Two Novel Peptide Toxins from the Spider Cyriopagopus longipes Inhibit Tetrodotoxin-Sensitive Sodium Channels.

Sodium channels play a critical role in the generation and propagation of action potentials in excitable tissues, such as nerves, cardiac muscle, and skeletal muscle, and are the primary targets of toxins found in animal venoms. Here, two novel peptide toxins (Cl6a and Cl6b) were isolated from the venom of the spider Cyriopagopus longipes and characterized. Cl6a and Cl6b were shown to be inhibitors of tetrodotoxin-sensitive (TTX-S), but not TTX-resistant, sodium channels. Among the TTX-S channels investigated, Cl6a and Cl6b showed the highest degree of inhibition against NaV1.7 (half-maximal inhibitory concentration (IC50) of 11.0 ± 2.5 nM and 18.8 ± 2.4 nM, respectively) in an irreversible manner that does not alter channel activation, inactivation, or repriming kinetics. Moreover, analysis of NaV1.7/NaV1.8 chimeric channels revealed that Cl6b is a site 4 neurotoxin. Site-directed mutagenesis analysis indicated that D816, V817, and E818 observably affected the efficacy of the Cl6b-NaV1.7 interaction, suggesting that these residues might directly affect the interaction of NaV1.7 with Cl6b. Taken together, these two novel peptide toxins act as potent and sustained NaV1.7 blockers and may have potential in the pharmacological study of sodium channels.

Mutational analysis of ProTx-I and the novel venom peptide Pe1b provide insight into residues responsible for selective inhibition of the analgesic drug target NaV1.7.

Management of chronic pain presents a major challenge, since many currently available treatments lack efficacy and have problems such as addiction and tolerance. Loss of function mutations in the SCN9A gene lead to a congenital inability to feel pain, with no other sensory deficits aside from anosmia. SCN9A encodes the voltage-gated sodium (NaV) channel 1.7 (NaV1.7), which has been identified as a primary pain target. However, in developing NaV1.7-targeted analgesics, extreme care must to be taken to avoid off-target activity on other NaV subtypes that are critical for survival. Since spider venoms are an excellent source of NaV channel modulators, we screened a panel of spider venoms to identify selective NaV1.7 inhibitors. This led to identification of two novel NaV modulating venom peptides (ß/µ-theraphotoxin-Pe1a and ß/µ-theraphotoxin-Pe1b (Pe1b) from the arboreal tarantula Phormingochilus everetti. A third peptide isolated from the tarantula Bumba pulcherrimaklaasi was identical to the well-known ProTx-I (ß/ω-theraphotoxin-Tp1a) from the tarantula Thrixopelma pruriens. A tethered toxin (t-toxin)-based alanine scanning strategy was used to determine the NaV1.7 pharmacophore of ProTx-I. We designed several ProTx-I and Pe1b analogues, and tested them for activity and NaV channel subtype selectivity. Several analogues had improved potency against NaV1.7, and altered specificity against other NaV channels. These analogues provide a foundation for development of Pe1b as a lead molecule for therapeutic inhibition of NaV1.7.

Cardiac protection of Bauhinia championii against reperfusion injury.

This study aims to investigate the protective effects of the Bauhinia championii (BC) against ischemia/reperfusion (I/R)-induced injury in an isolated heart model. Langendorff-perfused C57BL/6JNarl mice hearts were performed with 30 minutes ischemia and 60 minutes reperfusion by left anterior descending artery ligation. Before reperfusion, boiling water extracts of BC (10 mg/L) was pretreated for 15 minutes. During reperfusion, BC significantly decreased the occurrence of ventricular arrhythmias by lead II electrocardiogram (ECG). Electrophysiological effect of BC was further determined in isolated ventricular myocytes by whole-cell patch clamp technique. The underlying mechanism may result from its Na+ channel blocking activity characterized with reduced rise slope of action potential and Na+ current density. Moreover, BC dramatically reduced I/R-caused infarct size, which was accessed by 2,3,5-triphenyltetrazolium chloride (TTC) assay. Since BC decreased I/R-induced myoglobin release and oxidation of Ca2+ -calmodulin-dependent protein kinase, inhibition of myocardial necroptosis may account for the protective effects of BC on myocytes lose. This study indicated that BC may prevent I/R induced ventricular arrhythmias and myocyte death by blocking Na+ channels and decreasing necroptosis, respectively. Since most of the available antiarrhythmic remedies have unwanted adverse actions, BC could be a novel candidate for the treatment of myocardial infarction and ventricular arrhythmia.

