A synthetic peptide, derived from neurotoxin GsMTx4, acts as a non-opioid analgesic to alleviate mechanical and neuropathic pain through the TRPV4 channel

Abstract

Mechanical pain is one of the most common causes of clinical pain, but there remains a lack of effective treatment for debilitating mechanical and chronic forms of neuropathic pain. Recently, neurotoxin GsMTx4, a selective mechanosensitive (MS) channel inhibitor, has been found to be effective, while the underlying mechanism remains elusive. Here, with multiple rodent pain models, we demonstrated that a GsMTx4-based 17-residue peptide, which we call P10581, was able to reduce mechanical hyperalgesia and neuropathic pain. The analgesic effects of P10581 can be as strong as morphine but is not toxic in animal models. The anti-hyperalgesic effect of the peptide was resistant to naloxone (an μ-opioid receptor antagonist) and showed no side effects of morphine, including tolerance, motor impairment, and conditioned place preference. Pharmacological inhibition of TRPV4 by P10581 in a heterogeneous expression system, combined with the use of Trpv4 knockout mice indicates that TRPV4 channels may act as the potential target for the analgesic effect of P10581. Our study identified a potential drug for curing mechanical pain and exposed its mechanism.

Keywords: Mechanical pain; Mechanosensitive channel; Non-opioid analgesic; Pain; Peptide; TRPV4; Tolerance addiction.

Graphical abstract

Figure 1

The synthetic peptide P10581 reduced mechanical hyperalgesia in a dose-dependent manner when given intradermally (i.d.). (A) Sequence for P10581 compared to neurotoxin GsMTx4. (B) The structure of P10581 obtained by MD simulations based on the solution structure of GsMTx4 (PDB code 1TYK). (C) The effect of P10581 (i.d. 1200 ng/kg, 5 μL) on mechanical pain induced by pressure by using Randall–Sellito test. Normal saline (i.d. 5 μL) was used for control. The inset above shows the animal protocol. The decreased paw withdrawal threshold (PWT) induced by carrageenan (Carr. 1%, 5 μL) was tested 1.5 h after Carr. injection. BL: Baseline. (D) Evaluation of P10581 (i.d. 1200 ng/kg, 5 μL) on the baseline of the nociceptive mechanical threshold without inflamed. Morphine (i.d. 5 mg/kg) was used for positive control. The effect of GsMTx4 (i.d. 1200 ng/kg, 5 μL) was shown for comparison. n = 6 per group (except n = 10 for morphine). (E) The effects of peptides P10581 (left) and GsMTx4 (right) on mechanical hyperalgesia when low amounts of peptides were administered intradermally (i.d. 600 ng/kg, 5 μL). n = 6 per group. (F) Comparison of analgesic effects (ΔPWT) between P10581 and GsMTx4. ΔPWT = PWT_{(Post-Peptide)}–PWT_{(pre-Peptide)}. ΔPWT were obtained in (E). n = 6 per group. (G) Dose-dependent effects (ΔPWT) for P10581 compared with GsMTx4. Rats were subjected to Randall–Sellito tests 2 h after peptide injections. Solid lines are fits to the standard Hill equation. The IC₅₀ and Hill coefficient factors obtained were: 481.6 ± 71.2 ng/kg and 2.1 ± 0.6 for P10581, and 689.3 ± 21.5 ng/kg and 3.6 ± 0.2 for GsMTx4, respectively (n = 5 per group). The inset compared the IC₅₀ for the anti-hyperalgesic effect of P10581 vs. GsMTx4. (H) Comparison of the acute anti-hyperalgesic effect of P10581 injected intradermally vs. morphine, 0.4 μg (2 μg/kg, i.d.) and 200 μg (1 mg/kg, i.d.) per rat, respectively. Peptide/morphine was administered 1.5 h after Carr. injection, the PWT was tested 2 h after administration. The inset above shows the animal protocol. n = 5 per group. Right: the area under the curves (AUC, g·min) calculated from each rat. Data are expressed as mean ± SEM, ∗∗∗P < 0.001 and ∗∗P < 0.01 vs. saline. ^##P < 0.01, ^#P < 0.05, P10581vs. GsMTx4.

