The E15R point mutation in scorpion toxin Cn2 uncouples its

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The E15R point mutation in scorpion toxin Cn2 uncouples its depressant and excitatory activities on human NaV1.6 Mathilde R. Israel, Panumart Thongyoo, Jennifer R. Deuis, David J Craik, Irina Vetter, and Thomas Durek J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01609 • Publication Date (Web): 29 Jan 2018 Downloaded from http://pubs.acs.org on January 30, 2018

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Journal of Medicinal Chemistry

The E15R point mutation in scorpion toxin Cn2 uncouples its depressant and excitatory activities on human NaV1.6 Mathilde R. Israel,†,# Panumart Thongyoo,†,# Jennifer R. Deuis,† David J. Craik,† Irina Vetter,†,‡,* and Thomas Durek†,* †

Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD 4072, Australia School of Pharmacy, The University of Queensland, Woolloongabba, QLD 4102, Australia KEYWORDS: native chemical ligation, peptide synthesis, analgesic scorpion toxin, NMR, voltage-gated sodium channel ‡

ABSTRACT: We report the chemical synthesis of scorpion toxin Cn2; a potent and highly-selective activator of the human voltagegated sodium channel NaV1.6. In an attempt to decouple channel activation from channel binding, we also synthesised the first analogue of this toxin, Cn2[E15R]. This mutation caused uncoupling of the toxin’s excitatory and depressant activities, effectively resulting in a NaV1.6 inhibitor. In agreement with the in vitro observations, Cn2[E15R] is anti-nociceptive in mouse models of NaV1.6-mediated pain.

Centruroides noxious proved especially useful in dissecting the roles of the individual NaV isoforms in vivo because of its potent4 and highly selective activation of NaV1.6.6 These pharmacological properties make Cn2 a promising candidate for development of advanced NaV1.6-selective molecular probes with altered mechanism of action to explore their effect on NaV1.6 physiology.

INTRODUCTION: The generation and propagation of action potentials in neurons is realized through a well-orchestrated interplay of ion channels in which the family of voltage-gated sodium channels (NaV) plays a central role.1-2 Humans have at least nine functional NaV α subunits (NaV1.1–NaV1.9) that mediate specialized physiological processes in different tissues and electrically excitable cell types. Dissecting the physiological roles of individual subtypes has been hampered in the past because multiple individual NaV subtypes often co-localize and their central α subunits are structurally similar. The latter aspect in particular has also made it difficult to develop highly NaV– specific and subtype–selective modulators that could be used as molecular probes or as potential therapeutics in NaV -related diseases. Molecules modulating the activity of NaVs are abundant in the venoms of cone snails, spiders and scorpions and are noteworthy for their high potency and selectivity for individual Nav isoforms.3 These small disulfide-rich peptides bind to NaVs with high affinity and can interfere with specific steps of the NaV gating mechanism; for example by occluding the central pore or by interacting with one or more of the four voltage-sensing domains (VSDs) that control channel opening and closing. Using a range of NaV modulators and knockout animals, we recently demonstrated an important role for NaV1.6 in multiple peripheral pain pathways, particularly those involved in mediating cold and mechanical allodynia.4 Further exploration of the role of NaV1.6 in pain pathways and evaluation of NaV1.6 as a potential therapeutic target hinge on the availability of advanced molecular tools or model systems such as NaV1.6 knock-out animals (which are not viable) and/or NaV1.6selective molecular probes, such as 4,9-anhydro-tetrodotoxin that shows some NaV1.6 selectivity in vitro.5 In the abovementioned studies, the long-chain scorpion β-toxin Cn2 from

Scheme 1: Chemical synthesis of Cn2 by native chemical ligation. (i) native chemical ligation; (ii) folding and disulfide formation. The inset shows the primary structure of Cn2, with cysteines in bold and the ligation site (Glu28-Cys29) underlined. The inverted ‘E’ highlights the location of the point mutation (E15R).

RESULTS AND DISCUSSION: Scorpion β-toxin Cn2 is a polypeptide of 66 amino acid residues that is C-terminally carboxamidated and adopts a cysteine-stabilized αβ fold supported by four disulfide bonds.7-8 Cterminal carboxamidation is a posttranslational modification that is difficult to reproduce in bacterial expression systems and was recently shown to be important for high-affinity NaV1.4 binding of the related long-chain scorpion toxin Ts3.9

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Figure 1: Characterization of synthetic Cn2. a) uHPLC and MALDI-MS (inset) of synthetic wild-type Cn2 (calculated MW: 7584.49 Da (M+H+, monoisotopic composition). b) NMR Hα-chemical shift analysis derived from 2D TOCSY and NOESY spectra indicates correct folding of all synthetic analogs relative to native, venom derived Cn2 (PDB ID: 1CN2).7