Recombinant Production and Structure-Function Study of the Ts1 Toxin from the Brazilian Scorpion Tityus serrulatus.

An effective bacterial system for the production of ß-toxin Ts1, the main component of the Brazilian scorpion Tityus serrulatus venom, was developed. Recombinant toxin and its 15N-labeled analogue were obtained via direct expression of synthetic gene in Escherichia coli with subsequent folding from the inclusion bodies. According to NMR spectroscopy data, the recombinant toxin is structured in an aqueous solution and contains a significant fraction of ß-structure. The formation of a stable disulfide-bond isomer of Ts1, having a disordered structure, has also been observed during folding. Recombinant Ts1 blocks Na+ current through NaV1.5 channels without affecting the processes of activation and inactivation. At the same time, the effect upon NaV1.4 channels is associated with a shift of the activation curve towards more negative membrane potentials.

From identification to functional characterization of cyriotoxin-1a, an antinociceptive toxin from the spider Cyriopagopus schioedtei.

BACKGROUND AND PURPOSE: The NaV 1.7 channel is highly expressed in dorsal root ganglia of the sensory nervous system and plays a central role in the pain signalling process. We investigated a library prepared from original venoms of 117 different animals to identify new selective inhibitors of this target. EXPERIMENTAL APPROACH: We used high throughput screening of a large venom collection using automated patch-clamp experiments on human voltage-gated sodium channel subtypes and then in vitro and in vivo electrophysiological experiments to characterize the active peptides that have been purified, sequenced, and chemically synthesized. Analgesic effects were evaluated in vivo in mice models. KEY RESULTS: We identified cyriotoxin-1a (CyrTx-1a), a novel peptide isolated from Cyriopagopus schioedtei spider venom, as a candidate for further characterization. This 33 amino acids toxin belongs to the inhibitor cystine knot structural family and inhibits hNaV 1.1-1.3 and 1.6-1.7 channels in the low nanomolar range, compared to the micromolar range for hNaV 1.4-1.5 and 1.8 channels. CyrTx-1a was 920 times more efficient at inhibiting tetrodotoxin (TTX)-sensitive than TTX-resistant sodium currents recorded from adult mouse dorsal root ganglia neurons and in vivo electrophysiological experiments showed that CyrTx-1a was approximately 170 times less efficient than huwentoxin-IV at altering mouse skeletal neuromuscular excitability properties. CyrTx-1a exhibited an analgesic effect in mice by increasing reaction time in the hot-plate assay. CONCLUSIONS AND IMPLICATIONS: The pharmacological profile of CyrTx-1a paves the way for further molecular engineering aimed to optimize the potential antinociceptive properties of this peptide.

Polysaccharides from Portulaca oleracea L. regulated insulin secretion in INS-1 cells through voltage-gated Na+ channel.

The present study was undertaken to determine the involvement of voltage-gated Na+ channel (VGSC) and other mechanism related to insulin secretion in polysaccharides from Portulaca oleracea L. (POP)-induced secretion of insulin from insulin-secreting ß-cell line cells (INS-1) cells. Our results showed that the concentration of insulin both in culture medium and inside INS-1 cells were increased under the existing of different concentration of glucose by POP or TTX, respectively. However, the effect POP on insulin secretion and production were blocked by TTX, a VGSC blocker. Meanwhile, POP improved the mitochondrial membrane potential (Δψm), increased adenosine triphosphate (ATP) production, depolarized cell membrane potential (MP) and increased intracellular Ca2+ levels ([Ca2+]i). Furthermore, POP treatment increased the expression level of Nav1.3 and decreased the expression level of Nav1.7. TTX treatment decreased the expression level of Nav1.3 and Nav1.7. On the other hand, POP also elevated the survival of INS-1 cells. These results suggested that POP induced-secretion/production of insulin in INS-1 cells were mediated by VGSC through its change of function and subunits expression and subsequent VGSC- dependent events such as change of intracellular Ca2+ releasing, ATP metabolism, cell membrane and mitochondrial membrane potential, and also improvement of INS-1 cell survival. Meanwhile, our data indicated the potentiality of developing POP to be a drug for diabetes treatment and VGSC as a therapeutic target in diabetes treatment is valuable to be investigated further.

