In silico simulations and functional cell studies evidence similar potency and distinct binding of pacific and caribbean ciguatoxins

Vol.:(0123456789) 1 3 Exposure and Health https://doi.org/10.1007/s12403-022-00513-0 ORIGINAL PAPER In Silico Simulations andFunctional Cell Studies Evidence Similar Potency andDistinct Binding ofPacific andCaribbean Ciguatoxins SandraRaposo‑García1 · DavidCastro2· EmilioLence3· PabloEstévez2· JoséManuelLeão2· ConcepciónGonzález‑Bello3· AnaGago‑Martínez2· M.CarmenLouzao1· CarmenVale1· LuisM.Botana1 Received: 11 July 2022 / Revised: 21 September 2022 / Accepted: 22 September 2022 © The Author(s) 2022 Abstract Ciguatoxins (CTX) cause ciguatera poisoning, which is the most common reported human food poisoning related to natural marine toxins. Pacific ciguatoxins are the most abundant and studied CTX analogues; however, the growing distribution of Caribbean analogues and the limited data available on their biological effects make necessary to re-evaluate their relative potency. For decades, the guidelines established by regulatory agencies have assumed that the potency of the Caribbean CTXs were tenfold lower than the Pacific CTXs. We present here an integrated study involving Neuro-2a cells (the method used worldwide to test ciguatoxins), electrophysiological assays, and in silico simulations that evidence the similar cytotoxicity of Caribbean and Pacific ciguatoxins and their asymmetry binding within sodium channels. The binding mode of the toxins was first explored by molecular docking using the GOLD program and the resulting binary complexes were further studied by Molecular Dynamics simulation studies using the molecular mechanics force field AMBER. The simulation studies explain their distinct impact on the activation potential of the channel as experimentally observed and provide a detailed picture of the effects caused by these toxins on an atomic scale. Graphical Abstract Keywords Ciguatoxins· Sodium channels· Molecular modeling· Ciguatera poisoning· Toxicity Abbreviations CTX Ciguatoxin CP Ciguatera poisoning EFSA European food safety authority FAO Food and agriculture organization FDA Food and drug administration IC50 Half-maximal inhibitory concentration MD Molecular dynamics PM Pore module * Concepción González-Bello [email protected] * Ana Gago-Martínez [email protected] * Luis M. Botana [email protected] Extended author information available on the last page of the article S.Raposo-García et al. 1 3 TEF Toxicity equivalency factor VGSC Voltage-gated sodium channels VSM Voltage-sensing module WHO World health organization Introduction Ciguatoxins (CTX) are marine biotoxins produced by benthic dinoflagellates of the genera Gambierdiscus and Fukuyoa (Gomez etal. 2015; Holmes and Lewis 1994). On the base of their chemical structures, four different ciguatoxin groups were described: Caribbean CTXs (C-CTX1), Indian CTXs (I-CTX1), and Pacific ciguatoxins, which are divided into two different groups, CTX3C and CTX4A and their derivatives as summarized by the FAO/WHO (Food and Agriculture Organization and the World Health Organization) report (FAO 2020). Among these groups, Pacific ciguatoxins analogues (P-CTXs) are the most widely distributed, studied, and evaluated. The chemical structures of Pacific and Caribbean ciguatoxins are represented in Fig.1. Regarding C-CTXs, the studies performed until now have shown that the most commonly found analogues are the Caribbean Ciguatoxin-1 and -2, named as C-CTX1 and C-CTX2 (Estevez etal. 2019b). Ciguatoxin-producing dinoflagellates were initially found in Indo-pacific oceans and in the Caribbean Sea reviewed by Shmukler and Nikishin (Shmukler and Nikishin 2017); however, during the last decades, as a possible result of climate change (Ramilo etal. 2021; Xu etal. 2016) and anthropogenic activities, including the globalization of trade, these microalgal organisms have spread to European coasts (Botana 2016; FAO 2020; Otero etal. 2010), and this fact rises the need to fully revaluate the potency of Caribbean and Pacific ciguatoxin analogues. CTXs cause in humans the syndrome named ciguatera poisoning (CP) due to the ingestion of high levels of the toxin accumulated by carnivorous fish in their musculature, liver, and viscera after predating herbivorous fish, reviewed by Friedman etal. (2017) (Díaz-Asencio etal. 2019; FAO 2020; Friedman etal. 2017). Nowadays, more than 400 fish species are associated with CP in humans, with barracuda, red snapper, grouper, amberjack, sea bass, surgeonfish, and Fig. 1 Chemical structure and ring denotation of the principal Pacific and Caribbean ciguatoxins In Silico Simulations andFunctional Cell Studies Evidence Similar Potency andDistinct Binding… 1 3 moray eel as the main responsible of these intoxications (FAO 2020; Pérez-Arellano etal. 2005). Moreover, recently, other species of marine invertebrates such as bivalves, echinoderms, and gastropods were also reported as additional vectors of CP (FAO 2020; Rhodes etal. 2020; Silva etal. 2015). CP is characterized by cold allodynia, gastrointestinal, neurological, and cardiovascular symptoms, starting from 30min after the ingestion of contaminated fish to 24/48h and persist for weeks and even months (Friedman etal. 2007). Each year up to 500,000 CP cases might occur globally reviewed by Dickey (2008) (Díaz-Asencio etal. 2019; Dickey 2008; Inserra etal. 2017). In the last years CP has expanded to new regions such as Canary Islands, Madeira Islands, Selvagens archipelagos, New Zealand, and the Mediterranean Sea (Costa etal. 2018; EFSA 2010; Otero etal. 2010; Tudó etal. 2020). In 2004, CP was first confirmed after consumption of flesh from lesser amberjack (Seriola rivoliana) from the Canary Islands, a non-endemic region for CP (Pérez-Arellano etal. 2005) and later on associated with the presence of Caribbean ciguatoxins in Madeira and Canary Islands (Boada etal. 2010; Estevez etal. 2019c, 2020; Otero etal. 2010; Sanchez-Henao etal. 