The E15R Point Mutation in Scorpion Toxin Cn2 Uncouples Its

Jan 29, 2018 - β-Scorpion toxins bind to site-4 on NaVs, which has been mapped to the S3–S4 linker of domain II of the NaV α-subunit.(2) Binding c...
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Brief Article Cite This: J. Med. Chem. 2018, 61, 1730−1736

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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*,† †

Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland 4072, Australia School of Pharmacy, The University of Queensland, Woolloongabba, Queensland 4102, Australia

‡

S Supporting Information *

ABSTRACT: We report the chemical synthesis of scorpion toxin Cn2, a potent and highly selective activator of the human voltage-gated sodium channel NaV1.6. In an attempt to decouple channel activation from channel binding, we also synthesized 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 antinociceptive in mouse models of NaV1.6-mediated pain.

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that shows some NaV1.6 selectivity in vitro.5 In the abovementioned studies, the long-chain scorpion β-toxin Cn2 from 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 voltagesensing 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 knockout animals (which are not viable) and/or NaV1.6selective molecular probes such as 4,9-anhydro-tetrodotoxin © 2018 American Chemical Society

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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 C-Terminal 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 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 full-length 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 fragmentReceived: October 30, 2017 Published: January 29, 2018 1730

DOI: 10.1021/acs.jmedchem.7b01609 J. Med. Chem. 2018, 61, 1730−1736

Journal of Medicinal Chemistry

Brief Article

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 venom-derived Cn2 indicated excellent agreement, suggesting that the synthetic and venom-derived Cn2 molecules are structurally identical (Figure 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 NaV1.6-expressing HEK293 cells with EC50s of 72 nM (pEC50 7.14 ± 0.91) and 15.8 nM (pEC50 7.81 ± 0.20), respectively (Figure 2a). In line with native Cn2 previously reported, synthetic Cn2 (henceforth termed Cn2) at a concentration of up to 1 μM had no significant effect on sodium channel isoforms NaV1.1−NaV1.3, NaV1.5, NaV1.7, or NaV1.8 (Figure 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 NaV binding 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)-α-thioester and folded using the protocol established for wild-type toxin. NMR confirmed that the tertiary structure of this mutant is virtually unchanged from wild-type toxin (Figure 1b). With the two analogues in hand, we examined their electrophysiological properties at human NaV1.6. 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

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 Scheme 1. Chemical Synthesis of Cn2 by Native Chemical Ligationa

a

(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).

fragment via Boc-SPPS was straightforwar,d and the ligation with Cn2(29-66) proceeded smoothly, producing the fulllength 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 favor the native disulfide isomer and improve yields (Supporting Information (SI) Figure 5). The synthetic toxin was isolated by RP-HPLC in acceptable yield (30.8%), and its monoisotopic molecular weight, determined by high resolution MALDI-MS (7584.4 ± 0.2 Da, Figure 1a), was in excellent agreement with the theoretical value (7584.5 Da, taking into account C-terminal 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, Figure 8). The structural integrity of synthetic Cn2 was

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 analogues relative to native, venom derived Cn2 (PDB 1CN2).7 1731

DOI: 10.1021/acs.jmedchem.7b01609 J. Med. Chem. 2018, 61, 1730−1736

Journal of Medicinal Chemistry

Brief Article

Figure 2. Native and synthetic Cn2 activate NaV1.6 in the FLIPRTETRA membrane potential assay. (a) Synthetic or venom-derived Cn2 elicited Nav1.6-mediated increase in fluorescence normalized to baseline vehicle control (% Control) with EC50s of 15.8 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 (gray squares) or (c) 200 nM Cn2[E15R] (gray 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 (gray squares) or (d) 200 nM Cn2[E15R] (gray squares), fitted with single Boltzmann relationship (solid line).

