PBS containing methanol (1%) and PnTX-A (5.44 nmol/kg of mouse, n = 8 mice).

Test 1 Excitability Parameter 2 Control PnTX-A P Peak response (mV) 3.299 1.140 1.847 1.370 0.050 * ± ± Latency (ms) 3.359 0.065 3.349 0.106 0.893 T1 ± ± Stimulus (mA) for 50% max response 0.481 1.060 0.479 1.130 0.927 ± ± Stimulus-response slope 3.004 1.180 2.650 1.310 0.743 ± ± Resting slope 0.638 0.077 0.916 0.257 0.182 ± ± T2 Minimum slope 0.230 0.013 0.218 0.022 0.703 ± ± Hyperpolarizing slope 0.536 0.071 0.490 0.074 0.757 ± ± Strength-duration time constant (ms) 0.377 0.035 0.316 0.036 0.274 T3 ± ± Rheobase (mA) 0.333 1.070 0.327 1.130 0.861 ± ± TEd (10–20 ms) 47.680 2.570 45.250 4.930 0.700 ± ± TEd (peak) 48.640 2.320 45.240 4.110 0.555 ± ± TEd (40–60 ms) 39.440 3.000 38.080 1.980 0.826 ± ± TEd (90–100 ms) 36.000 3.010 35.230 1.810 0.878 ± ± TEd (accommodation) 12.590 1.230 10.170 2.890 0.441 T4 ± ± TEd (undershoot) 11.820 1.120 5.188 0.512 0.019 * − ± − ± TEh (10–20 ms) 97.600 4.780 86.230 2.660 0.318 − ± − ± TEh (20–40 ms) 132.000 8.430 123.900 7.160 0.686 − ± − ± TEh (90–100 ms) 159.20 12.60 150.300 4.910 0.753 − ± − ± TEh (overshoot) 13.350 1.460 7.238 1.450 0.079 ± ± Relative refractory period (ms) 2.349 1.090 2.532 1.100 0.632 ± ± T5 Superexcitability (%) 7.596 1.490 9.049 1.970 0.600 − ± − ± Subexcitability (%) 3.844 0.708 3.316 1.160 0.697 ± ± 1 T1 = Stimulus-response relationship, T2 = Current-threshold relationship (informed on accommodation capacity to depolarizing and hyperpolarizing currents), T3 = Strength-duration relationship (informed on nodal membrane potential), T4 = Threshold electrotonus and T5 = Recovery cycle. 2 TEd = Threshold electrotonus from depolarizing currents and TEh = Threshold electrotonus from hyperpolarizing currents. *: Signiﬁcant diﬀerence compared to control. Mar. Drugs 2019, 17, x FOR PEER REVIEW 6 of 17

Relative refractory period (ms) 2.349 ± 1.090 2.532 ± 1.100 0.632 T5 Superexcitability (%) −7.596 ± 1.490 −9.049 ± 1.970 0.600 Subexcitability (%) 3.844 ± 0.708 3.316 ± 1.160 0.697 1 T1 = Stimulus-response relationship, T2 = Current-threshold relationship (informed on accommodation capacity to depolarizing and hyperpolarizing currents), T3 = Strength-duration Mar. Drugsrelationship2019, 17, 306 (informed on nodal membrane potential), T4 = Threshold electrotonus and T5 = Recovery 6 of 17 cycle. 2 TEd = Threshold electrotonus from depolarizing currents and TEh = Threshold electrotonus from hyperpolarizing currents. *: Significant difference compared to control. 2.2. Eﬀects of PnTX-A and G on Isometric Twitch Tension In Vitro 2.2.PnTX-A Effects of (2.8–84 PnTX-A nM) and and G on PnTX-G Isometric (2.5–40 Twitch nM)Tension when in Vitro applied to isolated peroneal nerve-extensor digitorumPnTX-A longus (2.8–84 (EDL) nM) muscle and PnTX-G preparations (2.5–40 produced nM) when a concentration- applied to isolated and peroneal time-dependent nerve-extensor reduction ofdigitorum the peak force longus amplitude (EDL) ofmuscle muscle preparations twitches evoked produced by nerve a concentration- stimulation at 0.03and Hz.time-dependent Representative twitch-tensionreduction of the recordings peak force are amplitude shown in of Figure muscle4a. tw Theitches onset evoked of the by neuromuscularnerve stimulation block at 0.03 was Hz. fast withRepresentative the higher concentration twitch-tension of recordings PnTX-A used are show (84 nM),n in reachingFigure 4a. 50% The blockonset inof 5.1the neuromuscular0.85 min (n = 3), ± andblock 100% was block fast in with 16.5 the1.5 higher min (nconcentration= 3). The time of coursePnTX-A of used PnTX-A (84 nM), and PnTX-Greaching actions 50% block on the in relative5.1 ± ± isometric0.85 min twitch (n = 3), amplitude and 100% are block shown in 16.5 in Figure ± 1.5 4mina,b. (n Although = 3). The PnTX-Gtime course was of more PnTX-A active and than PnTX-G PnTX-A onactions nerve-evoked on the relative twitch isometric responses, twitch onset amplitude of the block are shown was about in Figure two-times 4a,b. Although slower with PnTX-G PnTX-G was as comparedmore active to PnTX-Athan PnTX-A (Figure on nerve-evoked4b,c). Further twitch experiments responses, are onset needed of the for block an understandingwas about two-times of the slowerslower onset with kinetics PnTX-G but as higher compared potency to PnTX-A of PnTX-G (Fig comparedure 4b,c). Further to PnTX-A. experiments Complete are and needed rapid for reversal an ofunderstanding PnTX-A induced of the neuromuscular slower onset blockkinetics was but obtained higher bypotency addition of PnTX-G of 100 µ comparedM 3,4-diaminopyridine to PnTX-A. (3,4-DAP)Complete to and the rapid medium reversal (Figure of 4PnTX-Aa,b). induced neuromuscular block was obtained by addition of 100 µM 3,4-diaminopyridine (3,4-DAP) to the medium (Figure 4a,b). (a) a5 50 mN

