Voltage-gated sodium (Na_v) channels initiate action potentials in most neurons, including primary afferent nerve fibres of the pain pathway. Local anaesthetics block pain through non-specific actions at all Na_v channels, but the discovery of selective modulators would facilitate the analysis of individual subtypes of these channels and their contributions to chemical, mechanical, or thermal pain. Here we identify and characterize spider (Heteroscodra maculata) toxins that selectively activate the Na_v1.1 subtype, the role of which in nociception and pain has not been elucidated. We use these probes to show that Na_v1.1-expressing fibres are modality-specific nociceptors: their activation elicits robust pain behaviours without neurogenic inflammation and produces profound hypersensitivity to mechanical, but not thermal, stimuli. In the gut, high-threshold mechanosensitive fibres also express Na_v1.1 and show enhanced toxin sensitivity in a mouse model of irritable bowel syndrome. Together, these findings establish an unexpected role for Na_v1.1 channels in regulating the excitability of sensory nerve fibres that mediate mechanical pain.

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.

Poor oral availability and susceptibility to reduction and protease degradation is a major hurdle in peptide drug development. However, drugable receptors in the gut present an attractive niche for peptide therapeutics. Here we demonstrate, in a mouse model of chronic abdominal pain, that oxytocin receptors are significantly upregulated in nociceptors innervating the colon. Correspondingly, we develop chemical strategies to engineer non-reducible and therefore more stable oxytocin analogues. Chemoselective selenide macrocyclization yields stabilized analogues equipotent to native oxytocin. Ultra-high-field nuclear magnetic resonance structural analysis of native oxytocin and the seleno-oxytocin derivatives reveals that oxytocin has a pre-organized structure in solution, in marked contrast to earlier X-ray crystallography studies. Finally, we show that these seleno-oxytocin analogues potently inhibit colonic nociceptors both in vitro and in vivo in mice with chronic visceral hypersensitivity. Our findings have potentially important implications for clinical use of oxytocin analogues and disulphide-rich peptides in general.

Loss-of-function mutations of Na_V1.7 lead to congenital insensitivity to pain, a rare condition resulting in individuals who are otherwise normal except for the inability to sense pain, making pharmacological inhibition of Na_V1.7 a promising therapeutic strategy for the treatment of pain. We characterized a novel mouse model of Na_V1.7-mediated pain based on intraplantar injection of the scorpion toxin OD1, which is suitable for rapid in vivo profiling of Na_V1.7 inhibitors. Intraplantar injection of OD1 caused spontaneous pain behaviors, which were reversed by co-injection with Na_V1.7 inhibitors and significantly reduced in Na_V1.7^−/− mice. To validate the use of the model for profiling Na_V1.7 inhibitors, we determined the Na_V selectivity and tested the efficacy of the reported Na_V1.7 inhibitors GpTx-1, PF-04856264 and CNV1014802 (raxatrigine). GpTx-1 selectively inhibited Na_V1.7 and was effective when co-administered with OD1, but lacked efficacy when delivered systemically. PF-04856264 state-dependently and selectively inhibited Na_V1.7 and significantly reduced OD1-induced spontaneous pain when delivered locally and systemically. CNV1014802 state-dependently, but non-selectively, inhibited Na_V channels and was only effective in the OD1 model when delivered systemically. Our novel model of Na_V1.7-mediated pain based on intraplantar injection of OD1 is thus suitable for the rapid in vivo characterization of the analgesic efficacy of Na_V1.7 inhibitors.

Spider venoms are a rich source of ion channel modulators with therapeutic potential. Given the analgesic potential of subtype-selective inhibitors of voltage-gated sodium (Na_V) channels, we screened spider venoms for inhibitors of human Na_V1.7 using a high-throughput fluorescent assay. Here, we describe the discovery of a novel Na_V1.7 inhibitor, μ-TRTX-Tp1a (Tp1a), isolated from the venom of the Peruvian green-velvet tarantula Thrixopelma pruriens. Recombinant and synthetic forms of this 33-residue peptide preferentially inhibited hNa_v1.7 > hNa_v1.6 > hNa_v1.2 > hNa_v1.1 > hNa_v1.3 channels in fluorescent assays. Na_V1.7 inhibition was diminished (IC₅₀ 11.5 nM), and the association rate decreased, for the C-terminal acid form of Tp1a compared to the native amidated form (IC₅₀ 2.1 nM), suggesting that the peptide C-terminus contributes to its interaction with hNa_V1.7. Tp1a had no effect on human voltage-gated calcium channels or nicotinic acetylcholine receptors at 5 μM. Unlike most spider toxins that modulate Na_V channels, Tp1a inhibited hNa_v1.7 without significantly altering the voltage-dependence of activation or inactivation. Tp1a proved to be analgesic by reversing spontaneous pain induced in mice by intraplantar injection in OD1, a scorpion toxin that potentiates hNa_v1.7. The structure of Tp1a as determined using NMR spectroscopy revealed a classical inhibitor cystine knot motif (ICK). The molecular surface of Tp1a presents a hydrophobic patch surrounded by positively charged residues, with subtle differences to other ICK spider toxins that might contribute to its different pharmacological profile. Tp1a may help guide the development of more selective and potent hNa_V1.7 inhibitors for treatment of chronic pain.