Figure 2

Synthetic short peptide P10581 reduces mechanical hyperalgesia in a dose-dependent manner when given intraperitoneally (i.p.). (A) Evaluation of i.p. injection of P10581 vs. GsMTx4 (270 μg/kg, 50 μL) on the baseline of the nociceptive mechanical threshold without inflamed. Morphine (i.p. 10 mg/kg) was used for positive control, n = 6 per group. (B) The effect of P10581 on mechanical pain induced by pressure on hindpaws. P10581 (i.p. 270 μg/kg, 50 μL) was administered intraperitoneally 1.5 h after the inflammatory model induced by Carr. (i.d. 1%, 5 μL). Normal saline (i.p. 50 μL) was used for control. n = 6 per group. (C) Dose-dependent effect for P10581 (i.p.) on mechanical hyperalgesia induced by Carr. (i.d. 2%, 50 μL) compared with GsMTx4. Rats were subjected to Randall–Sellito tests 3 h after peptide injection. n = 4–5 at per dose. (D) Comparison of the maximum anti-hyperalgesic effects (MaximΔPWT) between P10581 and GsMTx4. (E) Comparison of the acute anti-hyperalgesic effect of P10581 vs. morphine given intraperitoneally, 0.08 mg (400 μg/kg) and 0.5 mg (2.5 mg/kg) per rat, respectively. (F) The area under the curves (AUC, g·min) calculated from each rat. n = 4 per group. The peptide/drug was given intraperitoneally 1.5 h after Carr. (i.d. 2%, 5 μL). Rats were subjected to Randall–Sellito tests 1 h after peptide/drug treatment. Data are expressed as mean ± SEM, ∗∗∗P < 0.001 and ∗∗P < 0.01 vs. saline unless specified.

Figure 3

The synthetic peptide P10581 reduces neuropathic pain in a dose-dependent manner. Neuropathic pain was induced by chronic constriction nerve injury (CCI). (A) Rats were subjected to Randall–Sellito tests for neuropathic pain. Nerve injury lasted for 18 consecutive days as long as we tested (n = 10). (B) The animal protocol used for (C, D). (C) The effect of P10581 (i.d. 1.8 μg/kg, 5 μL) on neuropathic pain up to 14 days after CCI. n = 7 per group. (D) The same as in (C), but for the effect of GsMTx4 (i.d. 1.8 μg/kg, 5 μL), n = 6 per group. (E) Dose-dependent analgesic efficacies of P10581 on neuropathic pain obtained on Days 6, 7, 8, 10, and 14. n = 5–6 per dose. P10581 was administered intradermally 2 h before the rats were subjected to Randall–Sellito tests. (F) Summary of the IC₅₀ for the anti-hyperalgesic effects of P10581 on neuropathic pain on the days as indicated. (G) Summary of the maximum anti-hyperalgesic effects (MaxiΔPWT) of P10581 on neuropathic pain tested on the days as indicated. (H) Comparison of the dose-dependent analgesic effect for P10581 compared with peptide GsMTx4. (I) Comparison of the maximum anti-hyperalgesic effects (MaxiΔPWL) between P10581 and GsMTx4 on neuropathic pain. Rats were subjected to Randall–Sellito tests 2 h after peptides injection (i.d.) on Day 8 after CCI. n = 4–5 per dose. Data are expressed as mean ± SEM, ∗∗P < 0.01 and ∗P < 0.05, post-treatment vs. pre-treatment unless specified.