Thus, to realize the envisioned analogues, we developed a total chemical synthesis approach that permits milligram scale production of the relatively complex wild-type Cn2 and variants thereof. We initially attempted chemical synthesis of fulllength wild-type Cn2 by Fmoc solid-phase peptide synthesis. A trial TFA deprotection with concomitant peptide cleavage from the resin was conducted after coupling of Cys29 to evaluate peptide assembly. HPLC-MS analysis of the crude cleavage product indicated significant amounts of truncation and deletion side products, which prompted us to revise our plan to a fragment-based ligation strategy. The full-length sequence was divided into two segments, Cn2(1-28) and Cn2(29-66), which were joined by native chemical ligation at Glu28 and Cys29 (Scheme 1).10-11 Synthesis of the required Cn2(1-28)α−thioester fragment via Boc-SPPS was straightforward and the ligation with Cn2(29-66) proceeded smoothly, producing the full-length polypeptide in 44.8% yield. Folding and disulfide formation of the polypeptide required extensive optimization; guanidinium HCl at high concentrations (1.5 M) was essential during folding to prevent toxin from aggregating and precipitating, and relatively long folding times (72 h) were necessary to favour the native disulfide isomer and improve yields (SI Fig. 5). The synthetic toxin was isolated by RPHPLC in acceptable yield (30.8%) and its monoisotopic molecular weight, determined by high resolution MALDI-MS (7584.4 ± 0.2 Da, Fig. 1a), was in excellent agreement with the theoretical value (7584.5 Da; taking into account Cterminal amidation and formation of four disulfide bonds). HPLC comparison of the purified synthetic Cn2 toxin with native toxin isolated from C. noxious venom under identical conditions revealed excellent agreement, suggesting that the correct disulfide isomer had been obtained (SI Fig. 8). The structural integrity of synthetic Cn2 was further confirmed by 2D TOCSY and NOESY NMR spectroscopy, which allowed complete assignment of backbone proton resonances. Comparison of the Hα-chemical shifts to published values for venomderived Cn2 indicated excellent agreement, suggesting that the synthetic and venom-derived Cn2 molecules are structurally identical (Fig. 1b).7 To assess the effect of synthetically produced Cn2 on VGSC isoforms NaV1.1-1.8, a high-throughput fluorescence imaging plate reader (FLIPRTETRA) assay was used as previously described.12 Briefly, HEK293 cells expressing NaV1.1-1.8 in a monolayer were incubated with membrane

potential dye (Molecular Devices, Sunnyvale, CA) and the change in fluorescence was continuously measured prior to and during compound addition (FLIPRTETRA). Native and synthetic Cn2 concentration-dependently activated NaV1.6expressing HEK293 cells with EC50s of 72 nM (pEC50 7.14 ± 0.91) and 15.8 nM (pEC50 7.81 ± 0.20), respectively (Fig. 2a). In line with native Cn2 previously reported, synthetic Cn2 (henceforth termed Cn2) at a concentration of up to 100 nM had no significant effect on sodium channel isoforms NaV1.1NaV1.3, NaV1.5, NaV1.7 or NaV1.8 (Fig. 2b). Activity at the muscle specific NaV subtype, NaV1.4, has not been observed for native Cn2 at concentrations of 300 nM.6 However, our data show that synthetic Cn2 is a weak agonist for NaV1.4 in the FLIPR assay. Collectively, these data suggest that synthetic and venom-derived wild-type Cn2 are structurally and pharmacologically identical. Having established synthetic access to the wild-type toxin, we turned our attention to the design of Cn2 analogues with altered activity. Gurevitz and colleagues have shown that substituting a conserved glutamate in β-scorpion toxins Css4 or Bj-xtrIT with arginine (E15R) results in uncoupling of NaVbinding and NaV-activation.13-14 A similar strategy was recently adopted to β-scorpion toxin Ts1 to obtain NaV1.4 probes with reduced NaV excitatory activity.15 The corresponding Cn2 mutant, Cn2[E15R], was prepared using the same synthetic strategy used for wild-type toxin by replacing the N-terminal ligation fragment with Cn2(1-28, E15R)-αthioester and folded using the protocol established for wildtype toxin.

Figure 2: Native and synthetic Cn2 activate NaV1.6 in the FLIPRTETRA membrane potential assay. a) Synthetic or venomderived Cn2 elicited Nav1.6-mediated increase in fluorescence normalised to baseline vehicle control (% Control) with EC50s of


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15.8 nM and 72 nM, respectively. b) Cn2 had little effect on sodium channel isoforms NaV1.1–1.5, 1.7 and 1.8 at concentrations up to 100 nM but only elicited concentration-dependent changes in membrane potential in HEK293 cells expressing NaV1.6.

Figure 3: Effect of synthetic Cn2 (a, b) and Cn2[E15R] (c, d) on the electrophysiological properties of hNaV1.6 expressed in HEK293 cells. a, c) Current-voltage relationship in the absence (black circles) and presence of 10 nM Cn2 (grey squares) or c) 200 nM Cn2[E15R] (grey squares). All data presented as mean ± SEM. Inset, Cn2, but not Cn2[E15R], significantly increased current at membrane potentials of -60 to -40 mV (*= P < 0.05, paired t-test) (n=5). b, d) Voltage dependence of activation (G/V) and steady-state fast inactivation (I/Imax) of hNaV1.6 in the absence (black circles) and presence of 10 nM Cn2 (grey squares) or d) 200 nM Cn2[E15R] (grey squares), fitted with single Boltzmann relationship (solid line).

