Pharmacological characterisation of the highly NaV1.7 selective spider venom peptide Pn3a

Abstract

Human genetic studies have implicated the voltage-gated sodium channel Na_V1.7 as a therapeutic target for the treatment of pain. A novel peptide, μ-theraphotoxin-Pn3a, isolated from venom of the tarantula Pamphobeteus nigricolor, potently inhibits Na_V1.7 (IC₅₀ 0.9 nM) with at least 40-1000-fold selectivity over all other Na_V subtypes. Despite on-target activity in small-diameter dorsal root ganglia, spinal slices, and in a mouse model of pain induced by Na_V1.7 activation, Pn3a alone displayed no analgesic activity in formalin-, carrageenan- or FCA-induced pain in rodents when administered systemically. A broad lack of analgesic activity was also found for the selective Na_V1.7 inhibitors PF-04856264 and phlotoxin 1. However, when administered with subtherapeutic doses of opioids or the enkephalinase inhibitor thiorphan, these subtype-selective Na_V1.7 inhibitors produced profound analgesia. Our results suggest that in these inflammatory models, acute administration of peripherally restricted Na_V1.7 inhibitors can only produce analgesia when administered in combination with an opioid.

Figure 1. Isolation of the novel spider peptide μ-TRTX-Pn3a from the venom of Pamphobeteus nigricolor.

(a) Photo of the male Pamphobeteus nigricolor specimen from which crude venom was obtained. (b) Chromatogram resulting from fractionation of the crude venom using RP-HPLC (purple dashed line indicates acetonitrile gradient). Corresponding activity of each fraction to inhibit veratridine-induced Na_V1.3 responses is shown above (black circles). The arrow indicates the active peak. (c) Sequences of μ-TRTX-Pn3a and μ-TRTX-Pn3b identified by N-terminal sequencing.

Figure 2. Selectivity of μ-TRTX-Pn3a for hNa_V1.1-1.9 channels.

(a) Concentration-response curves and (b) comparative potency of Pn3a at hNa_V1.1-1.9 assessed by whole-cell patch-clamp experiments. Pn3a most potently inhibited Na_V1.7, with 40-fold selectivity over hNa_V1.1, 100-fold selectivity over hNa_V1.2, 1.3, 1.4 and 1.6, and 900-fold selectivity over Na_V1.5, Na_V1.8, and Na_V1.9. Data are presented as mean ± SEM, with n = 3–9 cells per data point. (c) Representative hNa_V1.1-1.9 current traces before (black) and after addition of Pn3a (red). Currents were obtained by a 20 ms pulse of −20 mV for Na_V1.1-1.7, a 20 ms pulse +10 mV for Na_V1.8, and a 40 ms pulse of −40 mV for Na_V1.9. Pn3a (10 nM) selectively inhibited peak current at hNa_V1.7 only.

Figure 3. Effect of μ-TRTX-Pn3a on the electrophysiological parameters of hNa_V1.1-1.8.

I-V curves before (black triangles) and after addition of Pn3a (white triangles). Pn3a (100 nM) inhibited peak current at all Na_V subtypes but caused a rightward shift in the I-V curve at Na_V1.7 only. G-V curves before (black circles) and after addition of Pn3a (white circles). Pn3a (100 nM) significantly shifted the V_1/2 of voltage-dependence of activation to a more depolarized potential at Na_V1.7 only (Δ + 21.3 mV). Voltage-dependence of steady-state fast inactivation curves before (black squares) and after addition of Pn3a (white squares). Pn3a (100 nM) caused small but significant shifts in the V_1/2 of steady-state fast inactivation at Na_V1.1, 1.2, 1.3, 1.4, 1.7 and 1.8. Data are presented as mean ± SEM, with n = 4–10 cells per data point.

Figure 4. Additional effects of μ-TRTX-Pn3a on the electrophysiological parameters of hNa_V1.7.

(a) Time course of peak current reduction upon application of (arrow) 100 nM, 30 nM or 10 nM Pn3a at Na_V1.7. Onset of block was measured with repetitive pulses to –20 mV every 20 s. Data are fitted with single-exponential functions and the time constant of block (τ) is indicated for each concentration. (b) Voltage-dependence of time-to-peak as a measure of activation kinetics at Na_V1.7. Pn3a (100 nM) delayed time to peak between −20 mV and 15 mV. (c) Voltage-dependence of fast inactivation time constants at Na_V1.7. Pn3a (100 nM) slowed the inactivation rate between −10 mV and 0 mV. (d) Rate of recovery from inactivation at Na_V1.7. Pn3a (100 nM) slowed the rate of recovery from inactivation. Data are fitted with single-exponential functions and the time constant of recovery (τ) is indicated. (e) Steady-state slow inactivation at Na_V1.7. Pn3a (100 nM) had no significant effect on the voltage-dependence of steady-state slow inactivation. Data are presented as mean ± SEM, with n = 4–6 cells per data point. Statistical significance was determined using two-way ANOVA, *P < 0.05 compared to control. (f) Representative ramp current elicited by a 50 ms depolarization from −100 to +20 mV at a rate of 2.4 mV/ms at Na_V1.7. Pn3a (100 nM) inhibited ramp currents. (g) Potassium currents before (black) and in the presence of Pn3a (coloured) elicited by depolarisations to 70 mV in hNa_v1.7/rK_v2.1 chimeras. Pn3a (300 nM) inhibited potassium currents in the DII hNa_V1.7/K_V2.1 and DIV hNa_V1.7/K_V2.1 chimeras. Scale bars: 50 ms (abscissa), 2 μA (DI, DII, DIV, K_V2.1) and 1 μA (DIII) (ordinate axis).

