µ-Conotoxins Targeting the Human Voltage-Gated Sodium Channel Subtype Na_V1.7

Abstract

µ-Conotoxins are small, potent, peptide voltage-gated sodium (Na_V) channel inhibitors characterised by a conserved cysteine framework. Despite promising in vivo studies indicating analgesic potential of these compounds, selectivity towards the therapeutically relevant subtype Na_V1.7 has so far been limited. We recently identified a novel µ-conotoxin, SxIIIC, which potently inhibits human Na_V1.7 (hNa_V1.7). SxIIIC has high sequence homology with other µ-conotoxins, including SmIIIA and KIIIA, yet shows different Na_V channel selectivity for mammalian subtypes. Here, we evaluated and compared the inhibitory potency of µ-conotoxins SxIIIC, SmIIIA and KIIIA at hNa_V channels by whole-cell patch-clamp electrophysiology and discovered that these three closely related µ-conotoxins display unique selectivity profiles with significant variations in inhibitory potency at hNa_V1.7. Analysis of other µ-conotoxins at hNa_V1.7 shows that only a limited number are capable of inhibition at this subtype and that differences between the number of residues in loop 3 appear to influence the ability of µ-conotoxins to inhibit hNa_V1.7. Through mutagenesis studies, we confirmed that charged residues in this region also affect the selectivity for hNa_V1.4. Comparison of µ-conotoxin NMR solution structures identified differences that may contribute to the variance in hNa_V1.7 inhibition and validated the role of the loop 1 extension in SxIIIC for improving potency at hNa_V1.7, when compared to KIIIA. This work could assist in designing µ-conotoxin derivatives specific for hNa_V1.7.

1. Introduction

Marine cone snail venom comprises a diverse mixture of disulfide-rich ion channel modulators called conotoxins [1]. µ-Conotoxins, characterised by a conserved cysteine framework (i.e., CC–C–C–CC), potently inhibit voltage-gated sodium (Na_V) channels through a pore blockage mechanism [2]. Although humans express nine different Na_V channel subtypes, µ-conotoxins have a preference for TTX-sensitive (TTX-s) subtypes, Na_V1.1–1.4, 1.6 and Na_V1.7, over TTX-resistant (TTX-r) subtypes Na_V1.5, 1.8 and 1.9 [3,4,5]. Of particular interest is their inhibitory potency at Na_V1.7, as this subtype has therapeutic potential for the treatment of pain [6,7]. However, the translation from hit to drug lead is hindered by the lack of specificity of µ-conotoxins for Na_V channel subtypes. Given the essential role of Na_V channels in the generation and propagation of action potentials, off-target inhibition of Na_V channels could lead to serious unwanted side-effects. Therefore, identifying µ-conotoxin residues contributing to potent and selective inhibition of human (h)Na_V1.7 channels would aid in the development of µ-conotoxin-derived therapeutics for diseases such as chronic pain.

Of the 22 µ-conotoxins characterised to date, only a few display potent inhibition of hNa_V1.7 [4,8]. We recently reported the discovery of SxIIIC, a novel µ-conotoxin that inhibits hNa_V1.7 with nanomolar potency, though non-selectively [9]. SxIIIC has high sequence homology with two other µ-conotoxins, SmIIIA and CnIIIC [10,11], both of which have been evaluated against mammalian Na_V channels, but only CnIIIC was characterised at human isoforms (hNa_V1.7 IC₅₀ 485 ± 94 nM) [4]. KIIIA is the only other µ-conotoxin reported to potently inhibit hNa_V1.7 (IC₅₀ 97–363 nM), although also in a non-selective fashion [8]. Several studies have used KIIIA to investigate residues essential for Na_V inhibition and identified several within the C-terminal α-helix of the peptide [8,12,13]. Those studies resulted in approaches for µ-conotoxin optimisation, including isolating the helical residues and downsizing KIIIA [14,15,16,17,18,19]. For example, Khoo and colleagues stabilised the KIIIA pharmacophore with a lactam helix, and while the selectivity profile across Na_V channel subtypes remained unaffected, the stabilised helix only yielded micromolar inhibitory potency [14]. In another example, a peptidomimetic based on the KIIIA pharmacophore residues was designed by Brady et al., but only showed micromolar inhibition of Na_V1.7 (species not reported) [15]. Other researchers have opted for a combination approach and designed ‘mini’ and ‘midi’ variants by combining two µ-conotoxins [16,17], or combining µ-conotoxins and spider toxins [18,19]; however, these too showed limited, non-selective inhibitory potency at hNa_V1.7.

In the present study, we used electrophysiology assays to characterise the hNa_V channel selectivity profiles of µ-conotoxins closely related to SxIIIC, including SmIIIA and KIIIA for Na_V1.7 (neuronal and pain related) over Na_V1.4 (muscle) sodium channel. Through structure-activity relationship analysis of hNa_V1.7-inhibiting µ-conotoxins, we discovered point mutations that affect potency and selectivity at hNav1.7. These findings could lead to the development of subtype-selective µ-conotoxin derivatives with potential for therapeutic development.

