Discovery of non-opioid peptides that selectively relieve mechanical pain in rodents through inhibition of TRPV4 channels.

Mechanical hyperalgesia is a primary symptom of clinical pain; there remains a therapeutic challenge for severe mechanical and chronic forms of neuropathic pain. The 34-amino-acid neuropeptide GsMTx4, isolated from Tarantula spatulata, was identified as a selective inhibitor of mechanosensitive channels and shown to reduce mechanical hyperalgesia and neuropathic pain in rats. We previously reported two types (type I and type II) of short peptides derived from GsMTx4 that mimicked the inhibitory action of GsMTx4 on a mechanosensitive BK (SAKca) channel. In this study, we investigated whether these short peptides alleviate mechanical hypersensitivity, a major symptom of neuropathic pain. The synthetic type I peptide 01 (Pept 01) was derived from loop2+loop3 of GsMTx4, whereas type II Pept 02, Pept 03 and, Pept 04 were derived from loop 2 of GsMTx4. Carrageenan-induced inflammatory pain was induced in rats and mice, while the chronic constriction nerve injury (CCI) model was established in rats. We showed that administration of short peptides (270 μg/kg, i.p.) selectively inhibited mechanical pain in rats but failed to impact thermal or cold hyperalgesia. Interestingly, the antihyperalgesic effects of these peptides were comparable to those of morphine; however, they were resistant to the μ-opioid receptor antagonist naloxone and lacked morphine-induced side effects, e.g., tolerance and conditioned place preference (CPP). Among them, Pept 03, which contained only one Trp (Trp1) at the head and an additional Arg (Arg11, corresponding to Arg18 in GsMTx4) at the end of the peptide, most potently alleviated mechanical hyperalgesia. Genetic deletion of the TRPV4 gene in mice mostly abolished the analgesic effect of Pept 03. In oocytes expressing TRPV4 channels, application of Pept 03 or Pept 01 inhibited GSK101- or hypotonicity-activated TRPV4 currents in a dose-dependent manner. These results suggest that TRPV4 channels may serve as a direct target for the short peptides in alleviating mechanical pain. This study identifies several natural toxin-based peptides as promising non-opioid analgesics, paving the way for the development of selective and potent painkillers to treat mechanical pain.