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Institute for Molecular Bioscience, Centre for Pain Research, The University of Queensland, Brisbane, Queensland 4072, AustraliaSchool of Pharmacy, The University of Queensland, Woolloongabba, Queensland 4103, Australia
Institute for Molecular Bioscience, Centre for Pain Research, The University of Queensland, Brisbane, Queensland 4072, AustraliaNational Cancer Institute, National Institutes of Health, Frederick, Maryland 21702, USA
Huwentoxin-IV (HwTx-IV) is a gating modifier peptide toxin from spiders that has weak affinity for the lipid bilayer. As some gating modifier toxins have affinity for model lipid bilayers, a tripartite relationship among gating modifier toxins, voltage-gated ion channels, and the lipid membrane surrounding the channels has been proposed. We previously designed an HwTx-IV analogue (gHwTx-IV) with reduced negative charge and increased hydrophobic surface profile, which displays increased lipid bilayer affinity and in vitro activity at the voltage-gated sodium channel subtype 1.7 (NaV1.7), a channel targeted in pain management. Here, we show that replacements of the positively-charged residues that contribute to the activity of the peptide can improve gHwTx-IV's potency and selectivity for NaV1.7. Using HwTx-IV, gHwTx-IV, [R26A]gHwTx-IV, [K27A]gHwTx-IV, and [R29A]gHwTx-IV variants, we examined their potency and selectivity at human NaV1.7 and their affinity for the lipid bilayer. [R26A]gHwTx-IV consistently displayed the most improved potency and selectivity for NaV1.7, examined alongside off-target NaVs, compared with HwTx-IV and gHwTx-IV. The lipid affinity of each of the three novel analogues was weaker than that of gHwTx-IV, but stronger than that of HwTx-IV, suggesting a possible relationship between in vitro potency at NaV1.7 and affinity for lipid bilayers. In a murine NaV1.7 engagement model, [R26A]gHwTx-IV exhibited an efficacy comparable with that of native HwTx-IV. In summary, this study reports the development of an HwTx-IV analogue with improved in vitro selectivity for the pain target NaV1.7 and with an in vivo efficacy similar to that of native HwTx-IV.
are involved in almost all aspects of human physiology, in particular in initiation and propagation of neurotransmission. To date, nine NaV subtypes (NaV1.1–1.9) have been described based on their distribution and physiological function (
). The NaV architecture is typified by four domains (domains I–IV), connected via intracellular loops of various lengths, with each domain containing six segments (S) such that S1–S4 form the voltage sensor domains, and S5 and S6 form the pore domain (Fig. 1A) (
), only highly-specific inhibitors would be of interest in order to avoid unwanted off-target effects.
In the search for novel modulators of these channels, gating modifier toxins (GMTs) discovered in spider venom have proven to be useful pharmacological probes and drug leads for conditions mediated by NaV function due to their high specificity and selectivity for the therapeutically-relevant sodium channel subtype NaV1.7 (
). However, huwentoxin-IV (HwTx-IV), a GMT found in the venom of the Chinese bird spider Haplopelma schmidti that acts selectively on tetrodotoxin-sensitive sodium channels, is known to have weak affinity for lipid membranes (
). These studies contributed to the perspective that the lipids surrounding NaVs and other voltage-gated ion channels can attract, concentrate, and manipulate the binding of GMTs near their active sites at the channels for improved potency (
A largely-unanswered question is how increased GMT–lipid affinity affects selectivity for NaV1.7. GMT selectivity for NaV1.7 is a desirable feature because of the reported toxicity of some of these peptides in vivo, probably associated with off-target effects (
). A difficulty with improving the selectivity of HwTx-IV, and similar GMTs, is that these peptides are attracted to a sequence of highly-conserved anionic amino acid residues (Glu-811, Asp-816, and Glu-818 (human NaV1.7 primary accession number Q15858)) located on the extracellular loop between S3 and S4 of domain II (Fig. 1C) (
) showed that R26A and K27A mutations of HwTx-IV resulted in some loss of potency at NaV1.7, accompanied by larger reductions in potency at NaV1.2. The R29A mutation resulted in no change in potency at NaV1.7, but a considerable reduction in potency at NaV1.2 (these studies were conducted on NaVs expressed in HEK293 cells) (
). The main difference between NaV1.7 and NaV1.2 at the putative HwTx-IV–binding site on S3 and S4 of DII is that NaV1.2 only has two anionic residues compared with three on NaV1.7 in the putative GMT-binding site (NaV1.2 contains Asn-816 in place of Asp-816) (Fig. 1C). Therefore, the reduction in potency for the Arg-29 analogue could be due to weakened electrostatic interactions between HwTx-IV and NaV1.2 (
). Based on these observations, we hypothesized that manipulation of the basic residues on loop 4 of HwTx-IV may aid in improving selectivity by modulating salt-bridge interactions between basic amino acids on loop 4 of the GMT and anionic amino acids in the conserved S3 and S4 extracellular loop of DII on off-target NaVs.