NMR confirmed that the tertiary structure of this mutant is virtually unchanged from wild-type toxin (Fig. 1b). With the two analogues in hand, we examined their electrophysiological properties at human NaV1.6.

10 nM Cn2, sodium current was observed at previously prohibitive potentials (Fig. 3a), with significantly increased current at pulses between -60 to -40 mV (P< 0.05; paired t-test). In contrast, Cn2 (10 nM) decreased current between potentials -10 mV and +50 mV, with peak whole cell current elicited by a pulse of 0 mV decreased by 47.3 ± 0.1%. The V1/2 of NaV1.6 activation for Cn2 treated cells (-24.1 ± 0.8 mV) was not significantly different from control (-24.4 ± 0.3 mV) (Fig. 3b). However, Cn2 significantly increased the slope factor at NaV1.6 (control: 4.8 ± 0.3; Cn2: 8.2 ± 0.8, p < 0.05) leading to a higher conductance at more hyperpolarised membrane potentials. Cn2 (10 nM) also caused a depolarizing shift in the V1/2 of NaV1.6 steady-state fast inactivation (∆V1/2: +10.2 mV) (Fig. 3b), which in combination with a shallower activation slope, led to an increase in window current. In effect, there was a 20% increase in channel availability at -35 mV in the presence of Cn2 compared with control. This excitatory (activating) Cn2 effect is coupled to a depressant activity as evidenced by the concomitant reduction in peak sodium conductance.

β-Scorpion toxins bind to site-4 on NaVs, which has been mapped to the S3-S4 linker of domain II of the NaV αsubunit.2 Binding causes a hyperpolarizing shift in the voltage dependence of channel activation while NaV inactivation kinetics are not affected.6, 16-18 To assess whether synthetically produced Cn2 retains these properties we obtained whole-cell patch clamp recordings from HEK293 cells expressing hNaV1.6. Cn2 caused a concentration-dependent decrease in peak current (obtained by recording resultant currents at pulses to 0 mV) with an EC50 of 6.2 nM (pEC50 8.2 ± 0.1) (SI Fig. 10). This value is in agreement with the predicted EC50 from Schiavon et al.,6 39.2 ± 3.7 nM. To test for the typical shift in the voltage dependence of activation associated with β scorpion toxins, current-voltage (I-V) curves were generated. This protocol includes a depolarizing prepulse to 0 mV in order to facilitate channel opening and toxin binding as described by the voltage sensor trapping model.16 Indeed, in the presence of



The electrophysiological effects of Cn2[E15R] on hNaV1.6 were evaluated at a toxin concentration of 200 nM, because preliminary studies indicated that at this concentration hNaV1.6 sodium current is inhibited by approximately 50%. In contrast to Cn2, 200 nM Cn2[E15R] did not elicit current at previously prohibitive potentials (Fig. 3c), but led to a depolarizing shift in the voltage-dependence of activation (Fig. 3d, V1/2: control -17.4 ± 0.2 mV; 200 nM Cn2[E15R], -11.4 ± 0.6 mV). In addition, Cn2[E15R] significantly increased the slope factor at NaV1.6 (control: 5.0 ± 0.2; Cn2 7.9 ± 0.6, p < 0.05), reducing conductance at more depolarised membrane potentials without changing conductance at hyperpolarized potentials. In line with these observations, Cn2[E15R] at 200 nM decreased peak current by 42.4 ± 1.5%. The V1/2 of steadystate fast inactivation of NaV1.6 was not significantly changed in the presence of 200 nM Cn2[E15R], but a proportion (~25%) of the Cn2[E15R] treated channels resisted inactivation altogether as evidenced by the current elicited at pulses between -40 and 0 mV (Fig. 3d).