Pharmacological characterization of potent and selective NaV1.7 inhibitors engineered from Chilobrachys jingzhao tarantula venom peptide JzTx-V.

Identification of voltage-gated sodium channel NaV1.7 inhibitors for chronic pain therapeutic development is an area of vigorous pursuit. In an effort to identify more potent leads compared to our previously reported GpTx-1 peptide series, electrophysiology screening of fractionated tarantula venom discovered the NaV1.7 inhibitory peptide JzTx-V from the Chinese earth tiger tarantula Chilobrachys jingzhao. The parent peptide displayed nominal selectivity over the skeletal muscle NaV1.4 channel. Attribute-based positional scan analoging identified a key Ile28Glu mutation that improved NaV1.4 selectivity over 100-fold, and further optimization yielded the potent and selective peptide leads AM-8145 and AM-0422. NMR analyses revealed that the Ile28Glu substitution changed peptide conformation, pointing to a structural rationale for the selectivity gains. AM-8145 and AM-0422 as well as GpTx-1 and HwTx-IV competed for ProTx-II binding in HEK293 cells expressing human NaV1.7, suggesting that these NaV1.7 inhibitory peptides interact with a similar binding site. AM-8145 potently blocked native tetrodotoxin-sensitive (TTX-S) channels in mouse dorsal root ganglia (DRG) neurons, exhibited 30- to 120-fold selectivity over other human TTX-S channels and exhibited over 1,000-fold selectivity over other human tetrodotoxin-resistant (TTX-R) channels. Leveraging NaV1.7-NaV1.5 chimeras containing various voltage-sensor and pore regions, AM-8145 mapped to the second voltage-sensor domain of NaV1.7. AM-0422, but not the inactive peptide analog AM-8374, dose-dependently blocked capsaicin-induced DRG neuron action potential firing using a multi-electrode array readout and mechanically-induced C-fiber spiking in a saphenous skin-nerve preparation. Collectively, AM-8145 and AM-0422 represent potent, new engineered NaV1.7 inhibitory peptides derived from the JzTx-V scaffold with improved NaV selectivity and biological activity in blocking action potential firing in both DRG neurons and C-fibers.

A novel µ-conotoxin from worm-hunting Conus tessulatus that selectively inhibit rat TTX-resistant sodium currents.

µ-conotoxins are a group of marine Conus peptides that inhibit sodium currents, so µ-conotoxins are valuable in sodium channel research and new analgesic drug discovery. Here, a novel µ-conotoxin TsIIIA was identified from a worm-hunting Conus tessulatus. TsIIIA was chemical synthesized according to its amino acid sequence GCCRWPCPSRCGMARCCSS and identified by mass spectrum. Patch clamp on rat dorsal root ganglion cells showed that 10 µM TsIIIA specifically inhibit TTX-resistant sodium currents but has no effect on TTX-sensitive sodium currents. TsIIIA inhibits TTX-resistant sodium currents by a dose-dependent mode with an IC50 of 2.61 µM. Further study showed 10 µM TsIIIA has no obvious effect on the current-voltage relationships, conductance-voltage relationships and voltage-dependence of steady-state inactivation of TTX-resistant sodium channels. Mice hotplate analgesic assay indicated that TsIIIA obviously increase the pain threshold at 0.5-4 h. In addition, TsIIIA has better analgesic effects than Ziconotide, indicating that TsIIIA was a valuable lead compound for development of new analgesic drug.

Old Weapons for New Wars: Bioactive Molecules From Cnidarian Internal Defense Systems.