) as a starting molecule to capitalize on the increased potency of this molecule at NaV1.7, which is accompanied by stronger affinity for the lipid bilayer compared with HwTx-IV. We previously postulated that the lipid bilayer can attract and concentrate gHwTx-IV near its binding site at NaV1.7 to increase its apparent potency (
), we were interested in examining whether point mutations to Arg-26, Lys-27, and Arg-29 of gHwTx-IV would improve selectivity while maintaining potency at NaV1.7 through interactions with the lipid bilayer. To this end, we synthetically produced HwTx-IV, gHwTx-IV, [R26A]gHwTx-IV, [K27A]gHwTx-IV, and [R29A]gHwTx-IV (Fig. 1D). We ensured correct folding of the analogues using NMR, examined lipid binding of the peptides using surface plasmon resonance (SPR), and the activity of all peptides on human NaV1.7 was assessed using automated patch-clamp electrophysiology. In addition, we were specifically interested in examining selectivity for NaV1.7 over off-target NaVs that possess high-sequence homology to NaV1.7 at the putative binding site for HwTx-IV; therefore, peptides were tested on human NaV1.1, NaV1.2, NaV1.3, NaV1.4, NaV1.5, and NaV1.6 for off-target activity (Fig. 1C). To examine the ability of HwTx-IV and the analogues to inhibit activity of the channels, the most potent analogue at NaV1.7 was determined using automated patch-clamp electrophysiology, and its potency at NaV1.1–1.7 was subsequently determined. The NaV1.1–1.7 selectivity profiles for HwTx-IV and all analogues were determined using a membrane potential assay on FLIPRTETRA (
). Taken together, this body of work demonstrates that for gHwTx-IV, mutations to amino acids involved in activity can improve selectivity for NaV1.7 and that the same modifications decrease affinity for lipid bilayers. The most potent analogue, [R26A]gHwTx-IV, was further tested for on-target activity in vivo using a mouse model of NaV1.7-mediated nociception, and it showed improved efficacy compared with gHwTx-IV and similar efficacy to HwTx-IV.
Oxidation and structural analysis of the peptides
To attain correctly folded peptides, oxidation of HwTx-IV was conducted over 16 h to obtain the oxidized peptide with the most thermodynamically favorable fold and a yield of 80% as described previously (Fig. S1) (
). However, repeated misfolding of [R29A]gHwTx-IV using thermodynamic approaches prompted the use of orthogonal oxidation (Fig. 2 and Figs. S1 and S2).
[R29A]gHwTx-IV was synthesized with S-trityl (Trt) protecting groups on Cys III and Cys VI, acetamidomethyl (Acm) protecting groups on Cys II and Cys V, and 4-methylbenzyl (4-MeBzl) protecting groups on Cys I and Cys IV. A slow–fast–slow kinetic approach was used to oxidize one disulfide bridge at a time (Fig. 2). DMSO was chosen for the slow oxidation steps (step 1, Cys III–Cys VI (85% yield), and step 3, Cys I–Cys IV (after cleavage of 4-MeBzl using hydrogen fluoride (20% yield)) because it offered a low-risk approach with no observed side reactions. I2 was used for the removal of Acm protecting groups from Cys II and Cys V (step 2) and the subsequent oxidation of the pair. We found that a 10-min reaction using 20 molar eq of I2 in 15% (v/v) methanol (MeOH), 42.5% (v/v) acetic acid (AcOH), and 42.5% (v/v) H2O was optimal to maximize the oxidized product (30% yield) and to minimize side reactions between I2 and the indole groups on the Trp residues in the peptide.
One-dimensional (1D) 1H NMR analysis of the peptides confirmed that they were folded (Fig. S1). Secondary chemical shift analysis obtained from the sequential assignment of two-dimensional (2D) TOCSY and NOESY spectra showed that the overall structure of the peptides was maintained (Fig. 3). Expected local differences in the backbones of the peptides are observed between gHwTx-IV and HwTx-IV at the E1G, E4G, F6W, and Y33W mutations as before (
), and between gHwTx-IV and the three analogues at R26A, K27A, and R29A (Fig. 3).
Affinity of GMTs for lipid bilayers
SPR was used to examine the affinity of the GMTs for lipid bilayers by measuring the amount of peptide bound to lipid (peptide/lipid (P/L mol/mol)) using zwitterionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and anionic POPC/POPS (4:1 molar ratio) model membranes. The rationale behind the choice of lipids for these studies has previously been discussed (
). Briefly, phospholipids containing phosphatidylcholine headgroups are abundant on the outer leaflet of mammalian cells, and phospholipids containing phosphatidylserine headgroups mimic the anionic environment surrounding membrane-embedded voltage-gated sodium channels (
), HwTx-IV had weak affinity for model lipid bilayers (all values are maximum P/L mol/mol ± S.E.) (POPC = 0.01 ± 0.01; POPC/POPS = negligible) compared with gHwTx-IV (POPC = 0.07 ± 0.02; POPC/POPS = 0.16 ± 0.03) (Fig. 4). [R26A]gHwTx-IV (POPC = 0.024 ± 0.003; POPC/POPS = 0.06 ± 0.01), [K27A]gHwTx-IV (POPC = 0.06 ± 0.01; POPC/POPS = 0.12 ± 0.01), and [R29A]gHwTx-IV (POPC = 0.05 ± 0.01; POPC/POPS = 0.06 ± 0.01) had intermediate affinities for the lipid bilayers compared with gHwTx-IV and HwTx-IV. We previously described the membrane-binding face of gHwTx-IV to include Gly-1, Gly-4, Trp-6, and Trp-33 among other residues on the hydrophobic patch (
). The results herein suggest that the positively-charged amino acid residues on loop 4 of the GMT also contribute to affinity for lipid bilayers and that substitution of these residues for alanine residues on gHwTx-IV reduces their affinity for lipid bilayers.