2. Results

2.1. Oxidation of Native µ-Conotoxins and Analogues

SxIIIC analogues including [Δ1,2]SxIIIC, [R16A]SxIIIC, [R16H]SxIIIC, [R16Q]SxIIIC, [D17A]SxIIIC, [R20A]SxIIIC, [R20E]SxIIIC, [R20H]SxIIIC, [R20Q]SxIIIC, [R20W]SxIIIC were produced using thermodynamic folding as the majority of these peptides produced one major isomer during folding as observed for native SxIIIC, and this major isomer was used for our electrophysiology studies (Figure S2). These peptides were not analysed further using NMR or high-resolution MS (HR-MS) for disulfide bond connectivity confirmation due to the similarities in RP-HPLC chromatogram compared to native SxIIIC suggesting native disulfide connectivity. SmIIIA, the KIIIA analogue [loop1R]KIIIA and SxIIIC analogues ([Δ7,8]SxIIIC, [Δ6–9]SxIIIC and [G8R]SxIIIC) were produced using regioselective folding as they did not form one major isomer during thermodynamic folding conditions (Figure S3). SxIIIC analogues [Δ1,2:R16H]SxIIIC, [Δ1,2:R20A]SxIIIC, [R16H:R20A]SxIIIC and [Δ1,2:R16H:R20A]SxIIIC) produced two main peaks during RP-HPLC analysis following thermodynamic folding (Figure S4) and both peaks were evaluated using 1D ¹H NMR (Figure S5) and compared to the NMR spectrum of native SxIIIC. When comparing the NMR spectrum of the SxIIIC peak 1 analogues (blue arrow in Figure S4), several additional or missing peaks were noted in amide region compared to the NMR spectrum of native SxIIIC. The 1D ¹H NMR of the second peak more closely resembled native SxIIIC suggesting native disulfide bond conformation (Figure S5), leading us to use the second peak (red arrow in Figure S4) for electrophysiology inhibitory potency evaluation.

2.2. Selectivity of µ-Conotoxins for hNa_V Channel Subtypes

µ-Conotoxin SmIIIA has been reported to only weakly inhibit mammalian Na_V1.7 [20,21], despite sharing ~90% sequence homology with the recently described non-selective hNa_V1.7 inhibitor SxIIIC [9]. KIIIA has also been shown to inhibit hNa_V1.7, albeit at various potencies probably due to the use of different species of Na_V1.7 and different recording systems [8,22]. To gain a comprehensive understanding of potency and selectivity of SmIIIA and KIIIA across human Na_V channel subtypes, we assessed the peptides by automated whole-cell patch-clamp electrophysiology in HEK293 cells expressing hNa_V1.1–1.7/β1 and CHO cells expressing hNa_V1.8/β3 and compared inhibitory potency with our results for SxIIIC in the same cell lines. Concentration-response curves (Figure 1A) were used to determine IC₅₀ values for the µ-conotoxins at each channel subtype (

Table 1. Summary of µ-conotoxin IC₅₀ values. IC₅₀ values displayed in nM ± SEM.

Subtype	SxIIIC a	SmIIIA	KIIIA
NaV1.1	132 ± 12	235 ± 15.0	136 ± 18
NaV1.2	364 ± 54	172 ± 49	186 ± 32
NaV1.3	89 ± 11	95 ± 9	>1000
NaV1.4	15 ± 11	14 ± 2	67 ± 15
NaV1.5	>1000	>1000	>1000
NaV1.6	125 ± 11	106 ± 37	762 ± 77
NaV1.7	152 ± 22	41 ± 4	379 ± 43
NaV1.8	>1000	>1000	>1000

n = 3–10 cells per data point. ^a Results from [9].

). Comparison of IC₅₀ values revealed differences in subtype selectivity between the three µ-conotoxins (Figure 1B). All three µ-conotoxins most potently inhibited hNa_V1.4, with SxIIIC (IC₅₀ 15 ± 11 nM) and SmIIIA (IC₅₀ 14 ± 2 nM) being significantly more potent than KIIIA (IC₅₀ 67 ± 15 nM). A similar pattern of inhibitory potency was observed for hNa_V1.3 and hNa_V1.6, while no differences in potency were seen at hNa_V1.2. At Na_V1.1, SxIIIC and KIIIA showed similar inhibitory potency, while SmIIIA was the least potent. KIIIA, SmIIIA and SxIIIC showed minimal to no inhibition of TTX-r hNa_V1.5 and SmIIIA and SxIIIC showed no inhibition of TTX-r hNa_V1.8 up to 1 µM (Table 1, Figure S6). Intriguingly, µ-conotoxin inhibitory potency at hNa_V1.7 was most varied, with a 9-fold difference between SmIIIA (IC₅₀ 41 ± 4 nM) and KIIIA (IC₅₀ 379 ± 43 nM) and a 4-fold difference between SmIIIA and SxIIIC (IC₅₀ 152 ± 22 nM). This makes SmIIIA, in our hands, the most potent µ-conotoxin inhibitor of hNa_V1.7 reported to date.