The renewed interest in the study of genes of immunity in Cnidaria has led to additional information to the scenario of the first stages of immunity evolution revealing the cellular processes involved in symbiosis, in the regulation of homeostasis and in the fight against infections. The recent study with new molecular and functional approach on these organisms have therefore contributed with unexpected information on the knowledge of the stages of capturing activities and defense mechanisms strongly associated with toxin production. Cnidarians are diblastic aquatic animals with radial symmetry; they represent the ancestral state of Metazoa, they are the simplest multicellular organisms that have reached the level of tissue organization.The Cnidaria phylum has evolved using biotoxins as defense or predation mechanisms for ensure survival in hostile and competitive environments such as the seas and oceans. From benthic and pelagic species a large number of toxic compounds that have been determined can have an active role in the development of various antiviral, anticancer and antibacterial functions. Although the immune defense response of these animals is scarcely known, the tissues and the mucus produced by cnidarians are involved in immune defense and contain a large variety of peptides such as sodium and potassium channel neurotoxins, cytolysins, phospholipase A2 (PLA2), acid-sensing ion channel peptide toxins (ASICs) and other toxins, classified following biochemical and pharmacological studies on the basis of functional, molecular and structural parameters. These basal metazoan in fact, are far from "simple" in the range of methods at their disposal to deal with potential prey but also invading microbes and pathogens. They could also take advantage of the multi-functionality of some of their toxins, for example, some bioactive molecules have characteristics of toxicity associated with a potential antimicrobial activity. The interest in cnidarians was not only directed to the study of toxins and venom, but also to the fact these animals have been suggested as source of new molecules potentially relevant for biotechnology and pharmaceutical applications. Here, we review the cnidarian type of toxins regarding their multifunctional role and the future possibility of drawing important applications in fields ranging from biology to pharmacology.

Biochemical and physiological characterization of a new Na(+)-channel specific peptide from the venom of the Argentinean scorpion Tityus trivittatus.

A new peptide with 61 amino acids cross-linked by 4 disulfide bridges, with molecular weight of 6938.12Da, and an amidated C-terminal amino acid residue was purified and characterized. The primary structure was obtained by direct Edman degradation and sequencing its gene. The peptide is lethal to mammals and was shown to be similar (95% identity) to toxin Ts1 (gamma toxin) from the Brazilian scorpion Tityus serrulatus; it was named Tt1g (from T. trivittatus toxin 1 gamma-like). Tt1g was assayed on several sub-types of Na(+)-channels showing displacement of the currents to more negative voltages, being the hNav1.3 the most affected channel. This toxin displays characteristics typical to the ß-type sodium scorpion toxins. Lethality tests and physiological assays indicate that this peptide is probably the most important toxic component of this species of scorpion, known for causing human fatalities in the South American continent.

Structure of membrane-active toxin from crab spider Heriaeus melloteei suggests parallel evolution of sodium channel gating modifiers in Araneomorphae and Mygalomorphae.

We present a structural and functional study of a sodium channel activation inhibitor from crab spider venom. Hm-3 is an insecticidal peptide toxin consisting of 35 amino acid residues from the spider Heriaeus melloteei (Thomisidae). We produced Hm-3 recombinantly in Escherichia coli and determined its structure by NMR spectroscopy. Typical for spider toxins, Hm-3 was found to adopt the so-called "inhibitor cystine knot" or "knottin" fold stabilized by three disulfide bonds. Its molecule is amphiphilic with a hydrophobic ridge on the surface enriched in aromatic residues and surrounded by positive charges. Correspondingly, Hm-3 binds to both neutral and negatively charged lipid vesicles. Electrophysiological studies showed that at a concentration of 1 µm Hm-3 effectively inhibited a number of mammalian and insect sodium channels. Importantly, Hm-3 shifted the dependence of channel activation to more positive voltages. Moreover, the inhibition was voltage-dependent, and strong depolarizing prepulses attenuated Hm-3 activity. The toxin is therefore concluded to represent the first sodium channel gating modifier from an araneomorph spider and features a "membrane access" mechanism of action. Its amino acid sequence and position of the hydrophobic cluster are notably different from other known gating modifiers from spider venom, all of which are described from mygalomorph species. We hypothesize parallel evolution of inhibitor cystine knot toxins from Araneomorphae and Mygalomorphae suborders.

Diterpene alkaloids from the roots of Aconitum moldavicum and assessment of Nav 1.2 sodium channel activity of aconitum alkaloids.