Figure 4

The analgesic effects of peptide P10581 do not develop analgesic tolerance in both inflammatory and neuropathic pain. (A) Animal protocol for repeated injections of peptide/drug after carrageenan for (B). (B) Repeated injections of P10581 (2 μg/kg, 5 μL, i.d.) did not reduce the analgesic effect in Carr. (i.d. 1%, 5 μL) inflammatory pain model, compared with morphine (i.d. 5 mg/kg). Peptide/morphine was given every 2 h for 12 h after. Rats were subjected to Randall–Sellito tests 1 h after peptide/drug injections. n = 6 per group. ∗∗∗P < 0.001, ∗∗P < 0.01 and ∗P < 0.05, peptide/drug vs. Saline. (C) Injection of CFA into mouse hindpaw results in a decreased mechanical PWT in the Randall–Sellito test. Inset showing the analgesic effect of P10581 (i.t. 2 μg/kg, 5 μL) after CFA. (D) Animal protocols for repeated injections of peptide/drug after CFA for (E). (E) Repeated intrathecal (i.t.) injections of P10581 (2 μg/kg, 5 μL, twice a day) did not induce tolerance compared with morphine in CFA pain model. Peptide/drugs were given twice a day (at 9:00 am and 5:00 pm) for 7 consecutive days from Day 4 after CFA. Rats were subjected to Randall–Sellito tests 1 h after peptide/drug injections at 9 am. n = 6 per group. (F) Animal protocol for repeated injections of peptide/drug after CCI for (G–I). (G) 9 days of consecutive repeated injections of morphine (i.d. 5 mg/kg, twice a day) developed tolerance on neuropathic pain in CCI, whereas P10581 (red circle, i.d. 7.2 μg/kg, 5 μL) mostly reversed the tolerance induced by morphine. The effect of P10581 on Day 16 (after CCI) was obtained 24 h after the final injection of morphine. n = 6 per group. (H) the same as in G but for the effect of P10581 (i.d. 8 μg/kg, 5 μL, twice a day). The effect of morphine on Day 16 (after CCI) was obtained 24 h after the final injection of P10581. n = 7 per group. (I) Comparison of the normalized analgesic effects (ΔPWL (%)) for P10581 vs. morphine (obtained from G and H). ΔPWT (%) = [(PWT_{(Post-Peptide/drug)}–PWT_{(Pre-Peptide/drug)})/(Maximum ΔPWT)] × 100. (J) The dose-dependent analgesic efficacies for P10581 that were obtained before (Red arrow in inset) vs. after (Green arrow in inset) 9 days of consecutive repetitive injections (twice a day, at 9:00 am and 5:00 pm). The dose-dependent effect of P10581 after repetitive injections was obtained 24 h after the final dose administration. n = 4–6 per dose. Solid lines are fit to the standard Hill equation. n = 4–6 per dose. (K) Summary of IC₅₀ obtained in (J). Data are expressed as mean ± SEM, ∗∗∗P < 0.001, ∗∗P < 0.01 and ∗P < 0.05 for (C–I), ^##P < 0.01, ^#P < 0.05, Post-treatment vs. pre-treatment. n.s. not significantly different.

Figure 5

Peptide P10581 does not produce conditioned place preference (CPP) and exerts potent naloxone resistance in rats. (A–D) Peptide P10581 did not exhibit CPP. (A) Schematic of the experimental design for training and test of CPP. (B) Representative tracking for Saline (left)-, Morphine (middle)-, and P10581 (right)-paired groups after CCP training (See Method). (C) Mice treated with P10581 (2 μg/kg, s.c. 100 μL), at which produced a maximum anti-hyperalgesic effect, did not produce a significant increase in preference (T2). Saline (100 μL. left)- and morphine (10 mg/kg, s.c. middle)-paired groups were shown for negative and positive controls, respectively. (D) Mice treated with P10581 (2 μg/kg, 100 μL, s.c. left) did not produce a significant increase in preference scores relative to the saline-treated group. Preference scores were calculated by subtracting the time spent on the drug-paired side during pre-conditioning from the time spent on the drug-paired side during postconditioning. n = 5 per group. (E, F) The analgesic effect of P10581 shows potent naloxone (Nal) resistance in rats. (E) P10581 (i.d. 2 μg/kg, 5 μL) on mechanical pain subjected to the Randall–Sellito tests, showed a large increase in response to PWT either in the absence or presence of naloxone. Note: morphine lost the anti-hyperalgesic effect on mechanical pain after co-application of naloxone. (F) The areas under the curves (AUC, g·min) calculated from each mouse. The peptide/morphine (1 mg/kg) was given 1 h after Carr. (i.d. 1%, 5 μL). Naloxone was injected subcutaneously (2 mg/kg, s.c.) 2 h after peptide/drug administration (to ensure the maximum anti-hyperalgesic effect of P10581). Note: peptide at 2 μg/kg (i.d.) can induce an analgesic effect against acute and inflammatory pain that can be as strong as morphine. Rats were subjected to the Randall–Sellito tests 15 min after naloxone was subcutaneously (s.c.) injected. n = 8–11 per group. Data are expressed as mean ± SEM, ∗∗∗P < 0.001 and ∗∗P < 0.01 vs. saline unless specified. n.s. not significant difference.