Potency and selectivity of the GMTs
Potency at human NaV1.7 was improved by each of the mutations compared with HwTx-IV, as determined by automated patch-clamp electrophysiology (Fig. 5A and Fig. S3A). As before (
), gHwTx-IV was ∼4-fold more potent than HwTx-IV (HwTx-IV IC50 = 35.7 ± 5.2 nm; gHwTx-IV IC50 = 8.1 ± 0.3 nm). Potencies of [K27A]gHwTx-IV and [R29A]gHwTx-IV were comparable with gHwTx-IV ([K27A]gHwTx-IV IC50 = 8.5 ± 2.0 nm; [R29A]gHwTx-IV IC50 = 7.7 ± 2.6 nm). Interestingly, [R26A]gHwTx-IV was nearly five times more potent than gHwTx-IV and 21-fold more potent than HwTx-IV at NaV1.7 ([R26A]gHwTx-IV IC50 = 1.7 ± 0.5 nm) (Fig. 5A and Table 1).
Table 1Potency of HwTx-IV, gHwTx-IV, and analogues on NaV1.7
Selectivity of [R26A]gHwTx-IV for human NaV1.7 over human NaV1.1–1.6 was then determined by automated patch-clamp electrophysiology (Fig. 5B and Fig. S3B). Although NaV1.8 and NaV1.9 are both validated pain targets (
). [R26A]gHwTx-IV showed 11- and 13-fold selectivity for NaV1.7 compared with NaV1.6 and NaV1.1, respectively. Selectivity was 27-fold greater at NaV1.2 and 64-fold greater at NaV1.3. The peptide showed no activity at NaV1.4 or NaV1.5 up to 300 nm; therefore, selectivity at both subtypes is at least 176-fold (Fig. 5B and Table 2). This results in an [R26A]gHwTx-IV selectivity profile of NaV1.7 > NaV1.6 > NaV1.1 > NaV1.2 > NaV1.3 ≫ NaV1.4 and NaV1.5.
Table 2Potency and selectivity of [R26A]gHwTx-IV on NaV1.1–1.7
Time constants of block (τ) were determined using automated patch-clamp electrophysiology for HwTx-IV, gHwTx-IV, and the most potent analogue at NaV1.7, [R26A]gHwTx-IV (Fig. 5C). Peptides at their respective IC90 concentrations (HwTx-IV, 100 nm; gHwTx-IV, 20 nm; [R26A]gHwTx-IV, 10 nm) were added to cells to achieve near-complete block, and the decrease in current was measured over a period of 20 min, during which all peptides reached a steady state. It is necessary to test each peptide at its IC90 because τ values are concentration-dependent. HwTx-IV inhibited the current with the shortest time constant of block (τ = 0.753 s), followed by gHwTx-IV (τ = 2.67 s) and then [R26A]gHwTx-IV (τ = 3.20 s) (Fig. 5C). This suggests that the mutations to HwTx-IV forming gHwTx-IV and [R26A]gHwTx-IV have some effect on the rate of binding to the channel. Time constants of block were also measured for the same three peptides at 100 nm to compare them at the same concentration (Fig. 5D), which showed similar τ values for all peptides (HwTx-IV, τ = 0.753 s; gHwTx-IV, τ = 1.48 s; and [R26A]gHwTx-IV, τ = 1.30 s).
A stepwise wash-off was performed with HwTx-IV, gHwTx-IV, and [R26A]gHwTx-IV at their respective IC90 values (Fig. 5C). After continuous wash-off for 30 min, gHwTx-IV showed the highest percentage of sustained inhibition (68.1% ± 7.6%). This is similar to the remaining inhibition of HwTx-IV (52.1% ± 13.3%), but [R26A]gHwTx-IV had a significantly lower percentage of remaining inhibition (28.7% ± 8.8%). This suggests that the mutations that make up gHwTx-IV do not affect the peptide's dissociation from the channel compared with HwTx-IV, whereas the R26A mutation has a significant effect on peptide dissociation.
IC50 values of the same three peptides were measured with holding potentials of both −90 mV and −55 mV to determine whether binding is state- or voltage-dependent, showing no significant difference (Fig. S4). This suggests that although there is strong evidence that HwTx-IV binds preferentially to the closed conformation of voltage-sensor DII, the modified toxins could experience some alterations in binding to the activated voltage-sensor, and unlike for some other GMTs (
), the activity of the peptides tested in this study appear not to be influenced by channel state or membrane potential.
A complete selectivity screen was performed for each peptide using the FLIPRTETRA membrane potential assay (Table 3, Fig. 6). The addition of veratridine is known to underestimate the potency of GMTs compared with electrophysiology measurements, as reported previously (
). Although this assay gives a median 15-fold underestimation of the potencies of GMTs compared with patch-clamp electrophysiology assays (as observed in this study) (Figs. S5 and S6 and Table S1), it is a lower-cost, high-throughput assay that can reliably be used to measure relative potencies and therefore selectivity. Overall, the observed trend of selectivity for NaV1.7 using both assays is in good agreement.