2020). Thus CP is a notifiable disease in the Canary Islands since the year 2015 (Sanchez-Henao etal. 2019) and the current situation in Europe points to an increasing expansion of CTXs (Canals etal. 2021; Diogène etal. 2021); in fact, human intoxications from imported fish have been recently described in Germany (Loeffler etal. 2022). The lack of information regarding the relative toxicity of Pacific and Caribbean ciguatoxins makes it difficult to establish their potency, a remarkable fact since their increasing appearance and distribution raises global public concerns regarding human health. In vivo studies carried out in the 90s evidenced a half lethal dose (LD50) for intraperitoneal P-CTX of 0.25μg kg−1 body weight (Vernoux and Lewis 1997) and 3.6μg kg−1 and 1μg kg−1, respectively, for C-CTX1 and C-CTX2 (Lewis etal. 1999, 1991). However, the mouse bioassay is currently no longer considered an appropriate method to detect ciguatoxins in fishery products due to poor specificity and ethical concerns (EFSA 2010). In the European Union (EU), seafood products which may contain ciguatoxins are not allowed to be placed on the market (EU 2019). In contrast, the United States Food and Drug Administration (FDA) has established a guidance level for Caribbean CTXs of 0.1μg kg−1 C-CTX1 equivalents and 0.01μg kg−1 for P-CTX1B (FAO 2020; FDA 2011) based on their intraperitoneal toxicity in mice which implies that Pacific ciguatoxins are tenfold more potent than their Caribbean congeners (Lewis etal. 1991; Vernoux and Lewis 1997). Nowadays there are several drawbacks which hinder the establishment of the real potency of these emerging marine toxins: (i) the data obtained so far are uncertain because the presence of CTXs in fish tissues is difficult to detect and quantify (Caillaud etal. 2010), (ii) absence of animal toxicity reports for these compounds, and (iii) CTX doses related with clinical symptoms of CP in humans are not well defined. These factors are aggravated by the lack of certified reference standards for the CTX group of toxins (EFSA 2010). Finally, lethality as reported with the mouse bioassay is not a useful value to quantify CTX potency since these toxins cause complex non-lethal symptoms and CP due to their effect on voltage-gated sodium channels (VGSC) specifically the domain IV of site 5 (FAO 2020). Although the effect of ciguatoxins is primarily on voltage-gated sodium channels (Catterall etal. 1992), these compounds can also alter potassium-gated voltage channels functioning (Hidalgo etal. 2002). The levels of C-CTX allowed in food are 1.8μg kg-1 in inedible fish, 0.9 to < 1.8μg kg-1 edible in small quantities, and edible with less than 0.9μg kg-1 (Lewis etal. 1999). The spread worldwide and the scarce reliable information about the biological effects of these emergent toxins in the same cellular model led to several international organizations such as FAO, WHO, and EFSA to highlight the need for a full re-evaluation of the toxicity and relative potencies of these toxins (EFSA 2010; FAO 2020). This work was designed to fully evaluate and compare the cytotoxicity of Pacific and Caribbean ciguatoxin analogues, as well as their effects on the functional activity of sodium channels. Besides, to achieve a better understanding of the experimental results, docking and molecular dynamics (MD) simulations have been used to explore, in atomic detail, the plausible binding mode of these toxins to the α subunit of VGSC, as well as to get an insight of the conformational changes promoted by these compounds. The molecular modeling results on Nav channels presented here suggest that both analogues bind to sodium channels in an opposite conformation but have similar toxicity as evaluated using internationally adopted methods to detect these toxins (Caillaud etal. 2010; Castro etal. 2020). Experimental Section Modeling andSimulation Studies Building oftheThree‑Dimensional Model of Nav1.6 Channel intheUnbound Form The three-dimensional structure of Nav1.6 channel was created using the AlphaFold method (Jumper etal. 2021) (UniProt accession code Q9UQD0) and further validated by Molecular Dynamics simulation studies. To this end, the protein was immersed in a cube of ~ 80,000 TIP3P water molecules and neutralized by addition of sodium ions (3) using S.Raposo-García et al. 1 3 the molecular mechanics force field ff14SB of AMBER 17 (Case etal. 2017). The residues 1–108, 427–735, and 1778–1980, which correspond to long and unfolded loops not involved in the membrane part of the channel, were deleted for the simulation. Computation of the protonation state of titratable groups at pH 7.0 was carried out using the H++ Web server (Gordon etal. 2005). As a result of this analysis, all histidine residues were considered in their neutral forms except H1678. The neutral histidine residues were protonated in ε position except H920 and H1354, for which the δ position was selected. Disulfide bridges were used between the following pairs of cysteine residues: C281/ C333, C324/C339, C906/C912, C944/C953, C1356/C1376, and C1721/C1736. The minimization of the protein and MD simulation of the resulting minimized structure were carried out following our previously described protocol (Vazquez- Ucha etal. 2021) and 200-ns simulations were performed. The cpptraj module in AMBER 17 was used to analyze the trajectories and to calculate the root-mean-square deviation of atomic positions (rmsd) of the protein during the simulation (Roe and Cheatham 2013). The molecular graphics program PyMOL (DeLano 2002) was employed for visualization and depicting protein structures. Building oftheThree‑Dimensional Models ofNav1.6@ ciguatoxin Binary Complexes (1) Ligand preparation: The geometries of ciguatoxins CTX1B, CTX3C, and C-CTX1 were minimized using a restricted Hartree–Fock (RHF) method and a 6-31G(d) basis set, as implemented in the abinitio program Gaussian 09 (Frisch etal. 2009). Partial charges were derived by quantum mechanical