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Thus, some (but not all) of the effects seen in wild-type Cn2 are significantly altered in the Cn2[E15R] mutant. First, Cn2[E15R] does not activate NaV1.6 at otherwise prohibitive membrane potentials whereas Cn2 does (Fig. 3a, c). Second, Cn2[E15R] causes a significant depolarizing (rightward) shift in the steady state activation curves, indicating that channels are more difficult to open (Fig. 3b, d). Third, Cn2[E15R] does not shift the V1/2 of Nav1.6 channel inactivation (whereas Cn2 does). In contrast, both Cn2 and Cn2[E15R] significantly reduce NaV1.6 peak current at nM concentrations (Fig. 3a, c). These data collectively show that the single amino acid (E15R) substitution not only causes an uncoupling of NaV binding and activation in scorpion β-toxins as previously reported,13-14 but, at least in the Cn2:hNaV1.6 studied here, also an uncoupling of excitatory (activating) and depressant (inhibitory) effects. Although the excitatory β-toxin effects on NaV gating have been satisfactorily explained by the voltage sensor trapping model,16 our findings are better rationalized by a ‘two-state voltage sensor trapping’ model proposed recently by Heinemann and colleagues to take into account the depressant effects observed for many of these toxins.19 In this model, Cn2 binds with similar affinities both resting and activated conformations of VSDII, macroscopically giving rise to the observed excitatory and depressant effects. Our data for Cn2[E15R] demonstrate uncoupling of these activities, suggesting that at the molecular level the mutation destabilizes the activated VSDII:toxin complex while not significantly affecting the resting VSD-II:toxin complex. Given that Cn2[E15R] only retains NaV1.6 depressant activity, we hypothesized that it would show antinociceptive properties in mouse models of NaV1.6-mediated pain.4 Intraplantar injection of Cn2 (10 nM) caused nocifensive behaviours in mice including flinching, lifting and shaking of the hindpaw (Fig. 4a). In contrast, 10 µM Cn2[E15R] failed to cause spontaneous pain behaviours when administered alone (Fig. 4a). Co-administration of Cn2 with Cn2[E15R] concentrationdependently reduced spontaneous pain behaviours evoked by Cn2 (pain behaviours/5 minutes, control (Cn2 alone): 73 ± 10; Cn2[E15R]: 52 ± 11 (0.2 µM) or 1 ± 1 (2 µM)) (Fig. 4b) suggesting that Cn2 and Cn2[E15R] compete for the same NaV1.6 binding site in vivo. Ciguatoxins (CTX) are non-selective sodium channel activators responsible for ciguatera fish poisoning.12, 20-21 Despite being non-selective, blocking either NaV1.6 or NaV1.7 abolishes the spontaneous pain induced by intraplantar injection of CTX.12 Cn2[E15R] significantly reduced nocifensive behaviours associated with CTX (pain behaviours: control 350 ± 35, Cn2[E15R] (10 µM) 63 ± 14; P < 0.001 unpaired t-test), confirming a distinct role for NaV1.6 in ciguatoxin-induced spontaneous pain (Fig. 4c). Finally, Cn2[E15R] was tested in the formalin mouse model, in which phase I is caused by direct activation of nociceptors and phase II is caused by inflammatory sensitization. Cn2[E15R] significantly reduced flinching associated with phase II (pain behaviours: control 232 ± 18, Cn2[E15R] (10 µM) 138 ± 15; P < 0.05 two-way ANOVA) (Fig. 4d). Thus, Cn2[E15R] not only competes with Cn2, but is a functional inhibitor of hNav1.6 in vivo in its own right. More broadly, our data also confirms an important, previously underappreciated role for NaV1.6 in peripheral nociception.

Figure 4: Cn2[E15R] causes NaV1.6 mediated analgesia in in vivo spontaneous pain models. a) Cn2 causes non-stimulus evoked pain behaviour when injected intraplantar, whereas Cn2[E15R] does not. b) Co-administration of Cn2[E15R] reduces pain behaviour caused by 10 nM Cn2 in a concentration dependent manner. c) Intraplantar injection of NaV activator CTX (5 nM) causes non-stimulus evoked pain behaviours that are significantly reduced by Cn2[E15R] treatment (**** = P < 0.001, unpaired t test, n=6-7) d) Effect of Cn2[E15R] on formalin induced pain behaviours; phase II pain behaviours were significantly reduced with intraplantar injection of 10 µM Cn2[E15R] (*= P < 0.05, unpaired t test, n = 3-13).

CONCLUSIONS:


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Journal of Medicinal Chemistry 7.1). After dissolution, the pH was re-adjusted to 6.9–7.0 and the mixture stirred under an argon atmosphere for 18 h. The reaction was quenched by adding TFA to a final concentration of 2% (v/v) and the product isolated by preparative HPLC (isolated yield: 20.0 mg, 2.6 µmol (44.8%); Cn2[1-66] observed mass 7596.0 ± 1.0 Da, calculated mass 7596.7 Da (average isotope composition, ESI-MS). 20 mg (2.6 µmol) of reduced Cn2 was dissolved in 5 mL of 6 M GdmHCl (pH ~ 5) to give a final peptide concentration of 4 mg/mL. The peptide solution was then added to 100 mL of oxidation buffer consisting of 100 mM Tris, 1.5 M GdmHCl, 10 mM reduced Lglutathione (GSH), 1 mM oxidized L-glutathione (GSSG), pH 8.1. Oxidation was performed at 22°C, monitored by HPLC and found to reach equilibrium after 72 hours. 2 mL of TFA was added to the oxidation mixture and the folded Cn2 purified by semi-preparative HPLC. Isolated yield: 6.5 mg, 0.8 µmol (30.8%). Cn2 observed mass 7588.3 ± 1.0 Da, calculated mass 7588.7 Da (average isotope composition). High-resolution MALDI-MS: Cn2 observed MW: 7584.41 ± 0.2 Da (M+H+); calculated MW: 7584.49 Da (M+H+, most abundant isotope composition). Cn2 (E15R) was prepared via the same protocol used for wild type toxin and afforded the analogue in similar yields (30-40% isolated yield for final folding step). High resolution ESI-MS: Cn2 (E15R) observed MW: 7610.62 ± 0.2 Da; calculated MW: 7610.56 Da (most abundant isotope composition, SI Fig. 7). All final products had a purity of ≥95% based on analytical uHPLC-MS analysis (Agilent 300SB-C18 column (2.1×50 mm, 1.8 µm, 300 Å), linear gradient of 15-45% of buffer B in buffer A over 12 min, 50°C, detection at 214/280 nm). NMR spectroscopy: Lyophilized Cn2 samples were dissolved in 90% H2O/10% D2O at a concentration of 0.2 mM and a pH of 3.6. Spectra were recorded at 303 K on a Bruker Avance 600 MHz spectrometer equipped with a cryogenically-cooled probe. Phase-sensitive mode using time-proportional phase incrementation for quadrature detection in the t1 dimension was used for all two-dimensional spectra. Excitation sculpting with gradients was used to achieve water suppression. NMR experiments included TOCSY using a MLEV-17 spin lock sequence with a 80 ms mixing time, and NOESY with a 200 ms mixing time. Spectra were recorded with 4096 data points in the F2 dimension and 512 increments in the F1 dimension. The t1 dimension was zero-filled to 1024 real data points, and the F1 and F2 dimensions were multiplied by a sine-squared function prior to Fourier transformation. The spectra were referenced to water at 4.715 ppm (303 K). All spectra were processed using TopSpin (Bruker) and manually assigned with CCPNMR using the sequential assignment protocol.23-24 Cn2 reference material, isolated from the venom of C. noxius Hoffmann, was a generous gift from Lourival Possani (Institute of Biotechnology, National Autonomous University of Mexico). Cell culture: Human embryonic kidney (HEK) cells with constitutive expression of NaV isoforms 1.1-1.8 were purchased from Scottish Biomedical (Glasgow, Scotland). Cells were maintained in minimal essential media (MEM) (Sigma-Aldrich, Australia) supplemented with 2mM L-glutamine and 10 % foetal bovine serum (FBS) (Assay Matrix, Ivanhoe, Australia). Cells lines were cultured with appropriate selection antibiotics; blasticidin and G418 for NaV1.1, 1.2, 1.3, 1.5, 1.6, 1.7 and 1.8; blasticidin, G418 and zeocin for NaV1.4. Each cell line was grown in 37°C in a humidified 5% CO2 incubator to 70-80% confluence, passaged every 3-4 days with TrypLE Express (Invitrogen). Fluorescent membrane potential assay: The activity of Cn2 at NaV1.1- NaV1.8 was assessed using a FLIPRTetra fluorescent membrane potential assay as previously described.12, 25 In brief, HEK293 cells stably expressing human NaV isoforms NaV1.1-NaV1.8 were plated on 384-well plates at a density of 10 000 – 15 0000 cells per well in normal growth medium (Minimal Essential Medium (MEM; Sigma-Aldrich, Australia). After 48 h culture at 37oC/5% CO2, growth medium was replaced with 20 uL of red membrane potential dye (Molecular Devices, Sunnyvale, CA) prepared in physiological salt solution (composition (in mM): NaCl (140), glucose (11.5), KCl (5.9), MgCl2 (1.4), NaH2PO4 (1.2), NaHCO3 (5), CaCl2 (1.8), 4-(2hydroxyethyl)-1-piperazineethane-sulfonic acid (HEPES) (10), pH

In summary, we successfully applied a native chemical ligation synthetic strategy to gain access to the highly NaV1.6selective scorpion toxin Cn2 and a pharmacologically unique analogue. Our data demonstrate that the excitatory and depressant activities in scorpion β-toxins can be uncoupled, suggesting that these activities are governed by distinct toxin pharmacophore residues. This work paves the way for rational design of NaV inhibitors based on β-scorpion toxins, which so far have been limited to a role as molecular tools due to their potent NaV-excitatory activity. We expect these molecules to be powerful probes for studying the physiological roles of NaV1.6 in health and disease.