2.3. A Limited Number of µ-Conotoxins Display Inhibitory Potency at hNa_V1.7

One of the challenges in comparing µ-conotoxin inhibitory potency between different studies is the differences in methods and species (human, mouse, and rat) used. While several µ-conotoxins have been evaluated at hNa_V1.7 channels [4,23,24,25], only a few studies have directly compared µ-conotoxin inhibitory potency at hNa_V1.7 in the same cell background. Therefore, we expanded our screen to analyse the hNa_V1.7 inhibition of other conotoxins by automated whole-cell patch-clamp electrophysiology. CnIIIC inhibited hNa_V1.7 with similar potency to KIIIA (IC₅₀ 546 ± 42 nM), whereas TIIIA, GIIIA, GIIIB and SIIIA showed no inhibitory potency at hNa_V1.7 up to concentrations of 1 µM (Figure 2). These results are consistent with previous studies where TIIIA and GIIIB (3 µM), assayed using two-electrode voltage-clamp electrophysiology, were inactive at hNa_V1.7 [25], and SIIIA (10 µM) only produced ~75% block at hNa_V1.7 when evaluated using whole-cell patch-clamp methods [24].

2.4. Charged Residues in Loop 3 Affect Selectivity for hNa_V1.7

Interestingly, except for SIIIA, all µ-conotoxins that potently inhibit hNa_V1.7 contain five residues within loop 3 (the region between the fourth and fifth cysteine residues), whereas those that were inactive only contained four residues (

Table 2. Comparison of µ-conotoxin sequences.

µ-Conotoxin	Sequence	Inhibitory Potency at hNaV1.7
GIIIA	RDCCTOOKKCKDRQCKOQ-RCCA *	No
GIIIB	RDCCTOORKCKDRRCKOM-KCCA *	No
GIIIC	RDCCTOOKKCKDRRCKOL-KCCA *	No
TIIIA	RHGCCKGOKGCSSRECROQ-HCC *	No
CnIIIC	GCCNGPKGCSSKWCRDHARCC *	Yes
KIIIA	CCN—CSSKWCRDHSRCC *	Yes
SIIIA	ZNCCNG—GCSSKWCRDHARCC *	No
SmIIIA	ZRCCNGRRGCSSRWCRDHSRCC *	Yes
SxIIIC	RGCCNGRGGCSSRWCRDHARCC *	Yes

* C-terminal amidation, Z-pyroglutamic acid, O-hydroxyproline.

). We were therefore interested in investigating the role of the charged residues within this loop in hNa_V1.7 inhibition and used SxIIIC as a scaffold to design analogues with either charge-conserving or charge-neutralising substitutions (Table S1). We used SxIIIC in preference over the more active SmIIIA µ-conotoxin due to the ease of synthesis of SxIIIC compared to SmIIIA, which has proved challenging to fold. Whole-cell patch-clamp electrophysiology experiments comparing inhibitory potency of the mutants at Na_V1.7 and Na_V1.4 (Figure 3) showed that replacement of Arg16 by Ala, [R16A]SxIIIC, resulted in a two-fold loss of inhibition (IC₅₀ 334.1 ± 25.5 nM), whereas replacement of Arg16 with a neutral sidechain in [R16Q]SxIIIC did not significantly affect the potency of SxIIIC (

Table 3. Summary of IC₅₀ values for SxIIIC analogues at hNa_V1.7 and hNa_V1.4. IC₅₀ displayed in nM ± SEM.

Analogue	hNaV1.7	Fold Difference to SxIIIC	hNav1.4	Fold Difference to SxIIIC
SxIIIC	152.2 ± 26.8	−	12.8 ± 13.0	−
[Δ1,2]SxIIIC	106.2 ± 19.4	ns	96.3 ± 13.0	↓7-fold
[Δ7,8]SxIIIC	313.6 ± 36.6	↓2-fold	−	−
[Δ6–9]SxIIIC	317.9 ± 100.0	↓2-fold	−	−
[G8R]SxIIIC	155.7 ± 18.9	ns	−	−
[Loop1R]KIIIA	305.1 ± 23.9	↓2-fold *	−	−
[R16A]SxIIIC	334.1 ± 25.5	↓2-fold	38.0 ± 13.0	↓3-fold
[R16H]SxIIIC	66.5 ± 2.5	↑2-fold	80.3 ± 24.7	↓6-fold
[R16Q]SxIIIC	151.3 ± 24.0	ns	99.5 ± 12.9	↓7-fold
[D17A]SxIIIC	123.5 ± 18.1	ns	14.6 ± 7.7	ns
[R20A]SxIIIC	142.3 ± 12.9	ns	64.8 ± 15.4	↓5-fold
[Δ1,2; R16H]	666.6 ± 211.6	↓4-fold	221.4 ± 26.9	↓17-fold
[Δ1,2; R20A]	481.8 ± 646.9	↓3-fold	222.3 ± 70.4	↓17-fold
[R16H; R20A]	888.5 ± 916.4	↓6-fold	247.8 ± 57.7	↓19-fold
[Δ1,2; R16H; R20A]	>1000	−	>1000	−

n = 3–4 cells per data point. ns—no significant difference. * compared to KIIIA. ↓ fold-decrease, and ↑ fold-increase in potency compared to SxIIIC.

