Discovery of a selective NaV1.7 inhibitor from centipede venom with analgesic efficacy exceeding morphine in rodent pain models

Abstract

Loss-of-function mutations in the human voltage-gated sodium channel NaV1.7 result in a congenital indifference to pain. Selective inhibitors of NaV1.7 are therefore likely to be powerful analgesics for treating a broad range of pain conditions. Herein we describe the identification of µ-SLPTX-Ssm6a, a unique 46-residue peptide from centipede venom that potently inhibits NaV1.7 with an IC50 of ∼25 nM. µ-SLPTX-Ssm6a has more than 150-fold selectivity for NaV1.7 over all other human NaV subtypes, with the exception of NaV1.2, for which the selectivity is 32-fold. µ-SLPTX-Ssm6a contains three disulfide bonds with a unique connectivity pattern, and it has no significant sequence homology with any previously characterized peptide or protein. µ-SLPTX-Ssm6a proved to be a more potent analgesic than morphine in a rodent model of chemical-induced pain, and it was equipotent with morphine in rodent models of thermal and acid-induced pain. This study establishes µ-SPTX-Ssm6a as a promising lead molecule for the development of novel analgesics targeting NaV1.7, which might be suitable for treating a wide range of human pain pathologies.

Keywords: chronic pain; drug discovery; peptide therapeutic.

Fig. 1.

Purification of Ssm6a from venom of the centipede S. subspinipes mutilans. (A) Lyophilized venom (2.0 mg) was dissolved in 0.1 M phosphate, pH 6.0 then fractionated on a C₁₈ RP-HPLC column (Left). Elution was performed at a flow rate of 1.5 mL/min using a gradient of acetonitrile in 0.1% trifluoroacetic acid. The peak indicated by an arrow was purified further by analytical C₁₈ RP-HPLC (Right) using a shallower acetonitrile gradient. (Inset) A photo by Yasunori Koide of S. subspinipes mutilans. (B) Sequence of transcript encoding Ssm6a. The signal peptide is shown in gray, the propeptide region is underlined, and the mature peptide is shown with white text on a black background. The 3′-UTR including the poly(A) tail is also shown. (C) Comparison of the primary structure of Ssm6a with other venom peptides reported to act on Na_V1.7, including protoxin-1 (27), ATX-II (39), and μ-conotoxin KIIIA (40).

Fig. 2.

Effect of Ssm6a on Na_V channel currents in rat DRG neurons. All current traces were evoked by a 50-ms step depolarization to −10 mV from a holding potential of −80 mV every 5 s. (A) Inhibition of TTX-s Na_V channel currents by 1 μM Ssm6a. (B) Concentration–response curve for block of TTX-s Na_V currents in DRG neurons by Ssm6a (n = 5). (C) Time course for block of TTX-s currents by Ssm6a and reversal of block by washing with external solution. (D) Current–voltage (I–V) relationship for TTX-s currents before and after application of 100 nM Ssm6a. (E) Ssm6a shifts the conductance–voltage relationship to more positive potentials (n = 5). (F) Ssm6a had no effect on the voltage-dependence of steady-state inactivation, which was estimated using a standard double-pulse protocol (n = 5). Data points are expressed as mean ± SE and curves are fits to either the Hill (B, D, E) or Boltzmann (F) equation.

Fig. 3.

Effect of Ssm6a on hNa_V1.1, hNa_V1.2, hNa_V1.6, and hNa_V1.7 expressed in HEK293 cells. Current traces were evoked by a 50-ms step depolarization to −10 mV from a holding potential of −80 mV every 5 s. Control currents are shown in black and current traces showing inhibition of hNa_V1.1 (A), hNa_V1.2 (B), hNa_V1.6 (C), and hNa_V1.7 (D) by the indicated concentrations of Ssm6a are shown in red. (E) Concentration-response curves for inhibition of hNa_V1.1, hNa_V1.2, hNa_V1.6, and hNa_V1.7 by Ssm6a (n = 5).

Fig. 4.

Effect of Ssm6a on current-voltage relationships. Ssm6a induced a depolarizing shift in the I-V curves for activation of (A) hNa_V1.1 (10.7-mV shift at 10 µM), (B) hNa_V1.2 (12.9-mV shift at 1 µM), (C) hNav1.6 (9.5-mV shift at 5 µM), and (D) hNav1.7 (13.5-mV shift at 20 nM). In contrast, the peptide had no effect on steady-state inactivation of these channels (A–D).

Fig. 5.

Analgesic effects of Ssm6a in mice. Ssm6a was more effective than morphine in attenuating nociceptive behavior (paw licking) during (A) phase I (0–5 min postinjection) and (B) phase II (15–30 min postinjection) following intraplantar injection of formalin. (C) Ssm6a and morphine were equally effectively in reducing the abdominal writhing induced by intraperitoneal injection of acetic acid. (D) Ssm6a and morphine were equally effectively in increasing the photothermal pain threshold in mice subjected to tail heating. Data points are mean ± SEM (n = 10). Statistically significant differences compared with the saline control group (calculated using a Student t test) are indicated by *P<0.05 and **P<0.01.

Fig. 6.

(A) Comparison of the stability of Ssm6a (●) and rat atrial natriuretic peptide in human plasma (▲). Datapoints are mean ± SD (n = 3). (B) Far-UV CD spectrum of Ssm6a showing minima characteristic of α-helical secondary structure at 208 and 222 nm. The helical content derived from θ₂₂₂ is 63%. (C) Thermal denaturation profile of Ssm6a in 0 (●), 4 (■), and 8 M (▲) urea. Solid lines are fits of a sigmoidal function to the data to obtain T_m values. (D–G) The duration of the analgesic effects of Ssm6a was determined by monitoring paw licking time during phase I (D) and phase II (E) in the formalin pain model, writhing movements during acid-induced pain (F), and tail withdrawal latency in the thermal pain model (G).