500 ms

a1 a2 a3 a4

15 mN 200 ms

Control PnTX-A + 3,4-DAP PnTX-A

(b) 1.1 (c) 1.1 1.0 1.0 0.9 0.9 0.8 0.8 0.7 0.7 0.6 0.6 0.5 0.5 0.4 0.4 Control (1% MetOH) 0.3 PnTX-A (28 nM) 0.3 PnTX-A (84 nM) PnTX-G (2.5 nM) 0.2 0.2 PnTX-G (10 nM) Relative twitchRelative amplitude PnTX-A (84 nM) + 3,4-DAP 0.1 Wash-out amplitude twitch Relative 0.1 PnTX-G (40 nM) 0.0 0.0 0 5 10 15 20 25 30 35 40 45 50 55 0 102030405060 Time (min) Time (min) FigureFigure 4. 4.PnTX-A PnTX-A blockade blockade of of nerve-evokednerve-evoked isometric twit twitchch tension tension without without modification modiﬁcation of ofdirectly directly elicitedelicited twitch twitch and and tetanus tetanus tension tension onon isolatedisolated mousemouse EDL muscles. muscles. (a ()a )(a1) (a1) Single Single twitch twitch tension tension recordingrecording under under control control conditions, conditions, (a2)(a2) MarkedMarked reduction in in twitch twitch am amplitudeplitude during during the the action action of of PnTX-APnTX-A (54 (54 nM), nM), (a3) (a3) Reversal Reversal ofof thethe blockadeblockade produced by by PnTX-A PnTX-A by by 3,4-DAP 3,4-DAP (100 (100 µM).µM). (a4,a5) (a4,a5) TwitchTwitch and and tetanus tetanus responses responses evokedevoked by by direct direct musc musclele stimulation stimulation at 0.03 at 0.03 and and 80 HZ, 80 HZ,respectively respectively in in an EDL muscle in which nerve-evoked contractions were completely blocked by 56 nM PnTX-A. (b,c) Time course and concentration dependence of PnTX-A and PnTX G eﬀects on nerve-evoked twitch responses, and the reversal by wash-out and 3,4-DAP (100 µM). After an equilibration period of 20 min PnTXs were applied at time 0. In (b) the wash-out of PnTX-A (84 nM), and the fast reversal of PnTX-A action by 3,4-DAP are shown. Note the slower onset kinetics of PnTX-G (c) as compared to PnTX-A (b) on nerve-evoked muscle contraction. Each value is expressed relatively to that obtained before addition of PnTXs. Mar. Drugs 2019, 17, x FOR PEER REVIEW 7 of 17

an EDL muscle in which nerve-evoked contractions were completely blocked by 56 nM PnTX-A. (b,c) Time course and concentration dependence of PnTX-A and PnTX G effects on nerve-evoked twitch responses, and the reversal by wash-out and 3,4-DAP (100 µM). After an equilibration period of 20 min PnTXs were applied at time 0. In (b) the wash-out of PnTX-A (84 nM), and the fast reversal of Mar. DrugsPnTX-A2019, 17action, 306 by 3,4-DAP are shown. Note the slower onset kinetics of PnTX-G (c) as compared to 7 of 17 PnTX-A (b) on nerve-evoked muscle contraction. Each value is expressed relatively to that obtained before addition of PnTXs. The relative potencies of PnTX-A and G were evaluated by concentration-response curves. CalculationThe relative of the meanpotencies inhibitory of PnTX-A concentration and G were producing evaluated 50% by reductionconcentration-response (IC50) of the isometriccurves. twitchCalculation response of wasthe ofmean 27.7 inhibitory nM for PnTX-A concentration and 11.3 nMproducing for PnTX-G 50% (Figurereduction5a,b). (IC The50) of action the ofisometric PnTX-A wastwitch reversible response by was washing of 27.7 out nM the for toxin PnTX-A from and the 11.3 bathing nM for medium PnTX-G within (Figure 120 5a,b). min. The Interestingly, action of inPnTX-A EDL muscles was reversible in which by full washing block of out nerve-evoked the toxin from contraction the bathing was producedmedium bywithin either 120 PnTX-A min. orInterestingly, PnTX-G, direct in EDL electrical muscles muscle in which stimulation full block couldof nerve-evoked still evoke contraction twitch and was tetanic produced contractions by either in responsePnTX-A toor single PnTX-G, and tetanicdirect electrical direct muscle muscle stimulation, stimulation respectively. could still Aevoke representative twitch and example tetanic is showncontractions in Figure in4 (a4,a5).response These to resultssingle indicateand tetanic that bothdirect PnTX-A muscle and stimulation, G exerted no respectively. direct action A on therepresentative contractile machinery example is of shown muscle in ﬁbers,Figure but4 (a4,a5) acted. These on neuromuscular results indicate transmission, that both PnTX-A as previously and G shownexerted for no other direct cyclic action imine on the toxins contractile [37–39]. machinery of muscle fibers, but acted on neuromuscular transmission, as previously shown for other cyclic imine toxins [37–39].

FigureFigure 5. 5.Concentration-response Concentration-response curves curves for for PnTX-A PnTX-A (a) ( anda) and PnTX-G PnTX-G (b) actions(b) actions on the on isometric the isometric twitch responsestwitch responses evoked byevoked nerve by stimulation nerve stimulation (closed (closed circles) circles) or by direct or by muscle direct muscle stimulation stimulation (open circles).(open Data points represent the mean S.D. of twitch response, after 60 min toxin exposure, relative to the circles). Data points represent the± mean ± S.D. of twitch response, after 60 min toxin exposure, relative respectiveto the respective controls controls of 3–6 EDL of 3–6 nerve-muscle EDL nerve-muscle preparations. preparations. The curves The curves were calculatedwere calculated from typicalfrom 2 sigmoidtypical non-linearsigmoid non-linear regression regression through data through points data (r =points0.989 (r and2 = 0.9220.989 forand PnTX-A 0.922 for and PnTX-A G, respectively). and G, Therespectively). dose required The to dose block required 50% of the to twitch block amplitude 50% of the (IC 50twitch) and nHamplitude were, respectively, (IC50) and 27.7nH nMwere, and 2.4respectively, for PnTX-A, 27.7 and nM 11.3 and and 2.4 2.7 for for PnTX-A, PnTX-G. and 11.3 and 2.7 for PnTX-G. 2.3. Effects of PnTX-A and G on Neuromuscular Transmission In Vitro 2.3. Effects of PnTX-A and G on Neuromuscular Transmission in Vitro To evaluate the action of PnTXs on synaptic transmission, intracellular recordings at single To evaluate the action of PnTXs on synaptic transmission, intracellular recordings at single neuromuscular junctions were performed from isolated EDL or FDB muscles. Experiments were neuromuscular junctions were performed from isolated EDL or FDB muscles. Experiments were first 2+ firstdone done in instandard standard Krebs-Ringer Krebs-Ringer so solutionlution containing containing physiological physiological Ca Ca2+ concentrationconcentration (2 (2mM). mM). IntracellularIntracellular recordings recordings at at junctionaljunctional regionsregions ofof musclemuscle fibers fibers revealed revealed that that single single nerve nerve stimulation stimulation alwaysalways triggered triggered an actionan action potential potential that sometimesthat sometimes dislodged dislodged the microelectrode the microelectrode due to the due contraction to the ofcontraction muscle fibers of muscle (Figure fibers6a). Muscle (Figure action6a). Muscle potential action triggered potential by triggered nerve stimulation by nerve stimulation had overshoots had of 21.3 1.61 mV when recorded at mean resting membrane potential of 69.3 .1.06 mV (n = 16, overshoots± of 21.3 ± 1.61 mV when recorded at mean resting membrane potential− of± −69.3 ±.1.06 mV 4(n di =ff erent16, 4 different EDL muscles). EDL muscles). As shown As shown in Figure in Figure6b, addition6b, addition of PnTX-Aof PnTX-A (84 (84 nM) nM) to to thethe standard physiologicalphysiological medium medium completely completely abolished, abolished, within with 30in min, 30 themin, generation the generation of muscle of actionmuscle potentials action uponpotentials nerve upon stimulation. nerve stimulation. In addition, In by addition, recordings by inrecordings junctional in areas, junctional these areas, experiments these experiments revealed the presencerevealed of the end-plate presence potentials of end-plate (EPPs) potentials of 3–5 mV (EPPs) amplitude of 3–5 thatmV amplitude were unable that to reachwere unable the threshold to reach for actionthe threshold potential for generation action potential in muscle generation fibers. Similarly, in muscle when fibers. using Similarly, a “floating” when microelectrode, using a “floating” it was possible to record muscle action potentials in FDB muscles without movement-bias due to contraction