). Interestingly, substituting Arg 16 with His [R16H]SxIIIC (His carries a neutral charge at physiological pH due to its low pKa value, 6.0) we observed a three-fold improvement in inhibition of hNa_V1.7 (IC₅₀ 66.5 ± 2.5 nM) compared to SxIIIC. We also assessed the neighbouring residue Asp17 and observed no significant change in inhibition with the Ala-replacement analogue [D17A]SxIIIC (IC₅₀ 123.5 ± 18.1 nM). Surprisingly, we also observed no difference for the Arg20 analogue [R20A]SxIIIC (IC₅₀ 142.3 ± 12.9 nM), despite the analogous residue in KIIIA (Arg 14—KIIIA numbering) having previously been reported to shift subtype selectivity in favour of hNa_V1.7 [8].

We next evaluated the inhibitory potency of these analogues, along with truncated SxIIIC, at hNa_V1.4 to observe differences in subtype selectivity. Compared to SxIIIC (IC₅₀ 12.8 ± 13.0 nM), the R16H and R16Q mutations significantly reduced potency at hNa_V1.4 (IC₅₀ 80.3 ± 24.7 nM and 99.5 ± 12.9 nM, respectively). Interestingly, this reduction gave IC₅₀ values equipotent to hNa_V1.7 (Figure 3). The same trend was observed for truncated [~1,2]SxIIIC and the [R20A]SxIIIC analogue, with both displaying reduced potency at hNa_V1.4 (IC₅₀ 96.3 ± 13.0 nM and 64.8 ± 15.4 nM, respectively) but retaining inhibitory potency at hNa_V1.7. Although [R16A]SxIIIC slightly lost potency at hNa_V1.4 (IC₅₀ 64.8 ± 15.4 nM; Table 3), this did not affect subtype selectivity (Figure 3). In contrast, the D17A mutation in SxIIIC did not affect inhibition of either hNa_V1.7 or hNa_V1.4 (IC₅₀ 14.6 ± 7.7 nM). Together these results reveal that several SxIIIC residues, including Arg1, Gly2, Arg16 and Arg20, may contribute to selectivity between hNa_V1.4 and hNa_V1.7.

2.5. Double and Triple SxIIIC Mutants

Given the minimal disruption to hNa_V1.7 potency caused by some SxIIIC mutations, we designed and synthesised double and triple mutant SxIIIC analogues (Table S1). Compared to native SxIIIC, all four analogues lost significant inhibitory potency at both subtypes (Figure 4). At hNa_V1.7, the double mutants [Δ1,2; R16H], [Δ1,2; R20A] and [R16H; R20A]SxIIIC lost 4-, 3- and 6-fold potency, respectively (Table 3), whereas at hNa_V1.4, the difference was much larger, with all three analogues decreasing potency by an average of 18-fold (Table 3). Notably, the triple mutant [Δ1,2; R16H; R20A] only displayed ~50% inhibition at hNa_V1.7 at 1 µM and was not active at hNa_V1.4 at 1 µM, the highest concentration tested (Figure 4 and Figure S7).

2.6. Key Structural Differences between hNa_V1.7 Inhibiting µ-Conotoxins

The main sequence differences of SxIIIC and SmIIIA compared to KIIIA are, a two-residue N-terminal extension and a four-residue loop 1 extension (Figure 5A). To explore the significance of these extensions, we compared the NMR solution structures (PDB: SxIIIC 6X8R, SmIIIA 1Q2J and KIIIA 2LXG). Notably, while the cysteine framework of ‘native’ KIIIA [27] differed from SxIIIC and SmIIIA, this does not appear to affect the overall structure of these µ-conotoxins. Comparison of secondary Hα chemical shift deviations from random coil values showed similar shifts between residues Arg13–Ala20 (Figure 5A), indicating conserved structural features across this region. The N-terminal half of the µ-conotoxins displayed the most variation. Backbone alignment of 3D NMR structures between Cys10 and Cys22 (SxIIIC numbering; Figure 5B) showed that the extra residues in loop 1 of SxIIIC and SmIIIA extend the top half of the structure. We propose that this section contributes to the increased potency of these two µ-conotoxins at hNa_V1.7. Furthermore, a substitution at Ala19 in SxIIIC to Ser19 in SmIIIA and KIIIA did not appear to affect the shifts for this residue. However, the neighbouring residue Arg20 (SxIIIC numbering) did show differences between the three µ-conotoxins and suggesting a different side-chain orientation of this residue.

2.7. N-Terminal Deletion Does Not Significantly Affect the Potency of SxIIIC

To assess the effects of N-terminal extensions observed in SxIIIC and SmIIIA compared to KIIIA, we designed the truncated SxIIIC analogue [Δ1,2]SxIIIC (Table S1). Due to difficulties synthesising native and modified SmIIIA, only SxIIIC and KIIIA analogues were pursued. In whole-cell patch-clamp electrophysiology experiments, we found that deletion of the N-terminal residues of SxIIIC did not significantly affect potency at hNa_V1.7 (IC₅₀ 106.2 ± 19.4 nM) compared to native SxIIIC (IC₅₀ 152.2 ± 26.8 nM) (p > 0.05) (Figure 6).