EXPERIMENTAL SECTION: Peptide synthesis: Peptides were synthesized by solid-phase peptide synthesis (SPPS). Peptide α-thioalkylesters corresponding to Cn2[1−28]-α-thioester (KEGYLVDKNTGCKYECLKLGDNDYCLRE-[S-CH2-CH2-CO]F), Cn2[1−28,E15R]-α-thioester (KEGYLVDKNTGCKYRCLKLGDNDYCLRE-[S-CH2-CH2-CO]L) were assembled by manual in situ neutralization Boc SPPS on pre-loaded PAM polystyrene resins as described recently.22 Cn2[29-66] (CKQQYGKGAGGYCYAFACW-CTHLYEQAIVWPLPNKRCSNH2) was assembled by automated Boc SPPS on a CSBio peptide synthesizer using MBHA polystyrene resin and standard protocols. The following side chain protecting groups were employed: Arg(Tos), Asp(OcHex), Asn(Xan), Cys(4-MeBzl), Glu(OcHex), Gln(Xan), His(Bom), Lys(2Cl-Z), Ser(Bzl), Thr(Bzl), Trp(Hoc) and Tyr(2Br-Z). After chain assembly, peptides were side chain-deprotected and simultaneously cleaved from the resin by treatment with anhydrous HF containing 10% (v/v) p-cresol and 5 eq. of Cys-OEt (per equivalent of His(Bom) residues) for 1 h at 0ºC. HF was evaporated under reduced pressure. The crude product was precipitated and washed with chilled diethylether, then dissolved in 50 % (v/v) aqueous acetonitrile containing 0.1 % trifluoroacetic acid (TFA) (v/v) and lyophilized. Peptides were purified by reversed-phase high-performance liquid chromatography (RP-HPLC) using preparative Vydac C8 (22 × 250 mm), semi-preparative Zorbax C3 (10 × 250 mm) and analytical Zorbax C18 (4.6 × 50 mm) columns. Preparative and semi-preparative peptide purifications were performed on Shimadzu Prominence LC30AT HPLC system using Phenomenex C18 RP-HPLC (21.2×250 mm, 15 µm, 300 Å) or Agilent C18 RP-HPLC (9.4×250, 5 µm, 300 Å) columns. Crude peptides were dissolved in a 10 % (v/v) acetonitrile/water mixture containing 0.05 % (v/v) TFA, before being loaded onto the column pre-equilibrated with 10 % of solvent B (90:10:0.1 – acetonitrile:water:TFA) in solvent A (0.1 % (v/v) TFA in water). Peptides were eluted using linear gradients of solvent B in solvent A, and fractions were collected across the expected elution time. Peptide purity and identity were assessed by HPLC on a Shimadzu Nexera LC-30AD uHPLC system equipped with an Agilent 300SBC18 column (2.1×50 mm, 1.8 µm, 300 Å) by using a linear gradient of 15-45% of buffer B in buffer A over 12 min at 50°C. Electrospray ionization mass spectrometry (ESI-MS) was performed on Shimadzu LCMS-2020 system or on a Shimadzu Nexera uHPLC coupled with an AB SCIEX 5600 mass spectrometer equipped with a Turbo V ion source. Matrix-Assisted Laser-Desorption Ionization-Time of Flight (MALDI)-TOF was conducted on an Applied Biosystems 4700 TOF/TOF Proteomics Analyzer using α-cyano-4-hydroxycinnamic acid (CHCA) as a matrix (8 mg/mL in 75% (v/v) acetonitrile, 0.05% (v/v) formic acid in water. Cn2[1−28]-α-thioester observed mass 3505.6 ± 0.4 Da, calculated mass (average isotope composition) 3505.9 Da; Cn2[1−28, E15R]-α-thioester observed mass 3498.8 ± 0.4 Da, calculated mass 3498.9 Da; Cn2[29-66] observed mass 4343.4 ± 0.5 Da, calculated mass 4344.0 Da. Fractions containing the desired product were pooled, lyophilized and stored at –20ºC. Peptide ligation and folding: We dissolved 25 mg of Cn2[29-66] MW: 4344.0, 5.8 µmol) and 22.9 mg of Cn2[1-28]-α-thioester, MW: 3505.9, 6.9 µmol) in 4 mL of ligation buffer (6M guanidinium HCl (GdmHCl), 200 mM Na-phosphate, 50 mM 4-mercaptophenylacetic acid (MPAA), 50 mM tris(2-carboxyethyl)phosphine (TCEP), pH