Figure 6

The analgesic effect of peptide P10581 was mostly abolished by Trpv4-gene deletion in mice. (A–H) Deletion of Trpv4-gene mostly abolished the analgesic effect of P10581: (A) Schematic diagram of Trpv4 knock-out mice and PCR verification strategies. (B) PCR verifications of KO-fragment deletion in Trpv4 KO mice. The 499 bp fragment band is generated from Trpv4 wild-type mice, and the 567 bp fragment band is the expected PCR result of the Trpv4 KO genotype. The primers used are listed in Table 1. (C) Injection of CFA in homozygous (Trpv4^−/−) mice decreased the mechanical PWT subjected to the Randall–Sellito test, which lasted for 10 days as long as we tested. n = 11. Inset compared the baseline (before CFA) response to mechanical PWT among wild-type (Trpv4^+/+) littermates, heterozygous (Trpv4^+/−), and homozygous (Trpv4^−/−) mice. n = 10–12. (D) The analgesic effects of P10581 (s.c. 2 μg/kg, 100 μL) on heterozygous (Trpv4^+/−) mice. The corresponding wild-type (Trpv4^+/+) littermates were used for controls. (E) The same as in (D), but for P10581 (s.c. 2 μg/kg, 100 μL) effects on homozygous (Trpv4^+/−) mice vs. WT littermates. (F) Comparison of analgesic effects (ΔPWT) induced by P10581 at 2 h post-injections among WT littermates and Trpv4-deficient mice. (G) The same as in (F), but obtained at 3 h post-injections. (H) Comparison of the analgesic effects (ΔPWT) of morphine (5 mg/kg) obtained at 2 h (left) or 3 h (right) post-injections. ΔPWT (g) = PWT _(Post-Drug)–PWT _(Pre-Drug). n = 4–5 per group. Data are expressed as mean ± SEM, ∗∗∗P < 0.0001 vs. saline. ∗∗P < 0.01 and ∗P < 0.01, Trpv4-deficient mice vs. WT littermates. n.s. not significant difference.

Figure 7

TRPV4 is inhibited by the peptide P10581. (A) Left: representative time course of whole-cell current recordings elicited in HEK293 that were transiently expressed with TRPV4. Right: representative ramp current traces taken at the time points at (1), (2), (3), and (4) from the time course recording. A ramp protocol elicited by a voltage ramp from −100 mV to +100 mV was used. Note, currents evoked by TRPV4 selective activator GSK101 (0.3 μmol/L) at +100 mV were inhibited by co-application of P10581 (5 μmol/L), further application of TRPV4 selective inhibitor GSK219 (1 μmol/L) did not produce further decreases in TRPV4 currents. Horizontal bars show the time durations of applications of GSK101, P10581, and GSK219 from both solutions. (B) Bar summarized the effect of P10581 on GSK101-activated TRPv4 whole-cell currents recorded at +100 mV. Further applied GSK219 did not produce further inhibition on the TRPV4 channel. n = 4–5 per group. (C, D) P10581 inhibits TRPV4 currents induced by hypotonicity. (C) Representative whole-cell current elicited in Xenopus oocytes that were transiently expressed with TRPV4. Note, TRPV4 current activated by hypotonicity was inhibited by P10581 (1 μmol/L). Currents were recorded by using standard two-electrode voltage clamp (TEVC) recording. (D) The normalized inhibitory effect of P10581 on TRPV4-induced currents by hypotonicity at +100 mV. Further applied TRPV4 selective inhibitor GSK219 did not produce further inhibition. (E, F) Dose-dependent inhibition effects of P10581 on TRPV4-channel. Solid line is fit to the standard Hill equation. The IC₅₀ and Hill coefficient factors obtained were: 5.2 ± 1.2 nmol/L and 0.92 ± 0.12 (n = 4–5 per dose). (G) Schematic diagrams of the effects of P10581 on the TRPV4 channel, showing that P10581 not only inhibits GSK101-evoked TRPV4 activation (a) but also inhibits the currents activated by hypotonicity. Data are expressed as mean ± SEM, ∗∗∗P < 0.001 vs. basal currents unless specified.