2.8. Loop 1 Truncation Reduces Potency of SxIIIC at hNa_V1.7

Interestingly, when we deleted residues within loop 1 of SxIIIC ([Δ7,8]SxIIIC), we observed reduced potency (IC₅₀ 313.6 ± 36.6 nM; Table 3) compared to native SxIIIC. As these IC₅₀ values were similar to KIIIA (IC₅₀ 383.5 ± 15.2 nM; Figure 6), which has a similarly truncated loop 1, these results suggest that the additional loop 1 residues in SxIIIC are required for increased inhibition of hNa_V1.7 compared to KIIIA. Furthermore, when we introduced an extra charged residue into loop 1 of SxIIIC ([G8R]SxIIIC) to mimic SmIIIA, we did not observe a significant change in potency (IC₅₀ 155.7 ± 18.9 nM; Table 3) compared to SxIIIC. This observation suggests that the additional charged residue in loop 1 of SmIIIA does not cause the 4.4-fold difference in inhibition of hNa_V1.7 observed between SxIIIC and SmIIIA. Unfortunately, previously mentioned difficulties in producing SmIIIA limited our ability to confirm this using SmIIIA analogues. Finally, when modifying KIIIA to include additional residues within loop 1 ([loop1R]KIIIA), we expected this analogue to mimic the inhibitory potency of SxIIIC. However, we observed no significant change in potency (IC₅₀ 305.1 ± 23.9 nM; Table 3) compared to native KIIIA, suggesting that either the disulfide connectivity of KIIIA is unfavourable for loop 1 extensions, or that this peptide binds in a different orientation.

3. Discussion

µ-Conotoxins are potent inhibitors of disease-relevant Na_V channels, yet their promiscuous activity across different subtypes has hindered their potential for therapeutic development, as off-target activity may lead to undesirable side effects. Due to an apparent lack of inhibitory potency of many µ-conotoxins at hNa_V1.7, only a few studies have analysed structure–activity relationships at this subtype. Here, we identified key features of µ-conotoxins of SxIIIC, SmIIIA and KIIIA that contribute to inhibition of hNa_V1.7, including loop length, and single point mutations that affect selectivity towards hNa_V1.4 without affecting potency at hNa_V1.7.

We initially assessed two µ-conotoxins, SmIIIA and KIIIA, by whole-cell patch-clamp methods, to evaluate their selectivity profiles for hNa_V channel subtypes. Compared to SxIIIC [9], these highly homologous µ-conotoxins displayed distinct selectivity profiles (

Table 4. Comparison of µ-conotoxin selectivity profiles of hNa_V channels.

µ-Conotoxin	Selectivity Profile
SxIIIC	1.4 > 1.3 > 1.6 ≈ 1.1 ≈ 1.7 > 1.2 >> 1.5 ≈ 1.8
SmIIIA	1.4 > 1.7 > 1.3 ≈ 1.6 > 1.2 > 1.1 >> 1.5 ≈ 1.8
KIIIA	1.4 > 1.1 > 1.2 > 1.7 > 1.6 > 1.3 >> 1.5 ≈ 1.8

). Interestingly, our results are not in agreement with mammalian selectivity profiles reported previously for SmIIIA [20] or KIIIA [12,30]. While these inconsistencies may be attributed to the use of different techniques (two-electrode voltage- or patch-clamp methods on oocytes vs. mammalian cells; manual vs. automated techniques), or the presence of different auxiliary β-subunits, the most plausible explanation is species differences (human, rat or mouse). The earlier studies used rat isoforms, as opposed to the human subtypes used in this study. Despite the high sequence homology between species, it is not uncommon for µ-conotoxins to display different inhibitory potency across species. Such interspecies variability highlights the need to study human isoforms when evaluating the therapeutic potential of µ-conotoxins. We showed that SmIIIA inhibits hNa_V1.7 more potently than both SxIIIC (~4-fold) and KIIIA (~10-fold). In our hands, SmIIIA is the most potent µ-conotoxin inhibitor of hNa_V1.7 (IC₅₀ 41 ± 4 nM) reported to date.