7.4) according to the manufacturer’s instructions and cells were incubated for 30 minutes at 37oC. Changes in fluorescence (excitation: 510-545 nm, emission: 565-625 nm) after addition of native and synthetic Cn2 were measured every second for 300 s using a FLIPRTetra fluorescent plate reader (Molecular Devices, Sunnyvale, CA) and analysed using Screenworks 3.1.1.4 (Molecular Devices). GraphPad Prism (Version 4.00, San Diego, California) was used to fit a 4parameter Hill equation with variable Hill slope to the data. All data, unless otherwise stated, are expressed as the mean ± standard error of the mean (SEM). Electrophysiology: The effect of Cn2 and analogues was assessed in whole cell recordings under voltage-clamp conditions using a QPatch 16-well automated electrophysiology platform (Sophion Bioscience, Ballerup, Denmark) and 16-well planar chip plates (Qplates, Sophion) with resistance of 2 ± 0.02 MΩ and diameter of 1 µm. HEK293 cells expressing human NaV1.6 were expanded in normal growth media into a T175 flask 48hrs prior to experiment and maintained at 37°C in humidified 5% CO2. At 70-80% confluence, cells were washed twice with Dulbecco’s phosphate-buffered saline prior to harvesting with 1 mL DetachinTM. Finally, cells were resuspended in 3 ml Ex-Cell ACF CHO media supplemented with 25 mM HEPES (Sigma-Aldrich, NSW, Australia) and placed on the Qpatch’s Qstirrer for 30 minutes prior to the assay. Positioning pressure was set at -60 mBar, holding potential -100 mV, minimum seal resistance 0.1 GΩ, holding pressure -20 mBar and currents were filtered at 25 kHz (8th order Bessel, cut off 5 kHz). Extracellular solution contained (in mM): NaCl 145, KCl 4, CaCl2 2, MgCl2 1, HEPES 10 and glucose 10; pH 7.4; osmolarity 305 mOsm. The intracellular solution contained (in mM): CsF 140, EGTA/CsOH 1/5, HEPES 10 and NaCl 10; pH 7.3 with CsOH; osmolarity 320 mOsm. Cells were incubated for 5 minutes before voltage protocols with working concentrations of peptides which were diluted in ECS containing 0.1% bovine serum albumin (BSA). All protocols contained a pre-pulse to 0 mV and 120 ms recovery at the holding potential to allow toxin binding and voltage sensor trapping consistent with previously described protocols.16 Current (I) – voltage (V) relationship before and after toxin application was assessed as follows, cells were held at -100 mV, (50 ms recovery) test pulses were recorded for 50 ms between -90 mV and +55 mV in 5 mV increments. Conductance-voltage relationships were obtained for voltage sweeps using G = I/(V-Vrev), Vrev being the reversal potential. Resulting data was fitted using a Boltzmann equation: GNa = GNa,max/1 + exp [(Vm – V1/2) / k] in GraphPad Prism; GNa is the voltage dependent sodium conductance, GNa,max is the maximal sodium conductance, Vm the membrane potential and k is the slope factor. Animal behavioural testing: Animal ethics approval was obtained from The University of Queensland Animal ethics committee. All experiments were conducted in accordance with local and national regulations and the International Associations for the Study of Pain guidelines for the use of animals. In order to assess evoked pain, Cn2, Pacific Ciguatoxin-1 (P-CTX-1) and formalin were used as previously described.4, 12, 26-28 Briefly, Cn2 was diluted in phosphate-buffered saline with 0.1% bovine serum albumin (BSA) to give final concentrations of 10 nM. Under isofluorane (3%) anaesthesia, 40 µL Cn2 was administered via shallow subcutaneous (intraplantar i.pl.) injection into the hindpaw of male C57BL/6 mice. Mice were allowed to recover in clear polyvinyl boxes and recorded for 20 minutes post injection. Following this, nocifensive behaviours including flinches, lifts and shakes were counted every 5-minute interval from a video recording by a blinded observer. In order to test the effect of the pharmacological modulator Cn2[E15R] (0.2 µM, 2 µM, 10 µM) on the induction of NaV1.6 spontaneous pain, 40 µL Cn2[E15R] was injected alone or co-administered with spontaneous pain inducing compounds (Cn2 (10 nM), P-CTX-1 (10 nM) and formalin (1%; v/v)).

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Analytical data for all peptides and supporting electrophysiological characterization material are available in pdf format. The Supporting Information is available free of charge on the ACS Publications website.

AUTHOR INFORMATION Corresponding Author * [email protected] or [email protected]

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. #: These authors contributed equally.

Funding Sources This work was supported by an Australian Research Council (ARC) Future Fellowship awarded to IV (FT130101215) and an ARC Laureate Fellowship to DJC (FL150100146). MRI is supported by an Australian Government Research Training Program Scholarship.

ACKNOWLEDGMENT We thank Lourival Possani for providing a sample of Cn2 isolated from C. noxius venom and Richard Lewis for providing CTX.

ABBREVIATIONS Cn2, Centruroides noxius toxin 2; CTX, Ciguatoxin; GdmHCl, guanidinium hydrochloride; HEPES, 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid; MBHA, 4methylbenzhydrylamine; MEM, minimal essential medium; MPAA, 4-mercaptophenylacetic acid; NaV1.X, voltage-gated sodium channel; TCEP, tris(2-carboxyethyl)phosphine; VSD, voltage-sensing domain