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, Figure 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 10 nM Cn2, sodium current was observed at previously prohibitive potentials (Figure 3a), with significantly increased current at pulses between −60 and −40 mV (P < 0.05; paired t-test). In contrast, Cn2 (10 nM) decreased current between potentials −10 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) (Figure 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 1732

DOI: 10.1021/acs.jmedchem.7b01609 J. Med. Chem. 2018, 61, 1730−1736

Journal of Medicinal Chemistry

Brief Article

a higher conductance at more hyperpolarized membrane potentials. Cn2 (10 nM) also caused a depolarizing shift in the V1/2 of NaV1.6 steady-state fast inactivation (ΔV1/2: +10.2 mV) (Figure 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. 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 (Figure 3c) but led to a depolarizing shift in the voltage-dependence of activation (Figure 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 depolarized 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 (Figure 3d). 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 (Figure 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 (Figure 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 (Figure 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 reported13,14 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 behaviors in mice including flinching, lifting, and shaking of the hindpaw (Figure 4a). In contrast, 10 μM Cn2[E15R] failed to

Figure 4. Cn2[E15R] causes NaV1.6 mediated analgesia in in vivo spontaneous pain models. (a) Cn2 causes nonstimulus evoked pain behavior when injected intraplantar, whereas Cn2[E15R] does not. (b) Co-administration of Cn2[E15R] reduces pain behavior caused by 10 nM Cn2 in a concentration dependent manner. (c) Intraplantar injection of NaV activator CTX (5 nM) causes nonstimulus evoked pain behaviors 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 behaviors; phase II pain behaviors were significantly reduced with intraplantar injection of 10 μM Cn2[E15R] (* = P < 0.05, unpaired t test, n = 3−13).

cause spontaneous pain behaviors when administered alone (Figure 4a). Co-administration of Cn2 with Cn2[E15R] concentration dependently reduced spontaneous pain behaviors evoked by Cn2 (pain behaviors/5 min; control (Cn2 alone), 73 ± 10; Cn2[E15R], 52 ± 11 (0.2 μM) or 1 ± 1 (2 μM)) (Figure 4b), suggesting that Cn2 and Cn2[E15R] compete for the same NaV1.6 binding site in vivo. Ciguatoxins (CTX) are nonselective sodium channel activators responsible for ciguatera fish poisoning.12,20,21 Despite being nonselective, blocking either NaV1.6 or NaV1.7 abolishes the spontaneous pain induced by intraplantar injection of CTX.12 Cn2[E15R] significantly reduced nocifensive behaviors associated with CTX (pain behaviors: control 350 ± 35, Cn2[E15R] (10 μM) 63 ± 14; P < 0.001 unpaired ttest), confirming a distinct role for NaV1.6 in ciguatoxininduced spontaneous pain (Figure 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 behaviors: control 232 ± 18, Cn2[E15R] (10 μM) 138 ± 15; P < 0.05 two-way 1733

DOI: 10.1021/acs.jmedchem.7b01609 J. Med. Chem. 2018, 61, 1730−1736

Journal of Medicinal Chemistry

Brief Article

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 −20 °C. Peptide Ligation and Folding. We dissolved 25 mg of Cn2[2966] (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 (6 M guanidinium HCl (GdmHCl), 200 mM Na-phosphate, 50 mM 4-mercaptophenylacetic acid (MPAA), 50 mM tris(2-carboxyethyl)phosphine (TCEP), pH 7.1). After dissolution, the pH was readjusted 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). Then 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 h. Then 2 mL of TFA was added to the oxidation mixture and the folded Cn2 purified by semipreparative 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, Figure 7). All final products had a purity of ≥95% based on analytical uHPLC-MS analysis (Agilent 300SB-C18 column (2.1 mm × 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. Phasesensitive mode using time-proportional phase incrementation for quadrature detection in the t1 dimension was used for all twodimensional 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 SB Drug Discovery (Glasgow, Scotland). Cells were maintained in minimal essential media (MEM) (Sigma-Aldrich, Australia) supplemented with 2 mM L-glutamine and 10% fetal 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).