As only a limited number of µ-conotoxins have been assessed at human Na_V isoforms, we explored the ability of µ-conotoxins GIIIA, GIIIB, TIIIA, CnIIIC and SIIIA to inhibit hNa_V1.7 by whole-cell patch-clamp methods. CnIIIC was found to be equipotent with KIIIA, whereas a lack of inhibitory potency was observed for the other four µ-conotoxins at the highest concentration tested (1 µM). The results for GIIIA, GIIIB and TIIIA were consistent with the homologous µ-conotoxin GIIIC, previously shown to be inactive at hNa_V1.7 [23]. While the results of SIIIA were expected [24], it is interesting that SIIIA (at 10 nmol, corresponding to 0.7 mg/kg) can produce an analgesic effect in a murine model of inflammatory pain [31]. Leipold et al. [3] introduced the Domain II pore loop of hNa_V1.7 into rNa_V1.4 and identified Asn889 (hNa_V1.7) as responsible for the reduced sensitivity of hNa_V1.7 to SIIIA. The corresponding residue in mNa_V1.7 is conserved, suggesting that this residue alone may not be responsible for species differences. Another possible explanation for the lack of activity of SIIIA at hNa_V1.7 may be the apparent low net charge (+1.7) compared to hNa_V1.7 inhibiting µ-conotoxins, which all have a net charge of +2.8 to +4.7 (Figure 7). While µ-conotoxins share key similarities such as a conserved disulfide network, the backbone sequences between these cysteines, referred to as loops 1–3, are varied. For example, GIIIA, GIIIB, GIIIC, PIIIA and TIIIA all have four residues in loop 3 (between Cys4–Cys6) and are classed in the M4-branch µ-conotoxins [32]. In contrast, SIIIA, KIIIA, CnIIIC, SmIIIA and SxIIIC have five residues in loop 3 and are classified as M5-branch µ-conotoxins [32]. M4-branch µ-conotoxins have an increased overall net charge (≥+4.8) compared to M5-branch µ-conotoxins (Figure 7), suggesting that overall net change may contribute to µ-conotoxins’ ability to inhibit hNa_V1.7.

To explore the role of residues in loop 3, we designed a series of µ-conotoxin analogues, opting to base our designs on SxIIIC, as opposed to the more potent SmIIIA, due to difficulties in folding the latter µ-conotoxin. Our experience in synthesising SmIIIA is consistent with literature that reports synthetic production of SmIIIA often yields multiple isomers when folded under thermodynamic conditions [33]. In the current study we used a regioselective approach for SmIIIA synthesis to ensure desired disulfide connectivity; however, despite successful direction of the disulfide bonds during regioselective SmIIIA synthesis, the yield was low, and purification proved surprisingly challenging considering the high sequence homology with SxIIIC. In contrast, SxIIIC could easily be oxidised thermodynamically, resulting in a single major isomer, or using regioselective oxidation. In addition to targeted Ala-mutations in SxIIIC, in lieu of an Ala-walk (replacing all non-Cys and Gly residues with Ala), which has been conducted for other µ-conotoxins [12,25,34,35], we were interested in exploring the consequences of different chemical properties at specific residues and thus employed rational design for our SxIIIC analogues.

Replacement of Arg16 of SxIIIC with an Ala ([R16A]SxIIIC) reduced potency two-fold at hNa_V1.7. Our results are in contrast to those of McArthur and colleagues, who found Ala-replacement of the equivalent residue in KIIIA ([R10A]KIIIA) increased inhibition at hNa_V1.7 [8]. That study did not report the disulfide connectivity of KIIIA, and activity studies lacked β-subunit expression, which has since been shown to affect the affinity of µ-conotoxins to Na_V channels [21]. Interestingly, we observed improved potency at hNa_V1.7 and decreased inhibition at hNa_V1.4 with a SxIIIC His-substitution at position 16, [R16H]SxIIIC. Whilst His theoretically carries a neutral charge at physiological pH, the charge state of this amino acid can be highly dependent on the environment created by surrounding amino acids and may carry a positive charge, which together with the additional bulk of the amino acid compared to the native Arg could explain the improved potency at Na_V1.7. Another analogue, [R20A]SxIIIC, also did not affect hNa_V1.7 inhibition and lost potency at hNa_V1.4; however, the peptide did not display altered subtype selectivity. These results are also in contrast to Arg20 KIIIA mutations previously reported [8] that shifted selectivity towards hNa_V1.7 from rNa_V1.2 and rNa_V1.4.

Using the three SxIIIC analogues that did not affect inhibition at hNa_V1.7 but reduced potency at hNa_V1.4, we designed and synthesised a series of double and triple mutants. None of the multiple SxIIIC mutant analogues improved potency at either channel tested (hNa_V1.4 and hNa_V1.7) or shifted selectivity in favour of hNa_V1.7. While these results were disappointing, they were not completely unexpected. µ-Conotoxins have naturally evolved to potently and selectively target Na_V channels, and the manipulation of such an evolutionarily refined natural product can be challenging. However, one explanation for the loss in potency may have been in choosing the incorrect isomer following thermodynamic oxidation (Figures S4 and S5). To assess if the folding contributed to the loss in inhibitory potency, we additionally tested the first eluting peak for each of the analogues against hNa_V1.4 and found them to be less active than the second eluting peak (Figure S7). In summary, none of the multiple mutant analogues improved the potency or selectivity of SxIIIC.

Given the potential clinical applications of selective Na_V1.7 inhibition, we compared the NMR structures of three µ-conotoxins with potent inhibition of hNa_V1.7, i.e., SxIIIC, SmIIIA and KIIIA. We identified two major differences, in the form of extensions in the N-terminal and loop 1 residues of SxIIIC and SmIIIA, both absent in KIIIA. As many residues within the highly conserved C-terminal region of the µ-conotoxins have been shown to be critical for µ-conotoxin inhibition [8,12,13,36], we proposed that these structural differences observed in the N-terminal region influenced the differences in the inhibitory potency at hNa_V1.7. To evaluate this, we designed an additional series of µ-conotoxins analogues using SxIIIC.