Table 3Fold selectivity of HwTx-IV, gHwTx-IV, and analogues determined by membrane potential assay
From the membrane potential assay, selectivity of [R26A]gHwTx-IV and [K27A]gHwTx-IV was 9-fold (comparable with 13-fold for [R26A]gHwTx-IV, as determined by patch-clamp electrophysiology), and [R29A]gHwTx-IV was 54-fold more selective for NaV1.7 than NaV1.1 (Table 2, Table 3). This was an improvement compared with the selectivity of gHwTx-IV (3-fold) and HwTx-IV (less than 1-fold) (Table 3). Similarly, [K27A]gHwTx-IV and [R29A]gHwTx-IV showed a 12- and 13-fold selectivity for NaV1.7 over NaV1.2 respectively (Table 3). The selectivity of [R26A]gHwTx-IV was the most improved at 41-fold (overestimated compared with 27-fold, as determined by patch-clamp electrophysiology) (Table 2, Table 3). gHwTx-IV had 1.5-fold selectivity for NaV1.7 over NaV1.2, and HwTx-IV showed almost equivalent potency for both NaV1.7 and NaV1.2 (Table 3). [R26A]gHwTx-IV and [R29A]gHwTx-IV had a 92-fold (overestimated compared with 64-fold from patch-clamp electrophysiology) and 273-fold selectivity (respectively) for NaV1.7 compared with NaV1.3 (Table 2, Table 3). In this case, the selectivity of [K27A]gHwTx-IV was 11-fold, which was less than the selectivity of gHwTx-IV (83-fold). HwTx-IV did not show inhibitory activity at NaV1.3 up to 10 μm in our hands, making it at least 41-fold more selective for NaV1.7 over NaV1.3 (Table 3), although an IC50 value of 338 nm was previously reported at rat NaV1.3 (
). In comparison with NaV1.7 to NaV1.4, [R26A]gHwTx-IV showed the greatest improvement in selectivity for NaV1.7, showing no inhibitory activity on NaV1.4 up to 10 μm, thus being at least 392-fold more selective (or at least 176-fold, from patch-clamp electrophysiology) (Table 2, Table 3). [R29A]gHwTx-IV was 309-fold more selective and [K27A]gHwTx-IV was 236-fold more selective for NaV1.7 than NaV1.4 (Table 3). By comparison, gHwTx-IV was 324-fold more selective and HwTx-IV was 41-fold more selective for NaV1.7 than for NaV1.4 (Table 3).
None of the peptides showed activity at NaV1.5; therefore, if IC50 values were set at an arbitrary value of 10 μm (Fig. 6), the selectivity of the peptides relative to NaV1.7 in ascending order is HwTx-IV (41-fold), [K27A]gHwTx-IV (50-fold), gHwTx-IV (136-fold), [R29A]gHwTx-IV (187-fold), and [R26A]gHwTx-IV (392-fold) (Table 3). Compared with NaV1.6, [R26A]gHwTx-IV and [R29A]gHwTx-IV were 56-fold (overestimated compared with 11-fold, determined by patch-clamp electrophysiology) and 55-fold more selective for NaV1.7, and [K27A]gHwTx-IV was 22-fold more selective for NaV1.7 over NaV1.6. gHwTx-IV and HwTx-IV had 9- and 24-fold selectivity, respectively, for NaV1.7 (Table 3).
These results show that a combination of mutations to HwTx-IV, including gHwTx-IV (E1G, E4G, F6W, and Y33W), and point mutations at the positively-charged residues in loop 4 lead to modified potency and selectivity of this GMT at NaV1.7.
Taking these results together with the GMT–lipid affinity studies, removal of positively-charged residues that contribute to the activity of gHwTx-IV reduces, but does not eliminate, affinity for the lipid bilayer. These mutations also improve the overall selectivity for NaV1.7 with respect to off-target NaVs containing a string of anionic residues at HwTx-IV's putative binding site (Fig. 1, C and D).
Activity of HwTx-IV, gHwTx-IV, and [R26A]gHwTx-IV in murine model of NaV1.7-mediated nociception
We were interested in examining whether the most selective peptide was efficacious in vivo; therefore, the effects of HwTx-IV, gHwTx-IV, and [R26A]gHwTx-IV were examined in a mouse model of NaV1.7-mediated nociception after intraplantar administration of OD1 (300 nm), an α-scorpion toxin that selectively impairs inactivation and enhances current from NaV1.7 (Fig. 7) (
). There is 100% homology between human and mouse NaV1.7 across the S3 and S4 helix sequence in DII, which includes the S3 and S4 extracellular loop previously described as the GMT-binding site, suggesting that a mouse model provides a suitable comparison for human NaV1.7 (UniProt ID Q15858 hNaV1.7 and Q62205 mNaV1.7) (Fig. S7). HwTx-IV significantly reduced nocifensive behaviors (cumulative nocifensive behavior count 132.0 ± 44.3, p < 0.01) compared with the control (OD1; 518.3 ± 59.1). There was a significant reduction in nocifensive behaviors for [R26A]gHwTx-IV (182.7 ± 10.0, p < 0.01), comparable with that of HwTx-IV, at the same dose (Fig. 7). There was a small but significant difference in nocifensive behavior for gHwTx-IV (308.0 ± 28.2, p < 0.05), although this peptide showed a delayed inhibitory effect during the first 10 min post-injection, with a nocifensive behavior count comparable with the control (Fig. 7). After this delay period, nocifensive behaviors were reduced to a similar level to HwTx-IV and [R26A]gHwTx-IV for the remaining 20 min of the experiment.