ANOVA) (Figure 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.

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CONCLUSIONS In summary, we successfully applied a native chemical ligation synthetic strategy to gain access to the highly NaV1.6-selective 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 NaVexcitatory activity. We expect these molecules to be powerful probes for studying the physiological roles of NaV1.6 in health and disease.

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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 preloaded PAM polystyrene resins as described recently.22 Cn2[29-66] (CKQQYGKGAGGYCYAFACW-CTHLYEQAIVWPLPNKRCS-NH2) 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 equiv of Cys-OEt (per equivalent of His(Bom) residues) for 1 h at 0 °C. HF was evaporated under reduced pressure. The crude product was precipitated and washed with chilled diethyl ether 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 mm × 250 mm), semipreparative Zorbax C3 (10 mm × 250 mm), and analytical Zorbax C18 (4.6 mm × 50 mm) columns. Preparative and semipreparative peptide purifications were performed on Shimadzu Prominence LC-30AT HPLC system using Phenomenex C18 RPHPLC (21.2 mm × 250 mm, 15 μm, 300 Å) or Agilent C18 RP-HPLC (9.4 mm × 250 mm, 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 mm × 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 a 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 1734

DOI: 10.1021/acs.jmedchem.7b01609 J. Med. Chem. 2018, 61, 1730−1736

Journal of Medicinal Chemistry

Brief Article

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 10000−150000 cells per well in normal growth medium (Minimal Essential Medium (MEM; Sigma-Aldrich, Australia). After 48 h culture at 37 °C/5% CO2, growth medium was replaced with 20 μL 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-(2-hydroxyethyl)1-piperazineethane-sulfonic acid (HEPES) (10), pH 7.4) according to the manufacturer’s instructions and cells were incubated for 30 min at 37 °C. Changes in fluorescence (excitation, 510−545 nm; emissionm, 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 analyzed using Screenworks 3.1.1.4 (Molecular Devices). GraphPad Prism (Version 4.00, San Diego, California) was used to fit a four-parameter 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 48 h 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 of Detachin. Finally, cells were resuspended in 3 mL of Ex-Cell ACF CHO media supplemented with 25 mM HEPES (Sigma-Aldrich, NSW, Australia) and placed on the Qpatch’s Qstirrer for 30 min prior to the assay. Positioning pressure was set at −60 mbar, holding potential −100 mV, minimum seal resistance 0.1 GΩ, and 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 min before voltage protocols with working concentrations of peptides which were diluted in ECS containing 0.1% bovine serum albumin (BSA). All protocols contained a prepulse 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 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 Behavioral 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. 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%) anesthesia, 40 μL of Cn2 was administered via shallow subcutaneous (intraplantar ipl) injection into the hindpaw of male C57BL/6 mice. Mice were allowed to recover in clear polyvinyl boxes and recorded for 20 min post injection. Following this, nocifensive behaviors including flinches, lifts, and shakes were counted every 5 min interval from a video recording

by a blinded observer. To test the effect of the pharmacological modulator Cn2[E15R] (0.2, 2, 10 μM) on the induction of NaV1.6 spontaneous pain, 40 μL of Cn2[E15R] was injected alone or coadministered with spontaneous pain inducing compounds (Cn2 (10 nM), P-CTX-1 (10 nM), and formalin (1%; v/v)).

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications Web site. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b01609. Analytical data for all peptides and supporting electrophysiological characterization material (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*For T.D.: phone, +61-7-334-62021; fax, +61-7-334-62090; Email, [email protected]. *For I.V.: E-mail, [email protected]. ORCID

David J. Craik: 0000-0003-0007-6796 Thomas Durek: 0000-0003-0686-227X Author Contributions ⊥

M.R.I. and P.T. contributed equally. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Notes

The authors declare no competing financial interest.

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

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

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REFERENCES

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