By creating a truncated N-terminal analogue, we found that the N-terminal residues of SxIIIC are not essential for inhibition of hNa_V1.7. However, truncated SxIIIC, which maintained inhibitory potency at hNa_V1.7, compared to native SxIIIC, displayed decreased potency at hNa_V1.4. The N-terminal extension of SxIIIC, and potentially SmIIIA, is likely to be essential for inhibition at other subtypes, as seen with µ-conotoxin KIIIB (identical to KIIIA but with a two-residue N-terminal extension) which inhibits rNa_V1.2 with 5-fold greater affinity than KIIIA [27]. These results are consistent with truncated SIIIA, which showed that these N-terminal residues played a non-essential role in rNa_V1.2 inhibition [34]. It could be suggested that these N-terminal residues play a role in guiding the µ-conotoxin to the binding site in different Na_V channel subtypes. In contrast to the N-terminal extension, we discovered that residues within loop 1 of SxIIIC influenced inhibition of hNa_V1.7. When loop 1 residues were removed to mimic KIIIA, the potency of SxIIIC reverted to IC₅₀ values similar to KIIIA. As many studies interested in optimising KIIIA for Na_V1.7 inhibition have focused on downsizing or reducing the µ-conotoxins to only retain residues contributing to the pharmacophore [14,15,16], our findings identify for the first time the importance of the residues within loop 1 of SxIIIC for potent inhibition of hNa_V1.7. Interestingly, when we reintroduced extra loop 1 residues into KIIIA, we did not observe the expected improved potency. By revisiting the NMR structure, we observed that although KIIIA had a different disulfide connectivity to SxIIIC, the overall conformation of the disulfide bonds did not change. This suggests that KIIIA with I–V, II–IV, III–VI connectivity might not be optimal for the addition of residues in loop 1 and this could be explored in future studies.

In addition to improved selectivity for Na_V1.7, which is essential to avoid severe systemic side effects, species selectivity of the µ-conotoxins currently limits preclinical efficacy test in relevant rodent models of pain. Accordingly, future work, such as modelling or structural studies using relevant channel states and subtypes, will be required to facilitate drug development efforts, although oral delivery of peptides remains a challenge that would likely limit µ-conotoxin-based therapeutics to parenteral administration.

In conclusion, we have identified that the number of residues within loops 1 and 3 of SxIIIC, SmIIIA and KIIIA contributes to potency at hNa_V1.7. Overall, the findings of this study provide valuable information for future design of hNa_V1.7 specific µ-conotoxin derived inhibitors.

4. Methods

4.1. Peptide Synthesis

KIIIA, SIIIA, SxIIIC and analogues including [Δ1,2]SxIIIC, [R16A]SxIIIC, [R16H]SxIIIC, [R16Q]SxIIIC, [D17A]SxIIIC, [R20A]SxIIIC, [R20E]SxIIIC, [R20H]SxIIIC, [R20Q]SxIIIC, [R20W]SxIIIC, [Δ1,2:R16H]SxIIIC [Δ1,2:R20A]SxIIIC, [R16H:R20A]SxIIIC and [Δ1,2:R16H:R20A]SxIIIC) were synthesised using methods described earlier [9]. Protecting groups trityl (Trt), acetamidomethyl (Acm) and 4,4′-dimethylsulfinylbenzhydryl (Msbh) were used for regioselective protection of SmIIIA and SxIIIC analogues ([Δ7,8]SxIIIC, [Δ6–9]SxIIIC and [G8R]SxIIIC) with connectivity Cys3–Cys15(Msbh)/Cys4–Cys21(Acm)/Cys10–Cys22(Trt). The KIIIA analogue [loop1R]KIIIA was produced with connectivity Cys1–Cys15(Msbh)/Cys2–Cys9(Acm)/Cys4–Cys16(Trt). All peptides were assembled on a CEM Liberty Prime automatic synthesiser (CEM, Matthews, NC, USA) using rink amide-AM resin. Peptides were cleaved from solid support and side chains simultaneously deprotected in 92.5% trifluoroacetic acid (TFA)/2.5% triisopropylsilane/2.5% water/2.5% 2,2-(ethylenedioxy)diethanethiol for 2 h at room temperature. Excess TFA acid was evaporated by N₂ flow, followed by peptide precipitation in ice-cold diethyl ether and centrifugation. Peptides were redissolved in solvent B (50% acetonitrile (ACN)/0.01% TFA) and lyophilised. TIIIA, GIIIA and GIIIB were synthesised as previously described [25,34]. A list of peptide sequences can be found in Supplementary Materials (Table S1).