NaV1.7 is a promising target for the development of novel therapeutics for the treatment of pain (
) can also influence affinity for lipid bilayers and selectivity for NaV1.7.
Point mutations to the positively-charged residues on loop 4 of gHwTx-IV reduce but do not eliminate affinity for model lipid bilayers (Fig. 4). This places [R26A]gHwTx-IV, [K27A]gHwTx-IV, and [R29A]gHwTx-IV in between gHwTx-IV and HwTx-IV with respect to affinity for neutral POPC and anionic POPC/POPS lipid bilayers. When affinity of HwTx-IV for model lipid membranes was first examined, this GMT showed no binding to lipid bilayers (
). We then engineered gHwTx-IV to increase affinity for model lipid membranes by reducing the anionic contribution to the overall surface charge of the peptide and by increasing the size of the hydrophobic patch (
). At the time, we proposed that the peptide may bind to the lipid bilayer using a membrane interaction face (Fig. 8A), consisting primarily of hydrophobic residues in a manner similar to ProTx-I and ProTx-II (
). The observed change in affinity for lipid bilayers of the three new analogues compared with gHwTx-IV suggests that Arg-26, Lys-27, and Arg-29 also contribute to the membrane-binding face residues important for affinity for the lipid bilayers, most probably through electrostatic interactions with anionic moieties on the lipid bilayer (Fig. 8A). In a study conducted by Henriques et al. (
), [E17K]ProTx-II showed increased affinity for POPC/POPS bilayers compared with the WT toxin. They propose that the introduction of a cationic residue increases the toxin's affinity for the anionic phospholipid headgroups (
), who found that a mutant of GsMTx4 with increased negative charge, [K15E]GsMTx4, showed an increase in POPC/1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) affinity.
[R26A]gHwTx-IV, [K27A]gHwTx-IV, and [R29A]gHwTx-IV displayed improved activity at NaV1.7 compared with activity of HwTx-IV at the same channel (Fig. 5). As before, gHwTx-IV was 4-fold more potent than HwTx-IV (
). [R29A]gHwTx-IV and [K27A]gHwTx-IV displayed similar potency compared with gHwTx-IV, and [R26A]gHwTx-IV was 21-fold more potent than HwTx-IV at NaV1.7 (Fig. 5 and Table 1). As mentioned, the basic residues on loop 4 of HwTx-IV have been proposed to form important interactions with anionic amino acids on the S3 and S4 loop of domain II on NaVs (
) made similar observations from their alanine scan, where activity at NaV1.7 and NaV1.5 was examined. The study showed that among other HwTx-IV analogues, [R26A]HwTx-IV, [K27A]HwTx-IV, and [R29A]HwTx-IV maintained minimal activity at NaV1.5, but [R26A]HwTx-IV and [K27A]HwTx resulted in loss of activity at NaV1.7 compared with HwTx-IV (
) and shown to be 2–3-fold selective for NaV1.7 over NaV1.1, NaV1.2, NaV1.3, and NaV1.6. Here, we have shown that gHwTx-IV mutations forming [K27A]gHwTx-IV and [R29A]gHwTx-IV do not negatively affect potency at NaV1.7, whereas [R26A]gHwTx-IV shows improved potency. This could be because potential losses to affinity for the channel may be compensated for by increased lipid membrane affinity. The lipid bilayer may anchor and concentrate these gHwTx-IV analogues in the lipid membrane near the channel active site. It is possible that previous studies did not see the same effect because those mutations (R26A, K27A, and R29A) were on HwTx-IV, a peptide with less affinity for the lipid bilayer compared with gHwTx-IV. The specific interactions between the basic residues on loop 4 of HwTx-IV and the NaV1.7 voltage-sensor domain remain unclear. Multiple binding models have been proposed using different methods, but no consensus has been reached (
). Additionally, because the mutations that make up gHwTx-IV and its analogues may alter the position of the peptide when binding, some interactions may differ.
Although gHwTx-IV has an increased affinity for the lipid bilayer compared with HwTx-IV, gHwTx-IV did not show an improved selectivity profile compared with HwTx-IV, with a comparable lack of selectivity against NaV1.2 and NaV1.1 and a reduction of selectivity against NaV1.3 and NaV1.6 when compared with NaV1.7 (Table 3). This lack of selectivity is most probably because gHwTx-IV is anchored in the lipid bilayer in a similar fashion for all the NaV channels, which is likely to be the same across the channel sub-types particularly in this study where all channels are studied in HEK293 cells. We propose that the improved selectivity profile for the new gHwTx-IV analogues comes from weakening some electrostatic interactions with the voltage-gated ion channels, combined with having the right amount of lipid affinity to anchor the peptides, contributing to an increase in potency at NaV1.7.