calculations using Gaussian 09, as implemented in the R.E.D. Server (version 3.0) (Vanquelef etal. 2011), according to the RESP model (Cornell etal. 1995). The missing bonded and non-bonded parameters were assigned, by analogy or through interpolation, from those already present in the AMBER database (GAFF) (Wang etal. 2006, 2004). (2) Docking studies: The binding mode of ciguatoxins CTX1B, CTX3C, and C-CTX1 were first explored by docking using the program GOLD 2020.2.0 (Jones etal. 1997) and the coordinates of Nav1.6 channel in the unbound form. The snapshot after 200ns of simulation was selected. The previously minimized ciguatoxin geometries were used as MOL2 files. Each ligand was docked in 25 independent genetic algorithm (GA) runs, and for each of these, a maximum number of 100,000 GA operations were performed on a single population of 50 individuals. Operator weights for crossover, mutation, and migration in the entry box were used as default parameters (95, 95, and 10, respectively) as well as the hydrogen bonding (4.0Å) and van der Waals (2.5Å) parameters. The position of M388 was used to define the active site, and the radius was set to 30Å. The GOLD scoring function was used. MD Simulations Studies ofNav1.6@ciguatoxin Binary Complexes (1) Minimization: Ligand coordinates obtained by docking were employed as starting point for MD simulations of the corresponding binary complexes. The complexes immersed in a cube of TIP3P water molecules and sodium ions were minimized in four stages: (1) initial minimization of the ligands (1000 steps, first half using steepest descent and the rest using conjugate gradient); steps (2), (3), and (4) were performed as steps (a), (b), and (c) in the minimization of the unbound form. A positional restraint force of 50kcal mol–1Å–2 was applied to those unminimized atoms during the first three stages (1–3). (2) Simulations: MD simulations of the Nav1.6@ciguatoxin binary complexes were performed as indicated for the unbound protein form (see above). For Nav1.6@CTX1B and Nav1.6@C-CTX1 complexes, simulations of 200ns were carried out, while for Nav1.6@CTX3C complex, 100-ns simulation was performed. Binding Free Energies Calculations The binding free energy for CTX1B, CTX3C, and C-CTX1 was calculated by the MM/PBSA approach implemented in Amber Tools 17 (Miller etal. 2012) ante-MMPBSA.py module was used to create topology files for the complex, enzyme, and ligands and binding free energies were calculated with the MMPBSA.py module. A single trajectory approach was used to calculate binding free energies considering only the last 100ns of the 200-ns MD trajectories. For the 100-ns MD trajectories, the last 80ns were considered. The Poisson–Boltzmann (PB) and Generalized Born (GB) implicit solvation models were employed. Both models provided similar results. Toxins andDrugs Used For the N2a-MTT assay and patch-clamp recordings, C-CTX1-purified solution (25.7ng/mL) and C-CTX1 Fish Tissue Reference Material (FTRM) (0.284 ± 0.019ng/g) were purified from an amberjack (Seriola spp.) from the Canary Islands (Spain) and were prepared at University of Vigo, in the framework of the project EuroCigua. For the LC–MS/MS analyses CTX1B, 52-epi-54-deox- yCTX1B, 54 deoxyCTX1B, CTX3C, CTX4A, and CTX4B reference materials were kindly provided by Professor Takeshi Yasumoto (Japan Food Research Laboratories, Japan). In Silico Simulations andFunctional Cell Studies Evidence Similar Potency andDistinct Binding… 1 3 C-CTX1 pure standard was kindly provided by Dr. Robert W. Dickey (University of Texas, USA) via Dr. Ronald Manger (Fred Hutchinson Cancer Research Center, Seattle, USA). CTX1B standard solution (4488ng/mL) was kindly provided by Professor Takeshi Yasumoto (Japan Food Research Laboratories, Japan). CTX3C standard solution (100ng) was purchased from FUJIFILM Wako Chemicals Europe GmBH (Neuss, Germany). The stocks standard solutions C-CTX1, CTX1B, and CTX3C used for N2a-MTT assay were diluted in methanol HPLC grade (MeOH, Merck KGaA, Darmstadt, Germany) until a concentration of 10pg/µL and were stored in dry conditions at −20°C. For electrophysiological recordings Pacific ciguatoxins, CTX3C as well as CTX1B, were purchased from Wako (FUJIFILM Wako Chemicals Europe GmbH, Neuss, Germany) and dissolved in dimethyl sulfoxide (DMSO) at a final concentration of 10μM (corresponding to 10,230ng/ mL of CTX3C and 11,113ng/mL of CTX1B), respectively. For experiments, 1-µM solutions (1023ng/mL CTX3C and 1111.3ng/mL CTX1B) were prepared in Locke’s buffer containing (in mM): 154 NaCl, 5.6 KCl, 1.3 CaCl2, 1 MgCl2, 10 HEPES, and 5.6 glucose, pH 7.4. The maximum solvent concentration used as a control was 0.1% DMSO and had no effect on voltage-gated sodium current amplitude. Similarly, C-CTX1 was dissolved in DMSO at a final concentration of 1μM (1141.4ng/mL C-CTX1) and dilutions were performed in Locke’s buffer, from an original solution of 25.7ng/mL. Sample Pretreatment C-CTX1 FTRM was extracted and purified following the conditions described by Estevez etal. (Estevez etal. 2019a). Briefly, Fish tissue (200g) was extracted twice with acetone (2 × 600mL) using an Ultraturrax® homogenizer. Acetone layers were combined (estimated volume of 1200mL) and evaporated to an aqueous residue using a multievaporator Syncore Polivap set at 45℃. The aqueous residue (estimated volume 150mL) was extracted twice with diethyl ether (2 × 200mL). The combined organic layers (400mL) were evaporated to a solid residue using a multievaporator Syncore Polivap set at 45℃. The solid residue was dissolved in 90% methanol (60mL) and defatted with twice the volume of n-hexane (120mL). The methanol residue was evaporated to dryness under reduced pressure and submitted to the purification step. The purification step was carried out using Solid-Phase Extraction (SPE) cartridges. An equivalent of 15g of fish tissue was loaded in each SPE cartridge. The solid residue from the extraction was dissolved in ethyl acetate (26mL) and aliquots of 2mL were loaded in a Florisil SPE cartridge (J. T. Baker, 500mg) previously conditioned with ethyl acetate (3mL). The cartridge was washed with ethyl acetate (3mL) and C-CTX1 was eluted with ethyl acetate:methanol 9:1 (v:v) (5mL). The toxic eluate was evaporated to dryness under a N2 stream at 40℃. The solid residue was reconstituted in 60% MeOH (26mL) and aliquots of 2mL were loaded in a C18 SPE cartridge (SUPELCLEAN, Supelco, 500mg) previously conditioned with 60% MeOH (3mL). The cartridge was washed with 60% MeOH (3mL) and C-CTX1 was eluted with 90% MeOH (5mL). The eluates from C18 SPE were combined and evaporated to dryness being filtered through 0.22-μm PVDF filter (Syringe Driver filter Unit, Millex®-CV 0.22μm, 13mm). LC–MS/MS Analysis LC–MS/MS analyses were performed following the conditions proposed by Estevez etal. (Estevez etal. 2019a) using an Agilent 1290 Infinity LC system coupled to an Agilent 6495 triple quad iFunnel (Agilent Technologies, Waldbronn, Germany). CTXs were separated using a C18 column (Poroshell 120- EC-C18, 3.0 × 50mm, 2.7µm, Agilent USA). The column temperature was set at 40ºC and the mobile phases for the chromatographic separation were (A) 0.1% formic acid and 5mM of ammonium formate in water and (B) MeOH. The gradient started at 78% of B with a linear gradient to 88% of B in 10min, holding 5min and increasing to 100% of B until 18min to wash the column. The gradient returned to 78% of B at 18min and the column was equilibrated during 4min prior to the next injection. The flow rate was set at 0.4mL/ min and the injection volume was 1 µL. The mass spectrometer operated in positive ionization mode (ESI+) monitoring by dynamic Multiple Reaction Monitoring (dMRM) the sodium adduct [M + Na]+ of the different CTXs as precursor and product ion at a collision energy (CE) of 40eV. Source and interface parameters were as the following: drying gas flow, 15 L/min, drying gas temperature 290°C, sheath gas flow 12 L/min, sheath gas temperature 400°C, nebulizer pressure 50 psi, fragmentor potential 380V, nozzle voltage 300V, and capillary voltage 5000V. The following toxins with reference material available were monitored (Table1): CTX1B, C-CTX1, 52-epi- 54-deoxyCTX1B, 54-deoxyCTX1B, CTX3C, CTX4A, and CTX4B. Human Cell Cultures (hNav1.6 HEK cells) Human embryonic kidney cell line (HEK293) stably expressing the human Nav1.6 alpha subunit of the sodium channels were kindly provided by Dr Andrew Powell (GlaxoSmithKline R&D, Stevenage, UK) cultured in DMEM/F12 medium supplemented with Glutamax, MEM non-essential S.Raposo-García et al. 1 3 amino acids solution (Gibco, 1% w/v), and 10% fetal bovine serum as previously described (Boente-Juncal etal. 2021). Geneticin (Gibco) was added at a final concentration of 0.4mg/mL to increase the expression of sodium channels. Cells were seeded at a density of 30,000 cell/mL and maintained at 37°C in a 5% CO2/95% O2 incubator until the cultures reached an 80% of confluence and placed at 30ºC for 24–48h before electrophysiological measurements (Burbidge etal. 2002). N2a‑MTT Assay Cytotoxicity assays were carried out using two different batches of Neuro-2a (N2a) cell line (ATCC® CCL-131), purchased from the American Type Culture Collection (LGC standards S.L.U., Barcelona, Spain) and numbered as 63649750 and 70005648. Base culture medium Roswell Park Memorial Institute (RPMI-1640, R8758, Sigma, Irvine, UK) was supplemented with 1% of 100mM sodium pyruvate (S8636, Sigma, Irvine, UK), 1% of 200mM l-Glu- tamine (G7513, Sigma, Irvine, UK), 1% of Penicillin–Streptomycin formed by 5,000 units and 5mg mL−1, respectively, (P4458, Sigma, St. Louis, MO, USA), and 10% or 5% v/v of fetal bovine serum (FBS, F2442, Sigma, St. Louis, MO, USA) to obtain an adequate complete growth media (RPMI-10 and RPMI-5, respectively). For passing or seeding, cells were unattached with trypsin EDTA solution (1x) (Sigma-Aldrich, St. Louis, MO, USA). Other reagents for N2a-MTT assay include phosphate-buffered saline (PBS), veratridine hydrochloride, ouabain octahydrate, DMSO, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), all of them purchased from Sigma-Aldrich (St. Louis, MO, USA). N2a cells were maintained in T75 flask (Corning, NY, USA) with 30mL of RPMI-10 at 37°C in a humidified atmosphere containing 5% CO2 and subcultured using a split 1:5 regarding initial cell culture every 48h. The experiments were done for three different cell passages per batch: passages 214/215/224 and 203/205/220 were selected in batch 63649750 and 70005648, respectively. For the cytotoxicity assay, methanol extracts of CTXs standards (C-CTX1, CTX3C, and CTX1B) were evaluated using the N2a-MTT assay protocol previously described by Castro etal., (2020). Briefly, 40,000 cells were seeded per well into 96-well assay plates (Corning, NY, USA) in 0.2mL of RPMI-5 and left to grow for 24h at 37°C under a humidified atmosphere enriched with 5% CO2. Then, cells were sensitized with a mixture 10:1 (v/v) of Ouabain/Veratridine solution (O/V). A good sensitivity for detection of CTX-like compounds was reached for a concentration of O/V which provokes a reduction of 20% in cell viability respect to non-sensitized cells. Ten serial dilutions were prepared from the CTXs stock standard solutions in RPMI-C, and sensitized cells were exposed 16h to each concentration level, ranging from 0.00019 to 0.019nM for C-CTX1, 0.0002 to 0.02nM for CTX1B, and 0.00021 to 0.021nM for CTX3C, in replicates of four wells. After this time, cell viability was estimated from absorbance due to formazan produced by the colorimetric MTT assay (Mosmann 1983). Absorbance readings were obtained with a multi-well scanning spectrophotometer (Thermo Fisher Scientific, Ratastie, Finland) using 530nm as testing wavelength and 630nm as reference wavelength. Absorbance data were expressed as percentage of cell viability relative to control. Resulting values were fitted to a sigmoidal four-parameter