(Figure6c). Action potentials recorded under this condition were not significantly di fferent from those successfully recorded with conventional microelectrodes (P < 0.05, n = 9). Additional experiments were performed using µ-conotoxin GIIIB (1.0–1.6 µM) which blocks specifically and irreversibly voltage-gated skeletal muscle Na+ channels [40]. Under this condition, full-sized EPPs could be evoked by nerve stimulation without contraction, as shown in a representative Mar. Drugs 2019, 17, x FOR PEER REVIEW 8 of 17 Mar. Drugs 2019, 17, x FOR PEER REVIEW 8 of 17 microelectrode, it was possible to record muscle action potentials in FDB muscles without movement- microelectrode, it was possible to record muscle action potentials in FDB muscles without movement- bias due to contraction (Figure 6c). Action potentials recorded under this condition were not bias due to contraction (Figure 6c). Action potentials recorded under this condition were not significantly different from those successfully recorded with conventional microelectrodes (P < 0.05, significantly different from those successfully recorded with conventional microelectrodes (P < 0.05, n = 9). n = 9). Mar. DrugsAdditional2019, 17, 306experiments were performed using µ-conotoxin GIIIB (1.0–1.6 µM) which blocks8 of 17 Additional experiments were performed using µ-conotoxin GIIIB (1.0–1.6 µM) which blocks specifically and irreversibly voltage-gated skeletal muscle Na+ channels [40]. Under this condition, specifically and irreversibly voltage-gated skeletal muscle Na+ channels [40]. Under this condition, full-sized EPPs could be evoked by nerve stimulation without contraction, as shown in a recordingfull-sized inEPPs Figure could6d. Further be evoked addition by of nerve PnTX-A stimulation markedly reducedwithout thecontraction, amplitude ofas theshown full-sized in a representative recording in Figure 6d. Further addition of PnTX-A markedly reduced the amplitude EPPsrepresentative (Figure6e). recording in Figure 6d. Further addition of PnTX-A markedly reduced the amplitude of the full-sized EPPs (Figure 6e). of the full-sized EPPs (Figure 6e).

Figure 6. PnTX-A and G block skeletal neuromuscular transmission. (a) Nerve-evoked muscle action Figure 6. PnTX-A and G block skeletal neuromuscular transmission. (a) Nerve-evoked muscle action potential recorded in aa junctionjunction ofof thethe EDLEDL musclemuscle uponupon nervenerve stimulation.stimulation. (b) EPP recorded 30 min potential recorded in a junction of the EDL muscle upon nerve stimulation. (b) EPP recorded 30 min after thethe addition addition of of PnTX-A PnTX-A (84 (84 nM) nM) to the to standard the standard Krebs-Ringer Krebs-Ringer solution. solution. (c) Muscle (c) actionMuscle potential action after the addition of PnTX-A (84 nM) to the standard Krebs-Ringer solution. (c) Muscle action recordedpotential withrecorded a “floating” with a “floating” microelectrode microelectrode in the FDB in muscle the FDB upon muscle nerve upon stimulation. nerve stimulation. (d) Full-sized (d) potential recorded with a “floating” microelectrode in the FDB muscle upon nerve stimulation. (d) EPPFull-sized recorded EPP after recorded treatment after with treatmentµ-conotoxin with GIIIBµ-conotoxin (1.6 µM) GIIIB in a junction(1.6 µM) of in the a FDBjunction muscle. of the(e) FDBEPP Full-sized EPP recorded after treatment with µ-conotoxin GIIIB (1.6 µM) in a junction of the FDB ofmuscle. reduced (e) amplitudeEPP of reduced recorded amplitude after the recorded action ofafter PnTX-A. the action Recordings of PnTX-A. in ( aRecordings) and (b) were in (a obtained) and (b) muscle. (e) EPP of reduced amplitude recorded after the action of PnTX-A. Recordings in (a) and (b) atwere a resting obtained membrane at a resting potential membrane of potential70 mV, and of −70 those mV, in and (c) those and (ind, e(c)) were and ( obtainedd,e) were atobtained68 and at were obtained at a resting membrane− potential of −70 mV, and those in (c) and (d,e) were obtained− at −6871 and mV, − respectively.71 mV, respectively. −−68 and −71 mV, respectively. Both PnTX-A and G significantly blocked (P < 0.05) by more than 80% full-sized EPP amplitudes, Both PnTX-A and G significantly blocked (P < 0.05) by more than 80% full-sized EPP amplitudes, recordedBoth inPnTX-A muscles and treated G significantly with µ-conotoxin blocked (P GIIIB < 0.05) as by shown more inthan Figure 80% 7full-sized. These resultsEPP amplitudes, indicate recorded in muscles treated with µ-conotoxin GIIIB as shown in Figure 7. These results indicate that thatrecorded these in toxins muscles block treated neuromuscular with µ-conotoxin transmission GIIIB as by shown reducing in Figure the amplitude 7. These ofresults EPPs. indicate It is worth that these toxins block neuromuscular transmission by reducing the amplitude of EPPs. It is worth noting notingthese toxins that no block significant neuromuscular change ontransmission the resting by membrane reducing the potential amplitude of muscle of EPPs. fibers It is wasworth detected noting that no significant change on the resting membrane potential of muscle fibers was detected in the inthat the no presence significant of PnTX-A change andon the G inresting the range membrane of concentrations potential of studied muscle ( Pfibers> 0.05; was n =detected12 and nin= the9, presence of PnTX-A and G in the range of concentrations studied (P > 0.05; n = 12 and n = 9, respectively).presence of PnTX-A and G in the range of concentrations studied (P > 0.05; n = 12 and n = 9, respectively). respectively).