We examined the in vivo efficacy of HwTx-IV, gHwTx-IV, and the most potent analogue, [R26A]gHwTx-IV, using a NaV1.7 engagement model based on intraplantar administration of the NaV1.7 agonist OD1 (
). HwTx-IV and [R26A]gHwTx-IV significantly decreased nocifensive behaviors compared with the control to a similar extent. Thus, the anti-nocifensive effects observed for [R26A]gHwTx-IV are comparable with those observed for other GMTs using the same model (
). Interestingly, gHwTx-IV had a delayed inhibitory effect, showing no reduction in nocifensive behaviors over the first 10 min post-injection, then causing a decrease in nocifensive behaviors comparable with HwTx-IV over the remainder of the experiment. A similar trend was observed for m3-HwTx-IV when tested in the same OD1 pain model at 100 nm (
). This suggests that gHwTx-IV and m3-HwTx-IV, which have similar mutations, may have slower on rates at 100 nm compared with HwTx-IV and [R26A]gHwTx-IV. To investigate the delayed inhibitory effect of gHwTx-IV, τ values were measured in vitro. τ values are concentration-dependent, and all in vivo experiments were conducted at 100 nm, so τ values were measured in vitro both at their individual IC90 concentrations and at 100 nm. All three peptides showed different τ values at their individual IC90 concentrations but similar values at 100 nm. At their individual IC90 concentrations, [R26A]gHwTx-IV had the highest τ value, followed by gHwTx-IV, and HwTx-IV had the lowest τ value. These results do not provide a clear explanation for the delayed in vivo activity of gHwTx-IV, as no apparent relationship exists between them. The in vitro order of potencies of the peptides was also not reflected in the in vivo experiments. Although [R26A]gHwTx-IV was 21-fold more potent than HwTx-IV, they showed similar potencies for the entire duration of the NaV1.7 engagement model, as did gHwTx-IV during the latter 20 min of the experiment.
Optimizing GMTs to access their targets in vivo remains an area for future work if these peptides are to be converted into efficacious drugs. Both HwTx-IV and the most selective analogues of ProTx-II remain toxic in animal models, resulting in death and possible histaminergic effects, respectively (
); therefore, an area for future investigations could be to examine whether the selectivity and efficacy of [R26A]gHwTx-IV is accompanied by a loss of neuromuscular toxicity in vivo or whether systemic dosing leads to motor side effects.
Taking the in vivo and in vitro data together, we have shown here that it is possible to engineer selective modulators of NaV1.7 while considering interactions with the lipid bilayers. The in vitro data give only a partial understanding of GMT behavior and further in vivo target engagement studies are therefore crucial to fully appreciate the biological activity of NaV1.7 modulators.
Here, we have considered all three components of the tri-molecular complex by investigating peptide–lipid bilayer as well as peptide–channel interactions in the quest to design potent and selective modulators of NaV1.7. As seen for [R26A]gHwTx-IV, [K27A]gHwTx-IV, and [R29A]gHwTx-IV, peptides do not require maximal affinity for the lipid bilayer to display an increase in potency at NaV1.7. Instead, for this suite of peptides, an intermediate affinity for lipid bilayers somewhere between gHwTx-IV and HwTx-IV is optimal for potency and selectivity. GMTs are probably adapted more for contact with the channel than for contact with the lipid bilayer (
); therefore, we propose that the tripartite relationship formed by gHwTx-IV and its analogues occurs such that the peptides are nestled between the extracellular loops formed by S3 and S4 and S1 and S2 and probably interact with the phospholipid headgroups in this region (Fig. 8B). We have previously discussed the possibility of this binding mode (
). Alternatively, the GMTs are first attracted to the anionic environment surrounding voltage-gated ion channels via electrostatic interactions, then concentrated in the lipid bilayer, and oriented externally near the S3- and S4-binding site on domain II of NaV1.7 (
), detailed studies on individual GMTs can produce novel insight and approaches to optimizing these peptides for use as potential drugs for pain and other NaV-dependent pathologies. We anticipate that new technologies, including cryogenic EM, will help us to overcome current limitations and allow future studies to focus on investigating the three components (GMTs, model lipid bilayers, and voltage-gated ion channels) simultaneously (
), providing a complete picture of the different interactions.