A new aconitane alkaloid, 1-O-demethylswatinine (1), was isolated from the root of Aconitum moldavicum together with the known compounds cammaconine (2), columbianine (3), swatinine (4), gigactonine (5), delcosine (6), lycoctonine (7), and ajacine (8). The structures were established by means of HRESIMS, 1D and 2D NMR spectroscopy, including 1H-1H COSY, NOESY, HSQC, and HMBC experiments, resulting in complete 1H-NMR chemical shift assignments for 1-4. The effects of the isolated compounds 4-8, together with eighteen other Aconitum diterpene and norditerpene alkaloids with different skeletal types and substitution patterns, were studied on Nav 1.2 channels by the whole-cell patch clamp technique, using the QPatch-16 automated patch clamp system. Pyroaconitine, ajacine, septentriodine, and delectinine demonstrated significant Nav 1.2 channel inhibition (57-42 %) at 10 µM concentration; several other compounds (acovulparine, acotoxicine, hetisinone, 14-benzoylaconine-8-O-palmitate, aconitine, and lycoctonine) exerted moderate inhibitory activity (30-22 %), while the rest of the tested alkaloids were considered to be inactive. On the basis of these results and by exhaustive comparison of data of previously published computerized QSAR studies on diterpene alkaloids, certain conclusions on the structure-activity relationships of Aconitum alkaloids concerning Nav 1.2 channel inhibitory activity are proposed.

Isolation and identification of a sodium channel-inhibiting protein from eggs of black widow spiders.

The eggs of black widow spider (L. tredecimguttatus) have been demonstrated to be rich in biologically active components that exhibit great research value and application foreground. In the present study, a protein toxin, named Latroeggtoxin-II, was isolated from the eggs using the combination of gel filtration, ion exchange chromatography and reversed-phase high performance liquid chromatography. Electrospray mass spectrometric analysis indicated that the molecular weight of the protein was 28.69 kDa, and Edman degradation revealed that its N-terminal sequence was ESIQT STYVP NTPNQ KFDYE VGKDY-. After being abdominally injected into mice and P. americana, the protein could make the animals especially P. americana display a series of poisoning symptoms. Electrophysiological experiments demonstrated that the protein could selectively inhibit tetrodotoxin-resistant Na(+) channel currents in rat dorsal root ganglion neurons, without significant effect on the tetrodotoxin-sensitive Na(+) channel currents. Using multiple proteomic strategies, the purified protein was shown to have only a few similarities to the existing proteins in the databases, suggesting that it was a novel protein isolated from the eggs of black widow spiders.

Isolation and characterization of an anti-insect ß-toxin from the venom of the scorpion Isometrus maculatus.

Im-3 was isolated from the venom of the scorpion Isometrus maculatus through several steps of HPLC fractionation based on the insect paralytic activity. Injecting Im-3 into crickets induced paralysis, but no toxicity was apparent in mice after an intracerebroventricular injection. Im-3 shares sequence similarity to scorpion ß-toxins that specifically affect insect sodium channels.

Isolation of a Nav channel blocking polypeptide from Cyanea capillata medusae - a neurotoxin contained in fishing tentacle isorhizas.

Jellyfish are efficient predators which prey on crabs, fish larvae, and small fish. Their venoms consist of various toxins including neurotoxins that paralyse prey organisms immediately. One possible mode of action of neurotoxins is the blockage of voltage-gated sodium (Na(v)) channels. A novel polypeptide with Na(v) channel blocking activity was isolated from the northern Scyphozoa Cyanea capillata (L., 1758). For that purpose, a bioactivity-guided multidimensional liquid chromatographic purification method has been developed. A neurotoxic activity of resulting chromatographic fractions was demonstrated by a bioassay, which based on the mouse neuroblastoma cell line Neuro2A. The purification process yielded one fraction containing a single polypeptide with proven activity. The molecular weight of 8.22 kDa was determined by matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-ToF MS). Utilising Laser Microdissection and Pressure Catapulting (LMPC) for the separation of different nematocyst types in combination with direct MALDI-ToF MS analysis of the intact capsules, the neurotoxin was found to be present in all types of fishing tentacle isorhizas (A-isorhizas, a-isorhizas, O-isorhizas) of C. capillata medusae.

Recombinant expression, purification, and characterization of scorpion toxin BmαTX14.

Long-chain and cysteine-rich scorpion toxins exhibit various pharmacological profiles for different voltage-gated sodium channel subtypes. However, the exploration of toxin structure-function relationships has progressed slowly due to the difficulty of obtaining synthetic or recombinant peptides. We now report that we have established an effective expression and purification approach for the novel scorpion toxin BmαTX14. BmαTX14 was over-expressed as inclusion bodies in Escherichia coli. The insoluble pellet was successfully transformed into active peptide by using a refolding procedure. One-step purification by reverse-phase HPLC was sufficient to generate chromatographically pure peptide. The yield of recombinant toxin reached 4mg from 1L LB medium. The pharmacological data further showed that BmαTX14 selectively inhibited the fast inactivation of mNa(v)1.4 (EC(50)=82.3±15.7nM) rather than that of rNa(v)1.2 (EC(50)>30µM), which indicates that BmαTX14 is a new α-like toxin. This work enables further structural, functional, and pharmacological studies of BmαTX14 and similar toxins.