logistic function with hill slope (4PL). From these dose–response curves, inhibitory concentrations (IC50), standard concentration required to cause a reduction of 50% in cell viability expressed in nM, were calculated with the SigmaPlot v.12.0 software. Electrophysiological Recordings Whole-cell voltage clamp recordings, achieved by gentle mechanical suction of the membrane patch, were performed in the HEK293 cell line expressing the human Nav1.6 alpha subunit as previously described (Burbidge etal. 2002). Recording electrodes were fabricated from borosilicate glass microcapillaries (outer diameter, 1.5mm), and the resistance was 5–10 MΩ. The progress of seal formation was evaluated by monitoring the Table 1 MRM transitions of the different CTXs monitored by LC–MS/MS Toxin Precursor ion [M + Na]+ (m/z)Product ion [M + Na]+ (m/z)Fragmentor (V) CE (eV) CAV (eV) CTX1B 1133.6 1133.6 380 40 4 C-CTX1 1163.7 1163.7 380 40 4 52-epi-54-deoxyCTX1B 1117.6 1117.6 380 40 4 54-deoxyCTX1B 1117.6 1117.6 380 40 4 CTX3C 1045.6 1045.6 380 40 4 CTX4A 1083.6 1083.6 380 40 4 CTX4B 1083.6 1083.6 380 40 4 In Silico Simulations andFunctional Cell Studies Evidence Similar Potency andDistinct Binding… 1 3 decrease in membrane resistance. Series resistance was compensated by about 70% with the internal compensation circuitry of the CV-7B current clamp and voltage clamp headstage. Cells were maintained at a holding potential (Vhold) of − 55mV and data were recorded after the membrane resistance has stabilized. Voltage-gated sodium currents were recorded using standard whole-cell voltage clamp recordings at room temperature with a Multiclamp 700B amplifier and digitalized with the Digidata 1440A data acquisition system (both from Axon Instruments, California, U.S.A.). Signals were sampled at 50kHz after low pass Bessel filtering at 10kHz. Leak currents were subtracted from active currents by the P/N leak protocol provided by the pClamp10 software. After establishing the whole-cell configuration, cells were allowed to stabilize for 5min before current recording protocols were initiated to ensure adequate equilibration between the internal pipette solution and the cell cytoplasm. Offline analysis was performed with the pClamp10 software (Axon Instruments). Intracellular electrode solution contained (in mM): 120 CsF, 15 NaCl, 10 EGTA, and 10 HEPES (pH 7.25 adjusted with CsOH), while the extracellular solution was Locke’s buffer. To obtain the concentration–response data each ciguatoxin was directly added to the recording chamber and in all the experiments the effect of CTXs on VGSC was evaluated 5min after bath application of each toxin concentration or the corresponding amount of solvent. To record sodium currents (INa) and the activation of voltagegated sodium channels voltage steps from − 80 to + 80mV (10mV increments, 17 steps and 0.15s duration) were applied. Data analysis was performed using GraphPad Prism 5. All data are expressed as means ± SEM of n determinations. Statistical comparisons were performed by one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test. P values < 0.05 were considered statistically significant. Results In the present work the binding of Caribbean and Pacific ciguatoxins to sodium channels was studied by an integrated approach that demonstrated similar toxic activity at the cellular level but different interactions with the protein that cause distinct effects on the activation voltage of sodium channels. Molecular Modeling Studies The binding mode of CTX1B, CTX3C, and C-CTX1 with the α subunit of the human Nav1.6 channel was investigated in silico. As no three-dimensional structure of this protein was available, a homology model was constructed using the AlphaFold method (Jumper etal. 2021) and further validated by Molecular Dynamics (MD) simulation studies. To this end, the protein was immersed in a truncated cube of TIP3P water molecules and neutralized by addition of sodium ions using the molecular mechanics force field AMBER 17 (Case etal. 2017). The minimization of the protein and the MD simulation (200ns) of the resulting three-dimensional structure was carried following our previously reported protocol (Vazquez-Ucha etal. 2021). The reported model in the unbound form is stable as no significant changes were observed during the whole simulation. The latter is easily visualized by the analysis of the root-mean-standard deviation (rmsd) of the whole-protein backbone, which showed relatively low values considering that it is a protein of about 2000 residues (Fig. S1). Fig. 2 Overall and detailed views of the three-dimensional model of the α subunit of the human Nav1.6 channel in the unbound form obtained by MD simulation studies. Snapshot taken after 200 ns of simulation is given. The four domains of the channel (I–IV) are shown in purple, orange, gray, and blue, respectively. Detailed views of domain IV, as well as the main hydrogen-bonding interactions (black-dashed lines and yellow shadow) of the inner part of the voltage-sensing module 4 (VSM4) are provided. Relevant side-chain residues are shown and labeled S.Raposo-García et al. 1 3 As shown in Fig.2, the α subunit of the human Nav1.6 channel consists of four homologous but not identical transmembrane domains (I–IV), each one composed by six transmembrane segments (S1–S6) that form (i) a voltage-sens- ing module (VSM) containing segments S1–S4; (ii) a pore module (PM) containing segments S5 and S6 and a P-loop that connects them; and (iii) the helical hairpin S4–S5 that connects both modules that seem to act as an elbow in the electromechanical motion of the VSM (Wisedchaisri etal. 2019). Four positive gating charges (arginine residues) that are exquisitely distributed along S4 with their side chains pointing toward segments S1–S3 seem to modulate the VSM function through polar interactions. Thus, these positively charged residues, which are stabilized on the same face of S4 through diverse hydrogen-bonding and electrostatic