Materials and methods
Peptides were assembled using Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry on a Rink amide resin (for C-terminal amidation) on a scale of 0.25 mmol/g using an automated Symphony peptide synthesizer (Gyros Protein Technologies Inc., Tucson, AZ), as described previously (
). Side-chain protecting groups for HwTx-IV, gHwTx-IV, [R26A]gHwTx-IV, and [K27A]gHwTx-IV were Arg(Pbf), Asp(tBu), Asn(Trt), Cys (Trt), Gln(Trt), Glu(tBu), Lys(Boc), Ser(tBu), Thr(tBu), Trp(Boc), and Tyr(tBu). Additionally, as described, Acm was used for Cys II and Cys V and 4-MeBzl for Cys I and Cys IV to facilitate orthogonal oxidation of [R29A]gHwTx-IV. Cleavage from resin and removal of side-chain protecting groups (except Acm and 4-MeBzl) was simultaneously achieved in 96% (v/v) TFA, 2% (v/v) triisopropylsilane, and 2% (v/v) H2O for 2.5 h followed by diethyl ether trituration prior to solubilizing the peptide in 45% (v/v) acetonitrile (ACN), 0.05% (v/v) TFA (solvent AB) followed by lyophilization. Reduced peptides were purified using reverse-phase HPLC (RP-HPLC) as before (
For [R29A]gHwTx-IV (Fig. 2), the Trt-protecting groups on Cys III and Cys VI were removed during cleavage, and the disulfide bridge between these free cysteines was formed by incubating the peptide (0.1 mg/ml) in 30% (v/v) DMSO/H2O for 24 h. The peptide was purified using previously described semi-preparatory methods (
) and lyophilized prior to removal of Acm protecting groups from Cys II and Cys V with simultaneous oxidation of the free cysteines (1 mg/ml) using 20 molar eq of I2 in 15% (v/v) MeOH, 42.5% (v/v) AcOH, and 42.5% (v/v) H2O. 0.1 m ascorbic acid was used to quench the reaction after 10 min, and the peptide was purified (
) and lyophilized. Finally, the 4-MeBzl groups were removed at 0 °C for 1 h using hydrogen fluoride for cleavage and p-cresol as a scavenger. The peptide was extracted from the cleavage solution using diethyl ether and extracted from the diethyl ether using phase separation in 45% (v/v) ACN, 0.05% (v/v) TFA, in H2O, and the Cys I–Cys IV pair was oxidized over 24 h by diluting to 0.05 mg/ml peptide, 22.5% (v/v) ACN, and 33% (v/v) DMSO in H2O. Each step of peptide oxidation was monitored using MALDI-TOF MS and analytical RP-HPLC as described previously (
HwTx-IV, gHwTx-IV, and the three analogues contain aromatic residues allowing for Nanodrop quantification using the following extinction coefficients: HwTx-IV ɛ280 = 7365 m−1·cm−1 and gHwTx-IV, [R26A]gHwTx-IV, [K27A]gHwTx-IV, and [R29A]gHwTx-IV ɛ280 = 17,430 m−1·cm−1.
). Spectra were obtained on a cryoprobe-equipped Bruker Avance III 600 MHz spectrometer (Bruker Biospin, Callerica, MA). TopSpin 3.5 (Bruker) was used to process the spectra, and sequential assignments were completed using CCPNMR analysis 2.4.1 (CCPN, University of Cambridge, Cambridge, UK) (
), model lipid bilayers were prepared using POPC and POPS (Avanti Polar Lipids, Alabaster, AL). SPR (Biacore 3000, GE Healthcare) was used to examine the amount of peptide bound to zwitterionic POPC and anionic POPC/POPS (4:1 molar ratio) deposited on an L1 sensor chip. Peptides were then injected onto the lipid surface and followed for the duration of association and dissociation. Data at 170 s (near the end of association) were collected, corrected for buffer contribution, and normalized using the assumption that 1 response unit = 1 pg/mm2 of lipid deposited (or peptide injected). As described previously (
), data were fitted to a one-site specific binding curve, and maximum P/L (mol/mol) was extrapolated from the line-of-best-fit using GraphPad Prism, version 8.0.2 (GraphPad Software Inc, San Diego, CA).
HEK293 cells heterologously expressing human NaV1.1, NaV1.2, NaV1.3, NaV1.4, NaV1.5, NaV1.6, and NaV1.7 containing α and β1 subunits (SB Drug Discovery, Glasgow, Scotland, UK) were maintained at 37 °C in a 5% CO2-humidified incubator within T75 flasks containing minimal essential medium Eagle's, supplemented with 10% (v/v) heat-inactivated fetal bovine serum, and 1% (v/v) GlutaMAX. Selection antibiotics included 600 μg/ml geneticin for all the cell lines, and 2 μg/ml blasticidin for NaV1.1, NaV1.3, NaV1.4, and NaV1.5. NaV1.2, NaV1.6, and NaV1.7 contained 4 μg/ml blasticidin. NaV1.4 also contained 500 μg/ml Zeocin. Replicating cells were subcultured at 70–80% confluence using 0.1% (v/v) trypLE express reagent (Thermo Fisher Scientific, Waltham, MA).
Potency and affinity determination by automated patch-clamp electrophysiology
Automated patch-clamp electrophysiology assays were performed as described previously using a QPatch-16 automated electrophysiology platform (Sophion Bioscience, Ballerup, Denmark) (
). In brief, HEK293 cells were dissociated with trypLE Express (Invitrogen) and suspended in Ham's F-12 with 25 mm HEPES, 100 units/ml penicillin-streptomycin, and 0.04 mg/ml soybean trypsin inhibitor, and then stirred for 30–60 min. Extracellular solution contained (mm) 70 NaCl, 70 choline chloride, 4 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, and 10 glucose adjusted to pH 7.4, 305 mosm. For subtypes 1.1, 1.2, 1.3, and 1.6, concentrations of NaCl were increased to 140 mm to adjust for smaller currents. Intracellular solution contained (mm) 140 CsF, 1 EGTA, 5 CsOH, 10 HEPES, and 10 NaCl adjusted to pH 7.3 with CsOH, 320 mosm.