4.2. Oxidation

Most SxIIIC analogues were oxidised thermodynamically to form disulfide bonds as described earlier [9]. However, due to difficulties obtaining the desired connectivity using thermodynamic oxidation, some peptides were oxidised using orthogonal protection of pairs of Cys residues. This regioselective oxidation was done as described previously [26,37], whereby linear peptides with Trt group removed were dissolved in acetic acid (2 mg/mL) and stirred at room temperature. To oxidise the first Cys pair, one equivalent of I₂ (dissolved in MeOH) was added dropwise until the reaction turned pale-yellow and the colour persisted. Five equivalents of free Trp were added to minimise side-reactions with iodine. Following 15 min of stirring, the reaction was diluted with water and hydrochloric acid to a final volume of 50% acetic acid and 1% hydrochloric acid. To remove the Acm group and form the second disulfide bond, eight equivalents of I₂ were added to the reaction and stirred for a further 30 min. The reaction was quenched with aqueous ascorbic acid until the solution became colourless. The intermediate product was isolated by RP-HPLC on a Shimadzu LC-20AT system equipped with an SPD-20A Prominence UV/VIS detector, and a FRC-10A fraction collector. Peptides were diluted to <2% acetic acid and loaded onto a Gemini, 5 µm C18 110 Å, 250 × 10 mm column (Phenomenex, Torrance, CA, USA). Peptides were purified using linear gradient between 5–25% solvent B over 40 min at 3 mL/min (solvent A: 0.05% TFA in H₂O; solvent B: 90% ACN/0.05% TFA in H₂O). Fractions containing the desired product were identified by electrospray ionisation-mass spectrometry (ESI-MS) and lyophilised. To remove the Msbh protecting groups and form the final disulfide bond, peptides were dissolved in TFA (1 mg/mL) and cooled on ice. Five equivalents of free Trp, dimethyl sulfate (1% v/v) and 20 equivalents of NaI were added and stirred for 15 min. The reaction was quenched by diluting the reaction with aqueous ascorbic acid (10 nM; 15 times the initial volume of TFA). The final product was isolated by RP-HPLC using a Gemini, 5 µm C18 110 Å, 250 × 5 mm column and a linear gradient 0–20% solvent B at 1 mL/min over 40 min. Fractions containing the desired product were identified by ESI-MS (Shimadzu LCMS-2020), lyophilised and stored at −20 °C. For analytical RP-HPLC and ESI-MS of the final products, see Figures S1–S4.

4.3. Cell Culture

HEK293 cells heterologously expressing hNa_V1.1–1.7/β1 (SB Drug Discovery, Glasgow, UK) or CHO cells heterologously expressing hNa_V1.8/β3 in a tetracycline-inducible system (ChanTest, Cleveland, OH, USA) were maintained in Minimum Essential Medium Eagle (M5650) supplemented with 10% v/v foetal bovine serum, 2 mM L-glutamine, and the selection antibiotics as recommended by the manufacturer. Cells were incubated at 37 °C with 5% CO₂ and split every 3–4 days when reaching 70–80% confluency using the dissociation reagent TrypLE Express (Thermo Fisher Scientific, Scoresby, VIC, Australia). Expression of hNa_V1.8 was induced by the addition of tetracycline (1 μg/mL) for 48 h prior to assays.

4.4. Whole-Cell Patch-Clamp Electrophysiology Assays

Automated whole-cell patch-clamp recordings were performed with a QPatch-16 automated electrophysiology platform (Sophion Bioscience, Ballerup, Denmark) as previously described [9].

The extracellular solution consisted of (in mM) 145 NaCl, 4 KCl, 2 CaCl₂, 1 MgCl₂, 10 HEPES, and 10 glucose, pH to 7.4 with NaOH (adjusted to 305 mOsm/L with sucrose). The intracellular solution consisted of (in mM) 140 CsF, 1 EGTA, 5 CsOH, 10 HEPES, and 10 NaCl, pH to 7.3 with CsOH (adjusted to 320 mOsm/L with sucrose). Peptides were diluted in extracellular solution with 0.1% bovine serum albumin and each concentration was incubated for 5 min. hNa_V1.1–1.7 currents were elicited by a 50 ms test pulse to −20 mV from a holding potential of −90 mV (repetition interval 20 s). hNa_V1.8 currents were elicited by a 50 ms test pulse to +10 mV from a holding potential of −90 mV (repetition interval 20 s) in the presence of TTX (1 µM) to inhibit endogenous TTX-sensitive current in CHO cells. Recordings were taken at ambient room temperature (22 °C). Peak current post-peptide addition (I) was normalised to peak current of buffer control (I₀). IC₅₀s were determined by plotting difference in peak current (I/I₀) and log peptide concentration. Concentration-response curves were fitted using the log (inhibitor) vs. response-variable slope (four parameters) Equation (1) to obtain pIC₅₀ values for each biological replicate.

y = 1/(1 + 10^((LogIC₅₀ − x) × HillSlope))

(1)

The corresponding IC₅₀s were used to compute mean ± standard error of the mean from at least n = 3 biological replicates.

5. Data Analysis

Data were analysed using GraphPad Prism 9.3.1. Statistical significance, defined as p < 0.05, was determined using One-way ANOVA with Tukey’s multiple comparison of IC₅₀ or pIC₅₀ values fitted for individual biological replicates as described above.