Figure 7. PnTX-A (84 nM) and PnTX-G (54 nM) block full-sized EPPs evoked by nerve stimulation, FigureFigure 7.7. PnTX-APnTX-A (84(84 nM)nM) andand PnTX-GPnTX-G (54(54 nM)nM) blockblock full-sizedfull-sized EPPsEPPs evokedevoked byby nervenerve stimulation,stimulation, while 100 nM PnTX-AK (containing an open form of the imine ring) had no action on EPP amplitudes. whilewhile 100100 nMnM PnTX-AKPnTX-AK (containing(containing anan openopen formform ofof thethe imineimine ring)ring) hadhad no action on EPP amplitudes. (a) Representative control EPP recorded in a junction from an EDL muscle that has been treated with ((aa)) RepresentativeRepresentative controlcontrol EPPEPP recordedrecorded inin aa junctionjunction fromfrom anan EDLEDL musclemuscle thatthat hashas beenbeen treatedtreated withwith µ-conotoxin GIIIB to prevent muscle action potentials generation. (b) EPP recorded during the action µµ-conotoxin-conotoxin GIIIBGIIIB toto preventprevent musclemuscle actionaction potentialspotentials generation.generation. ((bb)) EPPEPP recordedrecorded duringduring thethe actionaction of PnTX-G (20 nM). (c) Full-sized EPP recorded after 30 min of 100 nM PnTX-AK. (d) Graphs showing ofof PnTX-GPnTX-G (20(20 nM).nM). ((cc)) Full-sizedFull-sized EPPEPP recordedrecorded afterafter 3030 minmin ofof 100100 nMnM PnTX-AK.PnTX-AK. ((dd)) GraphsGraphs showingshowing control EPP values (mean ± SEM; n = 4–8 for each condition), the significant (*: P < 0.05) reduction of controlcontrol EPPEPP valuesvalues (mean (mean ± SEM;SEM; nn= = 4–84–8 forfor eacheach condition),condition), thethe signiﬁcantsignificant(*: (*:P P< < 0.05)0.05) reductionreduction ofof EPP amplitudes by PnTX-A± and G, and the lack of action of PnTX-AK (100 nM). EPPEPP amplitudesamplitudes byby PnTX-APnTX-A andand G,G, andand thethe lacklack ofof actionaction ofof PnTX-AKPnTX-AK (100 (100 nM). nM).

Further experiments were performed to determine whether miniature end-plate potentials

(mEPPs), which result from the release of a single quantum of ACh, are modiﬁed in amplitude and frequency by the PnTXs. Under control conditions, the mean amplitude of mEPPs recorded in standard Krebs-Ringer solution in unstimulated junctions from the EDL muscles was 0.92 0.03 mV (n = 6, ± Mar. Drugs 2019, 17, x FOR PEER REVIEW 9 of 17

Further experiments were performed to determine whether miniature end-plate potentials Mar.(mEPPs), Drugs 2019which, 17, 306result from the release of a single quantum of ACh, are modified in amplitude9 ofand 17 frequency by the PnTXs. Under control conditions, the mean amplitude of mEPPs recorded in fromstandard 6 diff Krebs-Ringererent muscles), solution addition in ofunstimulated 5 nM PnTX-A ju ornctions G to the from medium the EDL induced muscles a significant was 0.92 ± reduction 0.03 mV and(n = complete6, from 6 blockdifferent of mEPP muscles), amplitude addition within of 5 25nM min PnTX-A with bothor G toxinsto the (nmedium= 3, from induced 3 different a significant muscles inreduction each condition). and complete The block block of mEPPof mEPP amplitude amplitude was within fully reversible 25 min with upon both washing toxins (within (n = 303, min)from of3 thedifferent nerve-muscle muscles preparationsin each condition). with a The toxin-free block of medium. mEPP amplitude Estimations was of fully the frequency reversible of upon mEPPs washing when mEPP(within were 30 min) blocked of the by aboutnerve-mu 50%,scle but preparations still easily recognized with a toxin-free from the medium. basal electric Estimations noise, revealed of the thatfrequency the frequency of mEPPs was when not significantlymEPP were modifiedblocked by (Figure about8b). 50%, but still easily recognized from the basal electric noise, revealed that the frequency was not significantly modified (Figure 8b). (a) (b) (c) ) -1 1.0 2.0 0.4 mV 0.8 1.6 25 ms 0.6 1.2 0.4 * 0.8 0.2 0.4 Amplitude (mV) 0 * * 0 mEPP frequency (s Control PnTX-G PnTX-G

