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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, 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 voltage-gated sodium channel NaV1.6. In an attempt to decouple channel activation from channel binding, we also synthe- sised 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-nocicep- tive in mouse models of NaV1.6-mediated pain. INTRODUCTION: vitro.5 In the above-mentioned studies, the long-chain scorpion -toxin Cn2 from Centruroides noxious proved espe- The generation and propagation of action potentials in cially useful in dissecting the roles of the individual NaV neurons is realized through a well-orchestrated interplay of isoforms in vivo because of its potent4 and highly selective ion channels in which the family of voltage-gated sodium activation of NaV1.6.6 These pharmacological properties channels (NaV) plays a central role.1-2 Humans have at least make Cn2 a promising candidate for development of ad- nine functional NaV subunits (NaV1.1–NaV1.9) that medi- vanced NaV1.6-selective molecular probes with altered ate specialized physiological processes in different tissues mechanism of action to explore their effect on NaV1.6 phys- and electrically excitable cell types. Dissecting the physio- iology. logical roles of individual subtypes has been hampered in the past because multiple individual NaV subtypes often co- localize and their central 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 oc- cluding the central pore or by interacting with one or more of the four voltage-sensing domains (VSDs) that control channel opening and closing. Scheme 1: Chemical synthesis of Cn2 by native chemical ligation. (i) native chemical ligation; (ii) folding and disulfide for- Using a range of NaV modulators and knockout animals, mation. The inset shows the primary structure of Cn2, with cys- we recently demonstrated an important role for NaV1.6 in teines in bold and the ligation site (Glu28-Cys29) underlined. multiple peripheral pain pathways, particularly those in- The inverted ‘E’ highlights the location of the point mutation volved in mediating cold and mechanical allodynia.4 Further (E15R). exploration of the role of NaV1.6 in pain pathways and eval- uation of NaV1.6 as a potential therapeutic target hinge on RESULTS AND DISCUSSION: the availability of advanced molecular tools or model sys- Scorpion -toxin Cn2 is a polypeptide of 66 amino acid tems such as NaV1.6 knock-out animals (which are not via- residues that is C-terminally carboxamidated and adopts a ble) and/or NaV1.6-selective molecular probes, such as 4,9- cysteine-stabilized fold supported by four disulfide anhydro-tetrodotoxin that shows some NaV1.6 selectivity in 1 bonds.7-8 C-terminal carboxamidation is a posttranslational for high-affinity NaV1.4 binding of the related long-chain modification that is difficult to reproduce in bacterial ex- scorpion toxin Ts3.9 pression systems and was recently shown to be important 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 H-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 effect of synthetically produced Cn2 on VGSC isoforms a total chemical synthesis approach that permits milligram NaV1.1-1.8, a high-throughput fluorescence imaging plate scale production of the relatively complex wild-type Cn2 reader (FLIPRTETRA) assay was used as previously de- and variants thereof. We initially attempted chemical syn- scribed.12 Briefly, HEK293 cells expressing NaV1.1-1.8 in a thesis of full-length wild-type Cn2 by Fmoc solid-phase pep- monolayer were incubated with membrane potential dye tide synthesis. A trial TFA deprotection with concomitant (Molecular Devices, Sunnyvale, CA) and the change in fluo- peptide cleavage from the resin was conducted after cou- rescence was continuously measured prior to and during pling of Cys29 to evaluate peptide assembly. HPLC-MS anal- compound addition (FLIPRTETRA). Native and synthetic Cn2 ysis of the crude cleavage product indicated significant concentration-dependently activated NaV1.6-expressing amounts of truncation and deletion side products, which HEK293 cells with EC50s of 72 nM (pEC50 7.14 ± 0.91) and prompted us to revise our plan to a fragment-based ligation 15.8 nM (pEC50 7.81 ± 0.20), respectively (Fig. 2a). In line strategy. The full-length sequence was divided into two seg- with native Cn2 previously reported, synthetic Cn2 (hence- ments, Cn2(1-28) and Cn2(29-66), which were joined by forth termed Cn2) at a concentration of up to 100 nM had native chemical ligation at Glu28 and Cys29 (Scheme 1).10-11 no significant effect on sodium channel isoforms NaV1.1- Synthesis of the required Cn2(1-28)-−thioester fragment NaV1.3, NaV1.5, NaV1.7 or NaV1.8 (Fig. 2b). Activity at the via Boc-SPPS was straightforward and the ligation with muscle specific NaV subtype, NaV1.4, has not been observed Cn2(29-66) proceeded smoothly, producing the full-length for native Cn2 at concentrations of 300 nM.6 However, our polypeptide in 44.8% yield. Folding and disulfide formation data show that synthetic Cn2 is a weak agonist for NaV1.4 in of the polypeptide required extensive optimization; guani- the FLIPR assay. Collectively, these data suggest that syn- dinium HCl at high concentrations (1.5 M) was essential thetic and venom-derived wild-type Cn2 are structurally during folding to prevent toxin from aggregating and pre- and pharmacologically identical. cipitating, and relatively long folding times (72 h) were nec- Having established synthetic access to the wild-type essary to favour the native disulfide isomer and improve toxin, we turned our attention to the design of Cn2 ana- yields (SI Fig. 5). The synthetic toxin was isolated by RP- logues with altered activity. Gurevitz and colleagues have HPLC in acceptable yield (30.8%) and its monoisotopic mo- shown that substituting a conserved glutamate in -scor- lecular weight, determined by high resolution MALDI-MS pion toxins Css4 or Bj-xtrIT with arginine (E15R) results in (7584.4 ± 0.2 Da, Fig. 1a), was in excellent agreement with uncoupling of NaV-binding and NaV-activation.13-14 A similar the theoretical value (7584.5 Da; taking into account C-ter- strategy was recently adopted to -scorpion toxin Ts1 to ob- minal amidation and formation of four disulfide bonds). tain NaV1.4 probes with reduced NaV excitatory activity.15 HPLC comparison of the purified synthetic Cn2 toxin with The corresponding Cn2 mutant, Cn2[E15R], was prepared native toxin isolated from C. noxious venom under identical using the same synthetic strategy used for wild-type toxin conditions revealed excellent agreement, suggesting that by replacing the N-terminal ligation fragment with Cn2(1- the correct disulfide isomer had been obtained (SI Fig. 8). 28, E15R)-thioester and folded using the protocol estab- The structural integrity of synthetic Cn2 was further con- lished for wildtype toxin. firmed by 2D TOCSY and NOESY NMR spectroscopy, which allowed complete assignment of backbone proton reso- nances. Comparison of the H-chemical shifts to published values for venom-derived Cn2 indicated excellent agreement, suggesting that the synthetic and venom-derived Cn2 molecules are structurally identical (Fig. 1b).7 To assess the 2 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 normalised to baseline vehicle control (% Control) with EC50s of 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).

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