Search Results (7)

Aristotelia chilensis or “maqui” is a tree native to Chile used in the folk medicine of the Mapuche people as an anti-inflammatory agent for the treatment of digestive ailments, fever, and skin lesions. Maqui fruits are black berries which are considered a “superfruit” with notable potential health benefits, promoted to be an antioxidant, cardioprotective, and anti-inflammatory. Maqui leaves contain non-iridoid monoterpene indole alkaloids which have previously been shown to act on nicotinic acetylcholine receptors, potassium channels, and calcium channels. Here, we isolated a new alkaloid from maqui leaves, now called makomakinol, together with the known alkaloids aristoteline, hobartine, and 3-formylindole. Moreover, the polyphenols quercetine, ethyl caffeate, and the terpenes, dihydro-β-ionone and terpin hydrate, were also obtained. In light of the reported analgesic and anti-nociceptive properties of A. chilensis, in particular a crude mixture of alkaloids containing aristoteline and hobartinol (PMID 21585384), we therefore evaluated the activity of aristoteline and hobartine on Na_V1.8, a key Na_V isoform involved in nociception, using automated whole-cell patch-clamp electrophysiology. Aristoteline and hobartine both inhibited Nav1.8 with an IC₅₀ of 68 ± 3 µM and 54 ± 1 µM, respectively. Hobartine caused a hyperpolarizing shift of the voltage-dependence of the activation, whereas aristoteline did not change the voltage-dependence of the activation or inactivation. The inhibitory activity of these alkaloids on Na_V channels may contribute to the reported analgesic properties of Aristotelia chilensis used by the Mapuche people. Full article

µ-Conotoxins are small, potent, peptide voltage-gated sodium (Na_V) channel inhibitors characterised by a conserved cysteine framework. Despite promising in vivo studies indicating analgesic potential of these compounds, selectivity towards the therapeutically relevant subtype Na_V1.7 has so far been limited. We recently identified a novel µ-conotoxin, SxIIIC, which potently inhibits human Na_V1.7 (hNa_V1.7). SxIIIC has high sequence homology with other µ-conotoxins, including SmIIIA and KIIIA, yet shows different Na_V channel selectivity for mammalian subtypes. Here, we evaluated and compared the inhibitory potency of µ-conotoxins SxIIIC, SmIIIA and KIIIA at hNa_V channels by whole-cell patch-clamp electrophysiology and discovered that these three closely related µ-conotoxins display unique selectivity profiles with significant variations in inhibitory potency at hNa_V1.7. Analysis of other µ-conotoxins at hNa_V1.7 shows that only a limited number are capable of inhibition at this subtype and that differences between the number of residues in loop 3 appear to influence the ability of µ-conotoxins to inhibit hNa_V1.7. Through mutagenesis studies, we confirmed that charged residues in this region also affect the selectivity for hNa_V1.4. Comparison of µ-conotoxin NMR solution structures identified differences that may contribute to the variance in hNa_V1.7 inhibition and validated the role of the loop 1 extension in SxIIIC for improving potency at hNa_V1.7, when compared to KIIIA. This work could assist in designing µ-conotoxin derivatives specific for hNa_V1.7. Full article

Voltage-gated sodium (Na_V) channel subtypes, including Na_V1.7, are promising targets for the treatment of neurological diseases, such as chronic pain. Cone snail-derived µ-conotoxins are small, potent Na_V channel inhibitors which represent potential drug leads. Of the 22 µ-conotoxins characterised so far, only a small number, including KIIIA and CnIIIC, have shown inhibition against human Na_V1.7. We have recently identified a novel µ-conotoxin, SxIIIC, from Conus striolatus. Here we present the isolation of native peptide, chemical synthesis, characterisation of human Na_V channel activity by whole-cell patch-clamp electrophysiology and analysis of the NMR solution structure. SxIIIC displays a unique Na_V channel selectivity profile (1.4 > 1.3 > 1.1 ≈ 1.6 ≈ 1.7 > 1.2 >> 1.5 ≈ 1.8) when compared to other µ-conotoxins and represents one of the most potent human Na_V1.7 putative pore blockers (IC₅₀ 152.2 ± 21.8 nM) to date. NMR analysis reveals the structure of SxIIIC includes the characteristic α-helix seen in other µ-conotoxins. Future investigations into structure-activity relationships of SxIIIC are expected to provide insights into residues important for Na_V channel pore blocker selectivity and subsequently important for chronic pain drug development. Full article