Figure 8. PnTX-A and PnTX-G block of mEPP amplitude without affecting mEPP frequency. (a) Control Figure 8. PnTX-A and PnTX-G block of mEPP amplitude without affecting mEPP frequency. (a) mEPP amplitude (white column), after 10 min and 20 min of 5 nM PnTX-A action (blue columns), Control mEPP amplitude (white column), after 10 min and 20 min of 5 nM PnTX-A action (blue and 5 nM PnTX-G action (brown column). (b) Frequency of mEPP recorded under control conditions columns), and 5 nM PnTX-G action (brown column). (b) Frequency of mEPP recorded under control (white column) and after 10 min of 5 nM PnTX-A action (blue column) when mEPPs of reduced conditions (white column) and after 10 min of 5 nM PnTX-A action (blue column) when mEPPs of amplitude were still present. Note, under this condition, the lack of action of PnTX-A on mEPP reduced amplitude were still present. Note, under this condition, the lack of action of PnTX-A on frequency. (c) Examples of mEPPs recorded under control conditions and during the action of 5 nM mEPP frequency. (c) Examples of mEPPs recorded under control conditions and during the action of PnTX-G (brown traces). *: denotes significant differences compared to controls (P < 0.05). 5 nM PnTX-G (brown traces). *: denotes significant differences compared to controls (P < 0.05). Overall, our results show that synaptic transmission is strongly impaired by both PnTX-A and G, and suggestOverall, that our bothresults phycotoxins show that block,synaptic in atransmission reversible manner, is strongly the interactionimpaired by of both ACh PnTX-A quanta with and endplateG, and suggest nAChR. that both phycotoxins block, in a reversible manner, the interaction of ACh quanta with Toendplate determine nAChR. if PnTXs modified the sensitivity of nAChR to extracellularly applied ACh, an intracellularTo determine recording if PnTXs microelectrode modified the sensitivity was impaled, of nAChR as close to as extracellularly possible to the applied endplate ACh, area, an sointracellular that mEPPs recording with fast microelectrode time course could was be impaled, recorded. as Then, close anas iontophoreticpossible to the micropipette endplate area, filled so withthat AChmEPPs was with placed fast extracellularlytime course could in very be closerecorded. contact Th withen, an the iontophoretic muscle membrane, micropipette and to filled the intracellular with ACh microelectrodewas placed extracellularly in the endplate in very area. close Current contact pulses wi ofth 1the ms muscle duration membrane, at constant and intensity to the wereintracellular applied tomicroelectrode the pipette to in give the anendplate ACh potential area. Current of 5–8 mVpulses in amplitudeof 1 ms duration (Figure 9at, inset). constant After intensity stable AChwere potentialapplied to recordings, the pipette EDL to give preparations an ACh potential were perfused of 5–8 withmV in 100 amplitude nM PnTX-A, (Figure and 9, constant inset). After ACh pulsesstable wereACh deliveredpotential recordings, by iontophoresis EDL preparations each 20 or 30 were s. In theperfused presence with of 100 PnTX-A, nM PnTX-A, the amplitude and constant of the ACh potentialpulses were decreased delivered in a by time-dependent iontophoresis mannereach 20 soor after30 s. aboutIn the 10–14presence min of (n PnTX-A,= 3), no response the amplitude could beof evokedthe ACh by potential ACh, as decreased shown in thein a graphtime-dependent of Figure9. mann This resulter so after clearly about indicates 10–14 thatmin PnTX-A(n = 3), no blocks response the could be evoked by ACh, as shown in the graph of Figure 9. This result clearly indicates that PnTX- muscle-type α12β1δε nAChR subtype. A blocksAdditional the muscle-type experiments α12β were1δε nAChR performed subtype. to determine whether the spiroimine component in PnTX-AAdditional is essential experiments for the blocking were performed action of EPP to amplitudes.determine whether For this, the experiments spiroimine were component performed in withPnTX-A the syntheticis essential PnTX-AK for the blocking that includes action an of openEPP amplitudes. form of the imineFor this, ring experiments A. As shown were in Figureperformed7a,b, PnTX-AKwith the synthetic had no actionPnTX-AK on nerve-evoked that includes EPPan open amplitude form of at the concentrations imine ring A. atAs which shown PnTX-A in Figure exerted 7a,b, 80%PnTX-AK blockade had ofno EPP action amplitude. on nerve-evoked Similarly, EPP no significantamplitude at eff concentrationsect of PnTX-AK at (5.44 which nmol PnTX-A/kg of exerted mouse) was80% detectedblockade on of the EPP CMAP amplitude. recorded Similarly, from anesthetized no significant mice effectin vivo of PnTX-AK(n = 3, data (5.44 not shown).nmol/kg Theseof mouse) data indicatewas detected that theon spiroiminethe CMAP componentrecorded from constitutes anesthetized a critical mice structural in vivo (n factor = 3, data for the not action shown). of PnTXsThese in vitro and in vivo. Mar.Mar. DrugsDrugs 20192019,, 1717,, xx FORFOR PEERPEER REVIEWREVIEW 1010 ofof 1717

Mar. Drugs 2019, 17, 306 10 of 17 datadata indicateindicate thatthat thethe spiroiminespiroimine componentcomponent constitutesconstitutes aa criticalcritical structuralstructural factorfactor forfor thethe actionaction ofof PnTXsPnTXs inin vitrovitro andand inin vivo.vivo.

FigureFigure 9.9. TimeTimeTime course coursecourse of of theof thethe block blockblock of ACh-evoked ofof ACh-evokedACh-evoked potentials potentialspotentials by 100 byby nM 100100 PnTX-A nMnM PnTX-A appliedPnTX-A to appliedapplied a superficial toto aa superficialEDLsuperficial neuromuscular EDLEDL neuromuscularneuromuscular junction. Constant junction.junction. iontophoretic ConstantConstant ioiontophoreticntophoretic ACh pulses AChACh of 1 mspulsespulses duration ofof 11 msms were durationduration delivered werewere to deliveredthedelivered endplate toto regionthethe endplateendplate (detected regionregion by an intracellular(detected(detected byby microelectrode anan intracellularintracellular through microelectrodemicroelectrode mEPP recordings) throughthrough by mEPP amEPP high recordings)resistancerecordings) pipette byby aa highhigh containing resistanceresistance 1 M pipettepipette ACh hydrochloride. containingcontaining 11 M Inset:M AChACh typical hydrochloride.hydrochloride. control ACh-response Inset:Inset: typicaltypical obtained controlcontrol ACh-responsebeforeACh-response the addition obtainedobtained of PnTX-A beforebefore to thethe the additionaddition standard ofof Krebs-Ringer PnPnTX-ATX-A toto thethe solution. standardstandard The Krebs-RingerKrebs-Ringer resting membrane solution.solution. potential TheThe restingduring membrane recordings potential was 71.5 during1.5 mV. recordings was −71.5 ± 1.5 mV. resting membrane potential− during± recordings was −71.5 ± 1.5 mV. 2.4. Computational Modeling of PnTX-A and G Interactions with the Nicotinic Acetylcholine Receptor 2.4.2.4. ComputationalComputational ModelingModeling ofof PnTX-APnTX-A andand GG InteraInteractionsctions withwith thethe NicotinicNicotinic AcetylcholineAcetylcholine ReceptorReceptor Molecular docking calculations of PnTX-A and G were carried out at the interface α1-ε of mouse MolecularMolecular dockingdocking calculationscalculations ofof PnTX-APnTX-A andand GG werewere carriedcarried outout atat thethe interfaceinterface αα1-1-εε ofof mousemouse muscular nAChRs. Overall, the binding modes observed are similar to those described previously for muscularmuscular nAChRs.nAChRs. Overall,Overall, thethe bindingbinding modesmodes obseobservedrved areare similarsimilar toto thosethose describeddescribed previouslypreviously PnTX-A at the interface α1-δ of the Torpedo nAChR [31]. There are, however, significant differences in forfor PnTX-APnTX-A atat thethe interfaceinterface αα1-1-δδ ofof thethe TorpedoTorpedo nAChRnAChR [31].[31]. ThereThere are,are, however,however, significantsignificant the protein-ligand interactions that are described in detail below (Figure 10). differencesdifferences inin thethe protein-ligandprotein-ligand interactionsinteractions ththatat areare describeddescribed inin detaildetail belowbelow (Figure(Figure 10).10).