Figure 5. Solution structure of Pn3a.

(a) The family of the 20 lowest energy structures superimposed over the backbone with disulfide bonds shown in yellow (Protein Data Bank code 5T4R). (b) The lowest energy structure with β-strands shown as arrows and disulfide bonds in ball and stick representation.

Figure 6. Pn3a inhibits TTX-sensitive current in small-diameter DRG neurons and eEPSC in C-fibres from lamina I neurons.

(a) Percentage inhibition of sodium current by TTX and Pn3a in IB₄⁻, lightly IB₄⁺ and strongly IB₄⁺ small-diameter and large-diameter DRG neurons (n = 6–15). (b) Representative peak current vs time plot before and after addition of TTX and Pn3a in a small-diameter DRG neuron. (c) Representative peak current vs time plot before and after addition of TTX and Pn3a in a large-diameter DRG neuron (d) Representative current traces from a lamina I neuron indicating the position of the dorsal root evoked Aβ-, Aδ- and C-fibre currents pre- and post-treatment with Pn3a. (e) eEPSC amplitudes normalized to the baseline control for C-, Aδ-, Aβ-fibre currents in lamina I neurons treated with Pn3a (n = 7, 7 and 4, respectively). Data are presented as mean ± SEM. Statistical significance was determined using t-test compared to control, *P < 0.05.

Figure 7. Analgesic effects of selective Na_V1.7 inhibitors.

(a) Pn3a (i.p.) dose-dependently reversed spontaneous pain behaviours elicited by intraplantar injection of the Na_V1.7 activator OD1 in mice (over 10 min); n = 3–9 per group. (b) Time course of reversal of OD1-induced spontaneous pain behaviours by Pn3a (3 mg/kg i.p.); n = 6 per group. (c) Effect of Pn3a (3 mg/kg i.p.) and PF-04856264 (30 mg/kg i.p.) on noxious heat assessed using the hotplate test (50 °C) in mice; n = 6–16 per group. Dashed line represents time spent on the hotplate by Na_V1.7^Wnt knockout mice. (d) Time course of effect of Pn3a (3 mg/kg i.p.) and (e) PF-04856264 (30 mg/kg i.p.) on formalin-induced spontaneous pain behaviours in mice; n = 4–15 per group. Effect of Pn3a (3 mg/kg i.p.) and PF-04856264 (30 mg/kg i.p.) on (f) carrageenan-induced mechanical allodynia and (g) carrageenan-induced thermal allodynia in mice; n = 4–12 per group. Effect of Pn3a (0.3 nmoles i.t.) on (h) FCA-induced mechanical allodynia, (i) FCA-induced thermal allodynia, and (j) motor performance on the Rotarod in rats; n = 4–9 per group. Data are presented as mean ± SEM. Statistical significance was determined using t-test, one-way or two-way ANOVA with Dunnett’s post-test as appropriate, *P < 0.05 compared to control.

Figure 8. Selective Na_V1.7 inhibitors synergize with opioids to produce analgesia.

(a) Time course of effect and (b) total pain behaviours with Pn3a (3 mg/kg i.p.), oxycodone (1 mg/kg i.p.) or the combination, in Phase I (0–10 min) and Phase II (10–55 min) of the formalin model in mice; n = 7–11 per group. Effect of Pn3a (3 mg/kg i.p.), PF-04856264 (30 mg/kg i.p.), oxycodone (0.66 mg/kg i.p.), or the combinations on (c) carrageenan-induced mechanical allodynia and (d) carrageenan-induced thermal allodynia in mice; n = 4–12 per group. (e) Effect of Phlotoxin-1 (50 μg/kg i.p.) and thiorphan (20 mg/kg i.p.) alone or in combination on acute heat responses; n = 6 per group (f) Effect of phlotoxin 1 (50 μg/kg i.p.) and buprenorphine (50 μg/kg i.p.) alone or in combination on acute heat responses; n = 6 per group. Effect of Pn3a (3 mg/kg i.p.) and gabapentin (100 mg/kg i.p.) alone or in combination on (g) carrageenan-induced mechanical allodynia and (h) carrageenan-induced thermal allodynia in mice; n = 3–8 per group. Data are presented as mean ± SEM. Statistical significance was determined using one-way or two-way ANOVA with Dunnett’s post-test as appropriate, *P < 0.05 compared to control.