Na_V1.3 is a subtype of the voltage-gated sodium channel family. It has been implicated in the pathogenesis of neuropathic pain, although the contribution of this channel to neuronal excitability is not well understood. Tf2, a β-scorpion toxin previously identified from the venom of Tityus fasciolatus, has been reported to selectively activate Na_V1.3. Here, we describe the activity of synthetic Tf2 and assess its suitability as a pharmacological probe for Na_V1.3. As described for the native toxin, synthetic Tf2 (1 µM) caused early channel opening, decreased the peak current, and shifted the voltage dependence of Na_V1.3 activation in the hyperpolarizing direction by −11.3 mV, with no activity at Na_V1.1, Na_V1.2, and Na_V1.4-Na_V1.8. Additional activity was found at Na_V1.9, tested using the hNav1.9_C4 chimera, where Tf2 (1 µM) shifted the voltage dependence of activation by −6.3 mV. In an attempt to convert Tf2 into an Na_V1.3 inhibitor, we synthetized the analogue Tf2[S14R], a mutation previously described to remove the excitatory activity of related β-scorpion toxins. Indeed, Tf2[S14R](10 µM) had reduced excitatory activity at Na_V1.3, although it still caused a small −5.8 mV shift in the voltage dependence of activation. Intraplantar injection of Tf2 (1 µM) in mice caused spontaneous flinching and swelling, which was not reduced by the Na_V1.1/1.3 inhibitor ICA-121431 nor in Na_V1.9^-/- mice, suggesting off-target activity. In addition, despite a loss of excitatory activity, intraplantar injection of Tf2[S14R](10 µM) still caused swelling, providing strong evidence that Tf2 has additional off-target activity at one or more non-neuronal targets. Therefore, due to activity at Na_V1.9 and other yet to be identified target(s), the use of Tf2 as a selective pharmacological probe may be limited. Full article

Spider venom is a novel source of disulfide-rich peptides with potent and selective activity at voltage-gated sodium channels (Na_V). Here, we describe the discovery of μ-theraphotoxin-Pme1a and μ/δ-theraphotoxin-Pme2a, two novel peptides from the venom of the Gooty Ornamental tarantula Poecilotheria metallica that modulate Na_V channels. Pme1a is a 35 residue peptide that inhibits Na_V1.7 peak current (IC₅₀ 334 ± 114 nM) and shifts the voltage dependence of activation to more depolarised membrane potentials (V_1/2 activation: Δ = +11.6 mV). Pme2a is a 33 residue peptide that delays fast inactivation and inhibits Na_V1.7 peak current (EC₅₀ > 10 μM). Synthesis of a [+22K]Pme2a analogue increased potency at Na_V1.7 (IC₅₀ 5.6 ± 1.1 μM) and removed the effect of the native peptide on fast inactivation, indicating that a lysine at position 22 (Pme2a numbering) is important for inhibitory activity. Results from this study may be used to guide the rational design of spider venom-derived peptides with improved potency and selectivity at Na_V channels in the future. Full article

Millions of years of evolution have fine-tuned the ability of venom peptides to rapidly incapacitate both prey and potential predators. Toxicofera reptiles are characterized by serous-secreting mandibular or maxillary glands with heightened levels of protein expression. These glands are the core anatomical components of the toxicoferan venom system, which exists in myriad points along an evolutionary continuum. Neofunctionalisation of toxins is facilitated by positive selection at functional hotspots on the ancestral protein and venom proteins have undergone dynamic diversification in helodermatid and varanid lizards as well as advanced snakes. A spectacular point on the venom system continuum is the long-glanded blue coral snake (Calliophis bivirgatus), a specialist feeder that preys on fast moving, venomous snakes which have both a high likelihood of prey escape but also represent significant danger to the predator itself. The maxillary venom glands of C. bivirgatus extend one quarter of the snake’s body length and nestle within the rib cavity. Despite the snake’s notoriety its venom has remained largely unstudied. Here we show that the venom uniquely produces spastic paralysis, in contrast to the flaccid paralysis typically produced by neurotoxic snake venoms. The toxin responsible, which we have called calliotoxin (δ-elapitoxin-Cb1a), is a three-finger toxin (3FTx). Calliotoxin shifts the voltage-dependence of Na_V1.4 activation to more hyperpolarised potentials, inhibits inactivation, and produces large ramp currents, consistent with its profound effects on contractile force in an isolated skeletal muscle preparation. Voltage-gated sodium channels (Na_V) are a particularly attractive pharmacological target as they are involved in almost all physiological processes including action potential generation and conduction. Accordingly, venom peptides that interfere with Na_V function provide a key defensive and predatory advantage to a range of invertebrate venomous species including cone snails, scorpions, spiders, and anemones. Enhanced activation or delayed inactivation of sodium channels by toxins is associated with the extremely rapid onset of tetanic/excitatory paralysis in envenomed prey animals. A strong selection pressure exists for the evolution of such toxins where there is a high chance of prey escape. However, despite their prevalence in other venomous species, toxins causing delay of sodium channel inhibition have never previously been described in vertebrate venoms. Here we show that Na_V modulators, convergent with those of invertebrates, have evolved in the venom of the long-glanded coral snake. Calliotoxin represents a functionally novel class of 3FTx and a structurally novel class of Na_V toxins that will provide significant insights into the pharmacology and physiology of Na_V. The toxin represents a remarkable case of functional convergence between invertebrate and vertebrate venom systems in response to similar selection pressures. These results underscore the dynamic evolution of the Toxicofera reptile system and reinforces the value of using evolution as a roadmap for biodiscovery. Full article

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. Full article