Figure 10. Protein-ligand interactions in the docking complexes of PnTX-A (a) and PnTX-G (b) with FiguretheFigure mouse 10.10. Protein-ligandProtein-ligand nAChR at the α interactionsinteractions1-ε interface. inin Onlyththee dockingdocking amino acidscomplexescomplexes interacting ofof PnTX-APnTX-A through ((aa)) hydrogenandand PnTX-GPnTX-G bonds ((bb)) with with thethe mouse ligandmouse nAChR ornAChR involved atat thethe in αα toxin’s1-1-εε interface.interface. subtype OnlyOnly selectivity, aminoamino acids andacids in interaintera the sequencectingcting throughthrough alignment, hydrogenhydrogen are shown.bondsbonds withwith The thenumberingthe ligandligand oror of involvedinvolved amino acid inin toxin’stoxin’s residues subtypesubtype is thesame selectivitselectivit as iny,y, [ 31 andand]. inin thethe sequencesequence aalignment,lignment, areare shown.shown. TheThe numberingnumbering ofof aminoamino acidacid residuesresidues isis thethe samesame asas inin [31].[31]. In all cases, the hydrogen bond between the spiroimine group and the backbone oxygen of α1-Trp147,InIn allall cases,cases, which thethe is the hydrogenhydrogen signature bondbond of these betweenbetween classes thethe of spiroiminespiroimine toxins, is conserved. groupgroup andand Thethethe backbone hydrogenbackbone oxygen bondoxygen between ofof αα1-1- Trp147,theTrp147, carboxylate whichwhich isis group thethe signaturesignature of PnTX-A ofof thesethese and theclassesclasses side ofof chain toxins,toxins, of α isis1-Lys143 conserved.conserved. is conserved, TheThe hydrogenhydrogen but bond isbond not betweenbetween possible theforthe carboxylate PnTX-G,carboxylate in group whichgroup ofof the PnTX-APnTX-A carboxylate andand thethe is replacedsideside chainchain by ofof a αα vinyl1-Lys1431-Lys143 group. isis conserved,conserved, Another conserved butbut isis notnot possiblepossible interaction, forfor PnTX-G,observedPnTX-G, in forin whichwhich both PnTX-A thethe carboxylatecarboxylate and G, is made isis replacedreplaced by the side byby a chaina vinylvinyl of αgroup.group.1-Tyr195, AnotherAnother which conserved establishedconserved interaction,interaction, a hydrogen

Mar. Drugs 2019, 17, 306 11 of 17 bond with the oxygen from the tetrahydrofuran ring. The α1-Asp193 in Torpedo nAChRs corresponds to α1-Thr193 in mouse nAChRs, which is too short to interact with the hydroxyl group in position 15 of PnTX-A and G. Instead, we observe a hydrogen bond of this hydroxyl with the side chain of α1-Asp150. Overall, our in silico study of the interactions between PnTX-A and G and the interface α1-ε of mouse muscle-type nAChR highlights several important differences, but also common features, compared with the interaction of PnTX-A with Torpedo nAChRs that was previously described [31]. Thus, these various interaction patterns allow a fine tuning for the affinity and the specificity of different ligands against the nAChRs subtypes and their binding interfaces.

3. Discussion The present experimental results demonstrate, for the first time using a multimodal minimally-invasive in vivo electrophysiological approach, that synthetic PnTX-A and G, when injected locally to anesthetized mice, caused a time- and dose-dependent decrease of the CMAP recorded from the tail muscle in response to motor nerve stimulation. The CMAP block produced by both toxins in vivo was reversible within 6–8 h. Compared to other cyclic imine toxins, PnTX-A and G had a similar potency in vivo than GYM-A [36], but were less potent than 13-SPX-C [36] and 20-meSPX-G [37]. In vitro, PnTX-A and G blocked, in a reversible manner, nerve-evoked muscle contraction in the mouse peroneal nerve-EDL muscle preparation without affecting directly-elicited twitch or tetanic contractions. This is consistent with previous work done with other cyclic imine toxins like GYM-A [38], 13-SPX-C, and 13,19-didesmethyl spirolide C [39]. These results indicate that both PnTX-A and G affect neuromuscular transmission, but have no action on the contractile machinery of muscle fibers. This is in agreement with observations showing that 40 mM K+-induced muscle contractures were unaffected after treatment with PnTX-E, F and G [35]. A rank order of potency of PnTX-F > PnTX-G > PnTX-E has been reported in vitro on nerve-evoked twitch response in rat phrenic nerve-hemidiaphragm muscle [35]. In the present study, a rank order of potency PnTX-G > PnTX-A was obtained in the mouse peroneal nerve-EDL muscle. It is not possible to compare former and here-presented data on PnTX potency, since they have been done in different species and also in different muscles, and is well known that the diaphragm is more resistant to the action of neuromuscular blocking agents than limb muscles [41]. In contrast to previous studies with PnTX-E, F and G, extracted from dinoflagellates, which were performed in rat phrenic-cut-hemidiaphragm muscle, with an extracellular medium having a reduced K+ concentration [35]. Present intracellular recordings were performed in normal mouse EDL and FDB nerve-muscle preparations, equilibrated in normal Krebs-Ringer solution, and in some experiments a selective blocker of voltage-gated muscle Na+ channel was used for blocking muscle contraction. Analyses of the action of both PnTX-A and PnTX–G on neuromuscular transmission at single junctions of EDL and FDB revealed that both phycotoxins blocked the EPP amplitude. The block of EPP amplitude to subthreshold levels for action potential generation, at single junction, can explain the dose-dependent reduction in the CMAP observed in vivo by extracellular recordings in the caudal muscle. The reduction of the amplitude of spontaneous mEPP, without significant change in the mEPP frequency, and the block of full-sized EPPs and ACh-evoked potentials strongly indicate that PnTX-A and G exert a selective action at the postsynaptic level of the neuromuscular junction by blocking the interaction between ACh and the α12β1δε nAChR. Whether PnTXs have a presynaptic action was not directly investigated in our study. However, data obtained by measuring mEPP frequency, when there was about 50% block of mEPP amplitude, strongly suggests that PnTXs do not have a pre-synaptic action that would modify the quantal ACh release rate. Insight into the interaction between PnTX-A and PnTX-G and the muscle-type α12β1γδ nAChR was previously obtained in competition binding studies using [125I] α-bungarotoxin as a tracer. In those studies, both phycotoxins totally displaced, in a concentration-dependent the radiotracer [31,32]. Thus, PnTXs on the muscle-type α12β1γδ nAChR act as competitive antagonists. That 3,4-DAP was able to reverse the postsynaptic neuromuscular block Mar. Drugs 2019, 17, 306 12 of 17 produced by PnTX-A is an indication that PnTX-A may act also as a competitive antagonist on the + α12β1δε nAChR. 3,4-DAP is known to block a fast K current in motor nerve terminals, to greatly increase nerve-evoked quantal ACh release and to antagonize the competitive action of d-tubocurarine on the α12β1δε nAChR [42–44]. Taken together, the functional data obtained in the present study indicate that PnTX-A and PnTX-G block mouse neuromuscular transmission, which can explain the muscle paralysis and death via respiratory depression when administered in vivo in toxicity studies [6,23,30].