REFERENCES 1. Catterall, W. A. Voltage-Gated Sodium Channels at 60: Structure, Function and Pathophysiology. J. Physiol. 2012, 590 (11), 2577-2589. 2. Ahern, C. A.; Payandeh, J.; Bosmans, F.; Chanda, B. The Hitchhiker's Guide to the Voltage-Gated Sodium Channel Galaxy. J. Gen. Physiol. 2016, 147 (1), 1-24. 3. Stevens, M.; Peigneur, S.; Tytgat, J. Neurotoxins and Their Binding Areas on Voltage-Gated Sodium Channels. Front. Pharmacol. 2011, 2, 71. 4. Deuis, J. R.; Zimmermann, K.; Romanovsky, A. A.; Possani, L. D.; Cabot, P. J.; Lewis, R. J.; Vetter, I. An Animal Model of Oxaliplatin-Induced Cold Allodynia Reveals a Crucial Role for Nav1.6 in Peripheral Pain Pathways. Pain 2013, 154 (9), 1749-1757. 5. Rosker, C.; Lohberger, B.; Hofer, D.; Steinecker, B.; Quasthoff, S.; Schreibmayer, W. The TTX Metabolite 4,9-AnhydroTtx Is a Highly Specific Blocker of the NaV1.6 Voltage-Dependent Sodium Channel. Am. J. Physiol. Cell Physiol. 2007, 293 (2), C783C789. 6. Schiavon, E.; Sacco, T.; Cassulini, R. R.; Gurrola, G.; Tempia, F.; Possani, L. D.; Wanke, E. Resurgent Current and Voltage Sensor Trapping Enhanced Activation by a β-Scorpion Toxin Solely in NaV1.6 Channel. Significance in Mice Purkinje Neurons. J. Biol. Chem. 2006, 281 (29), 20326-20337. 7. Pintar, A.; Possani, L. D.; Delepierre, M. Solution Structure of Toxin 2 from Centruroides Noxius Hoffmann, a β-Scorpion Neurotoxin Acting on Sodium Channels. J. Mol. Biol. 1999, 287 (2), 359-367. 8. Vazquez, A.; Tapia, J. V.; Eliason, W. K.; Martin, B. M.; Lebreton, F.; Delepierre, M.; Possani, L. D.; Becerril, B. Cloning and Characterization of the cDNAs Encoding Na+ Channel-Specific Toxin-1 and Toxin-2 of the Scorpion Centruroides-Noxius Hoffmann. Toxicon 1995, 33 (9), 1161-1170.

ASSOCIATED CONTENT Supporting Information


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9. Dang, B. B.; Kubota, T.; Mandal, K.; Correa, A. M.; Bezanilla, F.; Kent, S. B. H. Elucidation of the Covalent and Tertiary Structures of Biologically Active Ts3 Toxin. Angew. Chem. Int. Ed. 2016, 55 (30), 8639-8642. 10. Dawson, P. E.; Muir, T. W.; Clark-Lewis, I.; Kent, S. B. Synthesis of Proteins by Native Chemical Ligation. Science 1994, 266 (5186), 776-779. 11. Dang, B. B.; Kubota, T.; Mandal, K.; Bezanilla, F.; Kent, S. B. H. Native Chemical Ligation at Asx-Cys, Glx-Cys: Chemical Synthesis and High-Resolution X-Ray Structure of Shk Toxin by Racemic Protein Crystallography. J. Am. Chem. Soc. 2013, 135 (32), 11911-11919. 12. Inserra, M. C.; Israel, M. R.; Caldwell, A.; Castro, J.; Deuis, J. R.; Harrington, A. M.; Keramidas, A.; Garcia-Caraballo, S.; Maddern, J.; Erickson, A.; Grundy, L.; Rychkov, G. Y.; Zimmermann, K.; Lewis, R. J.; Brierley, S. M.; Vetter, I. Multiple Sodium Channel Isoforms Mediate the Pathological Effects of Pacific Ciguatoxin-1. Sci. Rep. 2017, 7, 42810. 13. Karbat, I.; Ilan, N.; Zhang, J. Z.; Cohen, L.; Kahn, R.; Benveniste, M.; Scheuer, T.; Catterall, W. A.; Gordon, D.; Gurevitz, M. Partial Agonist and Antagonist Activities of a Mutant Scorpion βToxin on Sodium Channels. J. Biol. Chem. 2010, 285 (40), 3053130538. 14. Karbat, I.; Cohen, L.; Gilles, N.; Gordon, D.; Gurevitz, M. Conversion of a Scorpion Toxin Agonist into an Antagonist Highlights an Acidic Residue Involved in Voltage Sensor Trapping During Activation of Neuronal Na+ Channels. FASEB J. 2004, 18 (6), 683-689. 15. Kubota, T.; Dang, B. B.; Carvalho-de-Souza, J. L.; Correa, A. M.; Bezanilla, F. NaV Channel Binder Containing a Specific Conjugation-Site Based on a Low Toxicity β-Scorpion Toxin. Sci. Rep. 2017, 7, 16329. 16. Cestele, S.; Qu, Y.; Rogers, J. C.; Rochat, H.; Scheuer, T.; Catterall, W. A. Voltage Sensor-Trapping: Enhanced Activation of Sodium Channels by β-Scorpion Toxin Bound to the S3-S4 Loop in Domain II. Neuron 1998, 21 (4), 919-931. 17. Escalona, M. P.; Possani, L. D. Scorpion β-Toxins and Voltage-Gated Sodium Channels: Interactions and Effects. Front. Biosci. 2013, 18, 572-587. 18. Cestele, S.; Yarov-Yarovoy, V.; Qu, Y.; Sampieri, F.; Scheuer, T.; Catterall, W. A. Structure and Function of the Voltage

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Figure 1 49x13mm (300 x 300 DPI)

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Figure 4 (revised) 129x193mm (300 x 300 DPI)



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The E15R point mutation in scorpion toxin Cn2 uncouples its

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