interactions with the side-chain residues of helices S1–S3, undergo conformational changes as a response to the depolarization and repolarization of the membrane and would trigger longdistance motions in PM to open and close the pore (Ahern etal. 2016; Catterall etal. 2017). Once the model of the unbound form was validated, the binding modes of Pacific ciguatoxins CTX1B and CTX3C and Caribbean ciguatoxin C-CTX1 were studied by docking using the program GOLD 2020.2.0 (Jones etal. 1997) and the coordinates of Nav1.6 channel in the unbound form taken after 200ns of simulation. To define the docking region, it was considered that (i) as brevetoxins, CTXs bind to the S5 segment of domain IV of the channel (Cestele and Catterall 2000; FAO 2020) and (ii) the alanine scanning mutagenesis studies reported by (Konoki etal. 2019) on Nav1.2 channel showed that changes in the central site of segment S5, specifically residues I1654, G1655, L1656, L1657, and F1659, reduce the affinity of brevetoxin by two–threefold against the wild-type. This region of S5, which is conserved in the Nav1.6 channel, was therefore selected for binding. To assess the reliability of the GOLD-proposed binding mode, the highest score solutions for each CTX obtained by docking were further analyzed by MD simulation studies following a similar protocol as for the unbound form. Following the same protocol, the binding mode of brevetoxin BTX-3 was also explored for comparison. The outcomes of these simulation studies showed that all CTXs have in common the following features (Fig.3): (i) as brevetoxin, these compounds bind to the cleft created by segment S5 of domain IV, segment S6 of domain I, and the P-loop (P1) that connects segments S5 and S6 of domain IV (Fig. S2). The latter binding involves about half of the cyclic polyether skeleton, specifically moieties F–M for CTX1B and CTX3C and moieties A–I for C-CTX1. All CTXs showed to be very stable within the pocket as no significant changes were observed during the whole simulation (Fig. S3), either for the ligand (Fig. S4) or the protein backbone (Fig. S5); (ii) all CTXs interact by hydrogen bonding with at least one protein residue. CTX1B and C-CTX1 interact with the ɛ-amino group of K385 (S6, domain I) through the hydroxyl group in rings M and A, respectively, while for CTX3C this polar contact occurs between the main carbonyl group in G1584 and the NH group in W1584 and its hydroxyl group (Fig. S6). C-CTX1 establishes additional hydrogen-bonding interactions with residues N1485 and T1482, which are located on the helical hairpin close to segment S5; and (iii) all CTXs lie on the protein cleft thanks to numerous lipophilic interactions, mainly between the axial hydrogen atoms and methyl groups of the linkage between rings and the side-chain residues within the cleft. The latter interactions occur in great extent through both faces of the ciguatoxins polycyclic skeleton. Full details of the residues involved are provided in the supporting information (TableS1). While no significant differences on the overall binding mode of the two Pacific ciguatoxins were observed, except that CTX3C burrows deeper into the cleft than CTX1B as it lacks substituents on the terminal rings A and M (Fig.3A, B), the same does not occur for the Caribbean ciguatoxin (Fig.3C). Thus, the most relevant binding differences among these CTXs are as follows: (i) while Pacific ciguatoxins CTX1B and CTX3C bind to the α subunit of the human Nav1.6 channel with the ring M pointing toward its extracellular part and ring A toward the cytoplasm [head(IN) to tail(OUT)], C-CTX1 binds in the reverse manner, thus with ring A toward the extracellular part of the channel [tail(IN) to head(OUT)] and (ii) the overall extended conformation of ciguatoxins CTX1B and CTX3C causes binding of moieties A–E of the cyclic polyether skeleton to the interface formed by segment S3 and helical hairpin S4–S5; the curved shape of the Caribbean ciguatoxin C-CTX1 avoids the interaction with the latter site of the channel and facilitates further contacts between its moiety J–N with segment S5 as well as adjacent sites near the cytoplasm. Comparison of the conformation of domain IV of the channel upon CTXs binding with the obtained in the unbound form highlighted relevant differences in the relative arrangement of VSM4 and the helical hairpin S4–S5 in all complexes (Fig.4). Since the Pacific ciguatoxins CTX1B and CTX3C gave similar results, only the outcomes of ciguatoxins CTX1B and C-CTX1 are presented herein but additional information is given in the supporting information (Fig. S7). For the Nav1.6@CTX1B complex, the binding of a fragment of the ciguatoxin skeleton in the interface between segment S3 and linker S4–S5 causes an extended arrangement of the latter relative to segment S5, which implies a loss of the elbow conformation observed in the free form as well as a reduction in the overall plasticity of this region of the protein (Fig.4A, B). In addition, the protein site in segment S4 closest to the linker S4–S5 becomes less folded and segments S2 and S3 In Silico Simulations andFunctional Cell Studies Evidence Similar Potency andDistinct Binding… 1 3 move away from the PM4 domain. Helices S3 and S4, and somewhat less pronounced helix S2, twist mainly at their C-terminal part, which is closest to the cytoplasm. As a result of all these conformational changes, the position of the central gating charge in S4, R1626, is less controlled since the stabilizing partners, the side chains of residues N1558 (S2) and S1596 (S3), move away from its guanidinium group (Fig.4D, E). Thus, while for the unbound form, Fig. 3 Overall and detailed views of the three-dimensional models of the α subunit of the human Nav1.6 channel in complex with CTX1B (A), CTX3C (B), and C-CTX1 (C) obtained by MD simulation studies. Snapshots taken after 200ns of simulation are given. Domains I–IV are shown in purple, orange, gray, and blue, respectively. In the surface