4. Materials and Methods

4.1. Toxins and Chemicals PnTX-A, G and AK were synthesized, as previously reported [31]. The purity of PnTXs and analogue was over 98%, as judged by NMR data and HPLC performed for ﬁnal puriﬁcation (Supplementary data). The µ-conotoxin GIIIB was obtained from Alomone Labs (Alomone Labs, Jerusalem, Israel). All chemicals, including ACh hydrochloride and 3,4-DAP, were purchased from Sigma-Aldrich (Saint Quentin Fallavier, France). The in vivo experiments were performed using a stock solution of PBS (1X, 100 µL) added to 1% methanol (vehicle) and either 28 µM PnTX-A or 20 µM PnTX-G. PnTX-A was studied at doses of 0.54–5.44 nmol/kg of mouse. PnTX-G was studied at doses of 1.60–3.20 nmol/kg of mouse.

4.2. Animals Adult male and female Swiss mice (Mus musculus, 2–5 months of age and 23–28 g of body weight) were purchased from Janvier Elevage (Le Genest-Saint-Isle, France), and acclimatized at the CEA animal facility for at least 48 h before experiments. Live animals were treated according to the European Community guidelines for laboratory animal handling and to the guidelines established by the French Council on animal care “Guide for the Care and Use of Laboratory Animals” (EEC86/609 Council Directive – Decree 2001-131). In particular, they were housed four- to six-wise in cages with environmental enrichment, in a room with constant temperature and a standard light cycle of 12-h light/12-h darkness, and had free access to water and food. All experimental procedures on mice were approved by the Animal Ethics Committee of the CEA (project 17_088 authorized to E.B.) and by the French General Directorate for Research and Innovation (project APAFIS#2671-2015110915123958v4 authorized to E.B.).

4.3. Recordings from the Neuromuscular System of Anesthetized Mice In Vivo The multimodal excitability properties of the mouse neuromuscular system were assessed in vivo on females [weighting 26.4 1.4 g (n = 26)], under isoflurane (AErrane, Baxter S.A., Lessines, Belgium) ± anesthesia, by means of minimally-invasive electrophysiological methods using the Qtrac© software (Bostock H., Institute of Neurology, London, UK), as previously described [45]. Briefly, the CMAP was recorded using fine needle electrodes inserted into the tail muscle and connected to an amplifier (Disa EMG 14C13), in response to electrical stimulations delivered to the caudal motor nerve by two stimulators (A395, World Precision Instruments, Sarasota, FL, USA) via surface electrodes. To study the underlying mode of action of PnTX-A and G, intramuscular injections (5-µL maximal volume) of PBS containing 0.1–1% methanol and various doses of PnTX-A (from 0.54 to 5.44 nmol/kg of mouse) or PnTX-G (1.60 and 3.20 nmol/kg of mouse) were delivered with a 10-µL micro-syringe at the base of the mouse tail, between stimulation and ground electrodes. Similar injections (5 µL) were also done with PBS containing only 1% methanol to test an eventual effect of the vehicle associated with the highest dose of toxins studied. On-line recordings were initiated approximately 10 min before a given injection to observe the effects over time of PnTX-A, PnTX-G, and/or methanol on selected excitability parameters such as the CMAP amplitude and excitability threshold, registered continuously. To further identify Mar. Drugs 2019, 17, 306 13 of 17 the toxin underlying mechanism of action and duration of effects, five different excitability tests (stimulus-response, strength-duration and current-threshold relationships, as well as threshold electrotonus and recovery cycle) [46] were performed together before and various times (from 45 min to 12 h) after a given injection. As a whole, more than thirty parameters were determined from these five different excitability tests and analyzed, providing additional and complementary information on the functional status of ion channels, receptors and electrogenic pumps, as well as on membrane properties of the neuromuscular system [47,48].