Peptides were diluted in extracellular solution with 0.1% BSA. Assays were run with a holding potential of −90 mV pulsed to −20 mV for 50 ms at 0.05 Hz. Activity assays were performed with 5-min incubation periods after each peptide addition. To measure τ values, 100 nm HwTx-IV, 20 nm gHwTx-IV, and 10 nm [R26A]gHwTx-IV were added to cells and incubated for 20 min. Inhibition was quantified by measuring peak current, and all values were normalized to buffer control. Concentration–response curves were plotted with GraphPad Prism, version 8.0.2 (GraphPad Software Inc.), fitted with a four-parameter Hill equation with variable Hill coefficient. Time constants of block data were also plotted with GraphPad Prism, and τ values were calculated by fitting data to a one-phase decay. Data are presented as mean ± S.E.
Examination of subtype selectivity using FLIPR
The FLIPRTETRA plate reader (Molecular Devices, Sunnyvale, CA) was used to examine activity of the peptides at NaV channels as described previously (
). In brief, freshly-dissociated cells were plated into 384-well clear-bottom black-walled imaging plates (Corning, NY) at a density of 10,000–15,000 cells per well. After 48 h, the growth media were removed from the wells, and the cells were loaded with 20 μl per well red membrane potential dye (Molecular Devices) diluted in physiological salt solution (PSS; composition (mm): NaCl 140, glucose 11.5, KCl 5.9, MgCl2 1.4, Na2H2PO4 1.2, NaHCO3 5, CaCl2 1.8, HEPES 10 (pH 7.4)) according to the manufacturer's instructions for 30 min at 37 °C, 5% CO2. Peptides were diluted in PSS, 0.1% (w/v) BSA and added to the cells to achieve concentrations ranging from 10 μm to 0.3 nm and incubated for 5 min. NaV channels were stimulated using 60 μm veratridine, and changes to membrane potential were measured with a cooled CCD camera (excitation 515–545 nm, emission 565–625 nm) with reads taken every 1 s for 10 s before (baseline values) and for 300 s after addition of veratridine. PSS and 0.1% (w/v) BSA was used as a negative control. ScreenWorks 126.96.36.199 (Molecular Devices) was used to compute the area under the curve over 300 s, and the data were plotted and analyzed using GraphPad Prism, version 8.0.2 (GraphPad Software Inc.), to quantify inhibitory effects of the peptides. To calculate concentration–response curves, a four-parameter Hill equation with variable Hill slope was fitted to the data. All data are expressed as the mean ± S.E. and are representative of at least three independent experiments (three wells per experiment). Potency of each peptide at off-targets is compared with potency of the specific peptide at NaV1.7 (Table 3).
In vivo NaV1.7 target engagement
Animal ethics approval was obtained from the Animal Ethics Committee of University of Queensland. 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 Research. Male C57BL/6J mice aged 6–8 weeks (20–25 g) were housed in 12-h light/dark cycles with access to food and water ad libitum. To assess the in vivo effect of peptide analogues, an OD1-induced model of NaV1.7 target engagement was used as described previously (
Briefly, the NaV1.7-selective α-scorpion toxin OD1 (300 nm) was diluted in phosphate-buffered solution and 0.1% (w/v) BSA. Under brief and light anesthesia (3% (v/v) isoflurane), mice were administered OD1 (40 μl of 300 nm) via shallow intraplantar injection into the dorsal hind paw. Animals received OD1 alone (control, n ≥ 3) or were co-administered OD1 with gHwTx-IV or R26A[gHwTx-IV] (40 μl of 100 nm, n = 3). Following injection, mice were allowed to recover in polyvinyl boxes and were video recorded for 30 min post-injection. Spontaneous nocifensive behaviors (paw lifts, licks, shakes, and flinches) were counted by a blinded observer.
A. J. A., J. R. D., and C. I. S. conceptualization; A. J. A. and I. V. data curation; A. J. A. and P. T. formal analysis; A. J. A., P. T., A. M., H. N. T., J. R. D., M. R. I., K. L. M., and I. V. investigation; A. J. A., P. T., A. M., H. N. T., J. R. D., M. R. I., K. L. M., I. V., and C. I. S. methodology; A. J. A., P. T., A. M., J. R. D., I. V., and C. I. S. writing-original draft; A. J. A., P. T., A. M., H. N. T., J. R. D., M. R. I., K. L. M., D. J. C., I. V., and C. I. S. writing-review and editing; J. R. D., I. V., and C. I. S. supervision; I. V. and C. I. S. resources; I. V. and C. I. S. project administration; C. I. S. funding acquisition.
The authors from the Institute for Molecular Bioscience thank Olivier Cheneval for assistance with assembly of peptides and Dr. Johannes Koehbach and Dr. Thomas Durek for HF cleavage. We thank Zoltan Dekan (Institute for Molecular Bioscience) for discussions about orthogonal oxidation of disulfide-rich peptides. We also thank Dr. Sónia Henriques (School of Biomedical Sciences, Queensland University of Technology) for her expertise on peptide–lipid membrane interactions.
This work was supported by Australian National Health and Medical Research Council (NMHRC) Project Grant APP1080405 (to C. I. S.), Australian Research Council Future Fellow supported by ARC Grant FT160100055, ARC Australian Laureate Fellow supported by Australian Research Council Grant FL150100146, NHMRC Early Career Fellowship Grant APP1139961 (to J. R. D.), University of Queensland International Research Scholarships (to A. J. A., P. T., and H. N. T. T.), Australian Government Research Training Program Scholarships (to K. L. M., A. M., and M. R. I.), and by NHMRC Career Development Fellowship APP1162503 (to I. V.) The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.