representation, the ciguatoxins are highlighted in spheres. Detailed views of the main contacts between ciguatoxins and the transmembrane segments of the protein are provided. Relevant sidechain residues are shown and labeled. Hydrogen-bonding interactions are provided as dashed lines (red) and with a yellow shadow. Note how both Pacific ciguatoxins bind in a similar manner (head to tail), interacting with the transmembrane segments of domains I and IV: segments S5 (including the P-loops that connect S5 and S6, IV) and S3 (IV), the S4–S5 linker that connect VSM4 with the PM4, and segment S6 (I). On the contrary, C-CTX1 binds with ring A pointing toward the extracellular part of the channel (tail to head) and the interaction with the VSM4 (S3 and S4) is weaker than for Pacific ciguatoxins S.Raposo-García et al. 1 3 however, as far as we know this is the first study analyzing the effect of Caribbean and Pacific ciguatoxins in the same model and using three independent parameters. Besides, in this report the differences in the binding of the CTXs congeners to sodium channels were studied by MD simulation studies, suggesting that the binding of Pacific and Caribbean CTX to sodium channels alter the function of the pore. The results obtained clearly show that the three CTXs analogues caused a similar toxicity, but strikingly CTX3C and C-CTX1 exhibited the same potency, a fact that is at odds with the previously published data (EFSA 2010; Lewis etal. 1991; Vernoux and Lewis 1997). CTX1B had the highest cytotoxic effect and the greatest potency to inhibit sodium currents, followed by CTX3C and C-CTX1 which were very similar on their invitro effects. The higher potency obtained by the fish tissue extract may be due to the matrix effect, since as previously described (Castro etal. 2020) matrix effects can lead to changes in the cytotoxic effect of CTXs depending on the total lipid content of the fish specimens. In this study, the shift in the voltage activation of sodium currents, commonly caused by CTXs, was evaluated and Pacific analogues significantly shifted the voltage activation of sodium channels to more hyperpolarising potentials at concentrations of 1nM and higher. Again, this observation is in accordance with the previously described effects of ciguatoxins on sodium channels (Inserra etal. 2017; Strachan etal. 1999; Yamaoka etal. 2004). However, this fact could not be corroborated for the Caribbean analogue C-CTX1 nor the fish extract since, in this case, lower concentrations were used (from 0.0001nM to 1nM), due to the low availability of compounds. However, at 1nM, C-CTX1 did not modify the activation potential of sodium channels. The MD simulation studies performed with Pacific ciguatoxins suggested that the shift in the activation voltage of sodium channels caused by these toxins would be due to their interaction with the interface between VSM4 (segment S3) and the linker S4–S5 that connect the latter with segment S5. This binding, which proved to be similar for CTX1B and CTX3C, seems to promote relevant conformational changes in segments S2–S4, altering the ability of the positively charged residues (specifically R1626) to achieve the optimal conformation for establishing polar interactions with the counterion residues inside VSM. As a result of all these changes, the capacity of VSM to perform the required electromechanical motion as a response to the depolarization/repolarization of the membrane would be modified. These findings would agree with the role of the linker S4–S5 in the established “sliding helix” mechanism of voltage sensing reported by (Wisedchaisri etal. 2019) as the elbow that slide helix S4 through the channel for controlling the motion of the gated residues (arginines) for voltage sensor functions. On the other hand, the simulation studies carried out with the Nav1.6@C-CTX1 complex suggested that there is no direct interaction between the Caribbean ciguatoxin and this hinge zone of the channel, as well as no significant changes on the polar interactions network of the positively gated residues. This finding would also justify the experimentally observed lack of shifting on the voltage activation of the channel caused by this toxin. In this case, C-CTX1 seems to reduce the plasticity of the C-terminal part of helix S4, which is connected to the linker S4–S5, which would also compromise the intrinsic sliding of S4 for modulating the channel activity as for the Pacific ciguatoxins. Globally, despite the differences found in the binding mode of both types of ciguatoxins, either in its binding orientation in the channel Nav1.6 (head to tail and vice versa) or the specific binding pocket of part of its cyclic polyether skeleton, both CTXs seem to freeze the intrinsic motion of the VSM for activity, which would also trigger long-distance conformational changes in PM. The combined information provided in this work matches, from different approaches and technologies, the current observations regarding the toxicity of CTXs: (i) The calculated binding free energies of −63.5 ± 0.2kcal mol−1 and −61.9 ± 0.2kcal mol−1 for CTX1B and C-CTX1 explain the long-term effects of these toxins, as these energies are consistent with high-affinity ligands (high Kon and low Koff); (ii) the fact that the binding orientation of both types (head to tail and vice versa) generate different modes of voltage sensor activation explains why C-CTX do not modify the activation shift, and this is related to the different observed toxic profiles of P-CTX vs C-CTX. P-CTX is associated to more intense neurological symptoms, while C-CTX is associated to gastrointestinal effects (probably of neurological origin) and later peripheral nervous symptoms (Boucaud-Maitre etal. 2018); and (iii) the activation shift, also observed for other low toxicity molecules, such as gambierol, gambierone, or MTX3 (Raposo-Garcia etal. 2022), contributes to neuronal firing (observed specially in P-CTX toxicity), but Table 4 