Cymatherelactone and cymatherols A-C, polycyclic oxylipins from the marine brown alga Cymathere triplicata.

An investigation of the oxylipin chemistry of the temperate brown alga Cymathere triplicata led to the isolation of several secondary metabolites, cymatherelactone (1) and cymatherols A-C (2-4), the latter as their methyl ester derivatives (5-7), which contained cyclopentyl, cyclopropyl, epoxide and lactone rings. Their structures were elucidated using a combination of spectroscopic techniques and synthetic chemistry. Cymatherelactone (1), as well as R- and S-Mosher's esters of its seco acid, exhibited moderate sodium channel blocking activity.

Development and validation of a high-throughput double solid phase extraction-liquid chromatography-tandem mass spectrometry method for the determination of tetrodotoxin in human urine and plasma.

A sensitive analytical method for the determination of tetrodotoxin (TTX) in urine and plasma matrices was developed using double solid phase extraction (C18 and hydrophilic interaction liquid chromatography) and subsequent analysis by HPLC coupled with tandem mass spectrometry. The double SPE sample cleanup efficiently reduced matrix and ion suppression effects. Together with the use of ion pair reagent in the mobile phase, isocratic elution became possible which enabled a shorter analysis time of 5.5 min per sample. The assay results were linear up to 500 ng mL(-1) for urine and 20 ng mL(-1) for plasma. The limit of detection and limit of quantification were 0.13 ng mL(-1) and 2.5 ng mL(-1), respectively, for both biological matrices. Recoveries were in the range of 75-81%. To eliminate the effect of dehydration and variations in urinary output, urinary creatinine-adjustment was made. TTX was quantified in eight urine samples and seven plasma samples from eight patients suspected of having TTX poisoning. TTX was detected in all urine samples, with concentrations ranging from 17.6 to 460.5 ng mL(-1), but was not detected in any of the plasma samples. The creatinine-adjusted TTX concentration in urine (ranging from 7.4 to 41.1 ng µmol(-1) creatinine) correlated well with the degree of poisoning as observed from clinical symptoms.

DrTx(1-42), a C-terminally truncated analogue of drosotoxin, is a candidate of analgesic drugs.

Drosotoxin is an engineered tetrodotoxin-resistant (TTX-R) sodium channel-specific blocker with a non-toxic structural core (Zhu et al. Biochem Pharmacol 2010; 80:1296-302). Here, we report the discovery and functional characterization of a carboxyl-terminally truncated analogue of drosotoxin (named DrTx(1-42)) which selectively inhibited dorsal root ganglion (DRG) neuron TTX-R sodium current (I(Na)) with an IC(50) value of 1.74±0.07µM. Consistent with this effect, DrTx(1-42) significantly attenuates inflammatory hyperalgesia of mice in a formalin-induced pain model with stronger potency than indomethacin, a nonsteroidal anti-inflammatory and analgesic drug. Our mutational experiments indicate that the N-turn insertion is an essential functional determinant for the emergence of neurotoxicity from a non-toxic antifungal scaffold.

Animal peptides targeting voltage-activated sodium channels.

Throughout millions of years of evolution, nature has supplied various organisms with a massive arsenal of venoms to defend themselves against predators or to hunt prey. These venoms are rich cocktails of diverse bioactive compounds with divergent functions, extremely effective in immobilizing or killing the recipient. In fact, venom peptides from various animals have been shown to specifically act on ion channels and other cellular receptors, and impair their normal functioning. Because of their key role in the initiation and propagation of electrical signals in excitable tissue, it is not very surprising that several isoforms of voltage-activated sodium channels are specifically targeted by many of these venom peptides. Therefore, these peptide toxins provide tremendous opportunities to design drugs with a higher efficacy and fewer undesirable side effects. This review puts venom peptides from spiders, scorpions and cone snails that target voltage-activated sodium channels in the spotlight, and addresses their potential therapeutical applications.