4.4. Recordings from Isolated Mouse Nerve-muscle Preparations In Vitro Male and female mice were anesthetized with isoflurane inhalation before being euthanized by dislocation of the cervical vertebrae. In vitro assays on isometric twitch tension were performed on isolated EDL muscle, and those on synaptic transmission on isolated EDL and flexor digitorum brevis (FDB) muscles. Removal of muscles and dissections were performed within 20 min in an oxygenated Krebs-Ringer solution of the following composition (in mM): NaCl 150, KCl 5, CaCl2 2, MgCl2 1, glucose 11, and HEPES 5 (pH 7.4). For isometric twitch tension measurements, the EDL muscle with its attached peroneal nerve was carefully dissected and mounted in a silicone-lined bath filled with an oxygenated Krebs-Ringer solution. One of the EDL tendons was securely anchored onto the silicone-coated bath, while the other was attached via an adjustable stainless-steel hook to an isometric force transducer (FT03; Grass Instruments, West Warwick, RI, USA). Muscle twitches and tetanic contractions were evoked either by stimulating the motor nerve via a suction microelectrode (adapted to the diameter of the nerve), with supramaximal current pulses of 0.15 ms duration, at frequencies indicated in the text, delivered by the isolation unit of a stimulator (S-44 Grass Instruments) or by direct electrical stimulation through an electrode assembly build up in the silicone-coated bath and placed at a short distance along the length of the muscle. For each preparation investigated, the resting tension was adjusted with a mobile micrometer stage which allowed incremental variations of the muscle length in order to obtain maximal contractile responses. Signals from the isometric transducer were amplified, collected, and digitized with the aid of a computer equipped with a Digidata-1322A A/D interface board (Axon Instruments, Molecular Devices, Sunnyvale, CA, USA). Data acquisition and analysis were performed with the WinWCP v3.9.6 software program (John Dempster, University of Strathclyde, Scotland). Experiments were performed at constant room temperature (22 ◦C). For synaptic transmission measurements, either EDL or FDB nerve-muscle preparations were dissected and mounted in silicone-lined recording chambers with stainless mini-pins. The motor nerve was stimulated with a suction electrode, with pulses of 0.1 ms duration and supramaximal voltage (typically 3–8 V). Both internal and external platinum wires of the suction electrode were connected to the isolation unit of a stimulator (S-44 Grass Instruments). Intracellular recordings were performed with 1.2 mm (external diameter) borosilicate glass capillaries containing internal glass filaments (type GC120f; Clark Electromedical Instruments, Pangbourne, UK) pulled on a P-1000 puller (Sutter Instrument Company, Novato, CA, USA) and having a resistance of 6–12 MΩ when backfilled with a 3 M KCl solution. Conventional intracellular techniques were used to record the resting membrane potential, end-plate potential (EPP) and miniature end-plate potential (mEPP) with an Axoclamp-2B amplifier. “Floating” microelectrodes were made as described [49] and used to record action potential in contracting muscles. In some experiments, nerve-muscle preparations were incubated for 30–45 min with µ-conotoxin GIIIB (1.0–1.6 µM) to block muscle contraction upon nerve stimulation [40]. After muscle fiber impalement, synaptic potentials were digitized using a Digidata-1322A A/D interface board (Axon Instruments, Sunnyvale, CA, USA). For iontophoretic ACh application, high resistance pipettes (100–150 MΩ) made with borosilicate glass and filled with 1 M ACh hydrochloride were used. ACh efflux was induced by cationic-current pulses (1 ms duration) using a constant current generator that allowed the use of breaking currents to prevent spontaneous ACh efflux and avoid nAChR desensitization. Mar. Drugs 2019, 17, 306 14 of 17

4.5. Data and Statistical Analyses Sigmoid non-linear regressions through data points (correlation coefficient = r2) were used to calculate theoretical dose/concentration-response curves, according to the Hill equation (GraphPad Prism version 5): Rt/Rc = 1/[1 + ([toxin]/X) nH], (1) where Rt/Rc is the response recorded in the presence of a given toxin (Rt) and expressed as percentage of the value obtained in absence of toxin (Rc), [toxin] is the toxin concentration, X is the toxin dose (ID50) or concentration (IC50) necessary to inhibit 50% of the response, and nH is the Hill number. EPP amplitudes (exceeding 3 mV) were corrected for non-linear summation according to McLachlan and Martin 1981 [50]. In the present study we have used an “f“ factor of 0.8, and an equilibrium potential for ACh of 0 mV in the calculation of EPP amplitudes in mouse EDL neuromuscular preparations. Data are presented as means standard deviations (S.D.) of n different experiments. Differences ± between values were tested using the parametric two-tailed Student’s t-test (either paired samples for comparison within a single population or unpaired samples for comparison between two independent populations), and the one- or two-way analysis of variance (ANOVA for comparison between the means of independent populations) or the non-parametric Mann-Whitney U-test, depending on the equality of variances estimated using the Lilliefors’ test. Differences were considered statistically significant when P < 0.05.

4.6. Molecular Modeling

A homology model of the extracellular domain of mouse (α1)2β1δε nAChRs subtype was constructed using Modeller [51], and the Aplysia californica acetylcholine binding protein (AChBP) crystal structure as template (Protein Data Bank code 2WZY) [52]. Three-dimensional structures of PnTX-A and G were generated using Corina 3.6 (Molecular Networks GmbH, Erlangen, Germany, 2018). The docking procedure was carried out at the interface α1-ε in a similar manner as previously described for PnTX-A at the interface α1-δ of Torpedo nAChRs [31], using Gold (Cambridge Crystallographic Data Centre, Cambridge, UK) and the GoldScore scoring function. The binding site, deﬁned as a 20 Å radius sphere, was centred on the backbone oxygen atom of Trp147. All other parameters had default values. The receptor-ligand complexes images were produced using Pymol 2.2.0 (Schrödinger, LLC, New York, NY, USA, 2019).

5. Conclusions The study of multimodal excitability properties of mouse neuromuscular system in vivo revealed that PnTX-G is as efficient as PnTX-A to produce a reversible inhibition of CMAPs recorded from the tail muscle, without any significant modification of other excitability parameters. The block of muscle nAChR by PnTX-A and G, as shown in vitro, reduced the amplitude of EPPs to subthreshold levels for action potential generation in muscle fibers and explains the reduction of CMAP in vivo. Synaptic transmission in vitro was strongly impaired by both PnTX-A and G, since both phycotoxins blocked, in a reversible manner, the interaction of ACh quanta with endplate nAChR. Thus, PnTX-A and G are potent muscle-type nAChR antagonists. 3,4-DAP reversed the post-synaptic blockade produced by PnTX-A. Modeling studies revealed the molecular determinants responsible for the interaction of PnTXs with the muscle-type nAChR.

Supplementary Materials: The following are available online at http://www.mdpi.com/1660-3397/17/5/306/s1, Figure S1: 800 MHz 1H (a) and 200 MHz 13C (b) NMR spectra of PnTX-A recorded as a solution in CD3OD, Figure S2: 800 MHz 1H (a) and 200 MHz 13C (b) NMR spectra of PnTX-G triﬂuoroaceate recorded as a solution 1 13 in CD3OD, Figure S3: 500 MHz H (a) and 125 MHz C (b) NMR spectra of PnTX-AK recorded as a solution in CD3OD. Mar. Drugs 2019, 17, 306 15 of 17

Author Contributions: Conceptualization, E.B., B.I.I., A.Z., D.S. and J.M.; methodology and software, B.I.I.; validation and formal analysis, E.B., A.C., J.L. and J.M.; investigation, E.B., A.C., J.L. and J.M.; resources, A.Z.; writing—original draft preparation, E.B., B.I.I. and J.M.; writing—review and editing, E.B., B.I.I., R.A., D.S., A.Z. and J.M.; visualization, E.B. and J.M.; supervision and project administration, E.B., A.Z., D.S. and J.M.; funding acquisition, D.S., A.Z. and J.M. Funding: This research was funded by NIH/NIGMS grant GM R01-077379, and by the Interreg Atlantic program project (ALERTOX-NET-EAPA_317/2016). Acknowledgments: Patricia Villeneuve for excellent technical assistance. Conﬂicts of Interest: The authors declare no conﬂict of interest.

References

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