Comparison of the relative toxicity of CTX1B, CTX3C, and C-CTX1 using invitro techniques with the relative toxicity accepted by authorities Analogue INa inhibition N2a (B-63649750) N2a (B-70005648) EFSA Mean SD Mean SD CTX1B 1.00 1.00 –1.00 – 1.00 CTX3C 0.74 0.71 0.14 0.71 0.05 0.2 C-CTX1 0.74 0.73 0.13 0.63 0.05 0.1 In Silico Simulations andFunctional Cell Studies Evidence Similar Potency andDistinct Binding… 1 3 not to mice lethality, that seems to be mainly associated to peak currents, as evidenced by data shown in this paper. In view of these data, a full re-evaluation of the relative potencies of the CTXs-group of toxins should be pursued using purified compounds and the most sensitive techniques available to guarantee food safety and thus protect human and animal health. Given the TEF results shown in Table4 that we have obtained by independent studies with patch clamp in human cells and with mouse neuroblastoma, the current legal requirements that assign a tenfold lower potency for Caribbean ciguatoxins do need to be re-eval- uated. As shown in Table4, the mean functional relative potency of the ciguatoxin congeners used in this study was 1 for CTX1B, 0.73 ± 0.036 for CTX3C, and 0.71 ± 0.032 for C-CTX1, summarizing that the potencies of Pacific and Caribbean ciguatoxins are quite similar. One of the main concerns of this study, which is the same for any other study that uses Caribbean ciguatoxins, is the lack of a reference calibrant. Although the quantitation of C-CTX was done against an NMR-quantitated P-CTX, still clearly there is an urgent need of these certified toxins to eliminate quantitative uncertainties. Conclusion The results obtained in this study demonstrate that the Pacific analogue CTX1B is the most potent compound of this group, while CTX3C and C-CTX1 caused the same effect confirmed by the cytotoxicity assay and similar effects on the amplitude of sodium currents evaluated by electrophysiological recordings. Both in the cytotoxicity test and in the decrease on sodium current intensity the effect of both analogues CTX3C and C-CTX1 was only 0.3 times lower than that of CTX1B. These data are contrary to what is stated by EFSA and the American FDA, which established a relative potency of C-CTX1 10 times lower than Pacific analogues. Therefore, this study highlights the need to reevaluate the relative toxicities and potencies of ciguatoxins to establish safety limits for the presence of these emergent toxins in commercialized fish. In addition, docking analyses and simulation studies have demonstrated that the distinct binding mode of ciguatoxins with the channel Nav1.6 (head to tail and vice versa) justify their different impacts on the activation potential of sodium channels and confirm that this parameter will be more suitable to detect ciguatoxins in suspicious fish samples. Supplementary Information The online version contains supplementary material available at https:// doi. org/ 10. 1007/ s12403- 022- 00513-0 Acknowledgements The author’s thank Takeshi Yasumoto (Japan Food Research Laboratories) for kindly provided standards of Pacific ciguatoxins. Robert W. Dickey (University of Texas at Austin Marine Science Institute, USA) via Ronald Manger (Fred Hutchinson Cancer Research Center, Seattle, USA) for kindly provided standard of C-CTX1. We are also grateful to the Centro de Supercomputación de Galicia (CESGA) for use of the Finis Terrae computer. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. The research leading to these results has received funding from the following FEDER-co-funded grants. From Conselleria de Cultura, Educacion e Ordenación Universitaria, Xunta de Galicia, GRC (ED431C 2021/01, ED431C 2021/29, and the Centro singular de investigación de Galicia accreditation 2019–2022 ED431G 2019/03). From European Union Interreg AlertoxNet EAPA-317-2016, Interreg Agritox EAPA-998-2018, and H2020 778069-EMERTOX, and the EUROCIGUA project: “Risk Characterization of Ciguatera Fish Poisoning in Europe” GP/ EFSA/AFSCO/2015/03, co-funded by the European Food Safety Authority (EFSA). From Ministerio de Ciencia e Innovación PID2020-115010RB-I00/AEI/10.13039/501100011033. David Castro (D.C.) financial support for the PhD studies was obtained through EUROCIGUA project: Risk characterization of Ciguatera Fish Poisoning in Europe, framework partnership agreement GP/EFSA/ AFSCO/2015/03, co-funded by the EFSA. Pablo Estevez (P.E.) acknowledges financial support from the Xunta de Galicia (Regional Government, Spain) under grant ED481A-2018/207. Data Availability Enquiries about data availability should be directed to the authors. Declarations Conflict of interest The authors declare no financial or commercial conflict of interest. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. References Ahern CA, Payandeh J, Bosmans F, Chanda B (2016) The hitchhiker’s guide to the voltage-gated sodium channel galaxy. 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S.Raposo-García et al. 1 3 Authors and Affiliations SandraRaposo‑García1 · DavidCastro2· EmilioLence3· PabloEstévez2· JoséManuelLeão2· ConcepciónGonzález‑Bello3· AnaGago‑Martínez2· M.CarmenLouzao1· CarmenVale1· LuisM.Botana1 Sandra Raposo-García [email protected] David Castro [email protected] Emilio Lence [email protected] Pablo Estévez paeste[email protected] José Manuel Leão [email protected] M. Carmen Louzao mcar[email protected] Carmen Vale mdelcarmen.v[email protected] 1 Departamento de Farmacología, Farmacia y Tecnología Farmacéutica, Facultad de Veterinaria, Universidade de Santiago de Compostela, Campus Universitario s/n, 27002Lugo, Spain 2 Biomedical Research Center (CINBIO), Department ofAnalytical andFood Chemistry, University ofVigo, Campus Universitario de Vigo, 36310Vigo, Spain 3 Centro Singular de Investigación en Química Biolóxica E Materiais Moleculares (CiQUS), Departamento de Química Orgánica, Universidade de Santiago de Compostela, Jenaro de la Fuente s/n, 15782SantiagodeCompostela, Spain