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.

Some venomous cone snails feed on small fishes using an immobilizing combination of synergistic venom peptides that target Kv and Nav channels. As part of this envenomation strategy, δ-conotoxins are potent ichtyotoxins that enhance Nav channel function. δ-Conotoxins belong to an ancient and widely distributed gene superfamily, but any evolutionary link from ancestral worm-eating cone snails to modern piscivorous species has not been elucidated. Here, we report the discovery of SuVIA, a potent vertebrate-active δ-conotoxin characterized from a vermivorous cone snail (Conus suturatus). SuVIA is equipotent at hNaV1.3, hNaV1.4 and hNaV1.6 with EC50s in the low nanomolar range. SuVIA also increased peak hNaV1.7 current by approximately 75% and shifted the voltage-dependence of activation to more hyperpolarized potentials from -15 mV to -25 mV, with little effect on the voltage-dependence of inactivation. Interestingly, the proximal venom gland expression and pain-inducing effect of SuVIA in mammals suggest that δ-conotoxins in vermivorous cone snails play a defensive role against higher order vertebrates. We propose that δ-conotoxins originally evolved in ancestral vermivorous cones to defend against larger predators including fishes have been repurposed to facilitate a shift to piscivorous behaviour, suggesting an unexpected underlying mechanism for this remarkable evolutionary transition

Given the important role of voltage-gated sodium (NaV) channel-modulating spider toxins in elucidating the function, pharmacology, and mechanism of action of therapeutically relevant NaV channels, we screened the venom from Australian theraphosid species against the human pain target hNaV1.7. Using assay-guided fractionation, we isolated a 33-residue inhibitor cystine knot (ICK) peptide (Ssp1a) belonging to the NaSpTx1 family. Recombinant Ssp1a (rSsp1a) inhibited neuronal hNaV subtypes with a rank order of potency hNaV1.7 > 1.6 > 1.2 > 1.3 > 1.1. rSsp1a inhibited hNaV1.7, hNaV1.2 and hNaV1.3 without significantly altering the voltage-dependence of activation, inactivation, or delay in recovery from inactivation. However, rSsp1a demonstrated voltage-dependent inhibition at hNaV1.7 and rSsp1a-bound hNaV1.7 opened at extreme depolarizations, suggesting rSsp1a likely interacted with voltage-sensing domain II (VSD II) of hNaV1.7 to trap the channel in its resting state. Nuclear magnetic resonance spectroscopy revealed key structural features of Ssp1a, including an amphipathic surface with hydrophobic and charged patches shown by docking studies to comprise the interacting surface. This study provides the basis for future structure-function studies to guide the development of subtype selective inhibitors.

Voltage-gated sodium (Na_V) channels play a fundamental role in normal neurological function, especially via the initiation and propagation of action potentials. The Na_V1.1 subtype is found in inhibitory interneurons of the brain and it is essential for maintaining a balance between excitation and inhibition in neuronal networks. Heterozygous loss-of-function mutations of SCN1A, the gene encoding Na_V1.1, underlie Dravet syndrome (DS), a severe pediatric epilepsy. We recently demonstrated that selective inhibition of Na_V1.1 inactivation prevents seizures and premature death in a mouse model of DS. Thus, selective modulators of Na_V1.1 might be useful therapeutics for treatment of DS as they target the underlying molecular deficit. Numerous scorpion-venom peptides have been shown to modulate the activity of Na_V channels, but little is known about their activity at Na_V1.1. Here we report the isolation, sequence, three-dimensional structure, recombinant production, and functional characterization of two peptidic modulators of Na_V1.1 from venom of the buthid scorpion Hottentotta jayakari. These peptides, Hj1a and Hj2a, are potent agonists of Na_V1.1 (EC₅₀ of 17 and 32 nM, respectively), and they present dual α/β activity by modifying both the activation and inactivation properties of the channel. NMR studies of rHj1a indicate that it adopts a cystine-stabilized αβ fold similar to known scorpion toxins. Although Hj1a and Hj2a have only limited selectivity for Na_V1.1, their unusual dual mode of action provides an alternative approach to the development of selective Na_V1.1 modulators for the treatment of DS.

Velvet ants (Hymenoptera: Mutillidae) are a family of solitary parasitoid wasps that are renowned for their painful stings. We explored the chemistry underlying the stings of mutillid wasps of the genus Dasymutilla Ashmead. Detailed analyses of the venom composition of five species revealed that they are composed primarily of peptides. We found that two kinds of mutillid venom peptide appear to be primarily responsible for the painful effects of envenomation. These same peptides also have defensive utility against invertebrates, since they were able to incapacitate and kill honeybees. Both act directly on cell membranes where they directly increase ion conductivity. The defensive venom peptides of Dasymutilla bear a striking similarity, in structure and mode of action, to those of the ant Myrmecia gulosa (Fabricius), suggesting either retention of ancestral toxins, or convergence driven by similar life histories and defensive selection pressures. Finally, we propose that other highly expressed Dasymutilla venom peptides may play a role in parasitisation, possible in delay or arrest of host development. This study represents the first detailed account of the composition and function of the venoms of the Mutillidae.

Voltage-gated sodium (Na_V) channels are pore-forming transmembrane proteins that play essential roles in excitable cells, and they are key targets for antiepileptic, antiarrhythmic, and analgesic drugs. We implemented a heterobivalent design strategy to modulate the potency, selectivity, and binding kinetics of Na_V channel ligands. We conjugated μ-conotoxin KIIIA, which occludes the pore of the Na_V channels, to an analogue of huwentoxin-IV, a spider-venom peptide that allosterically modulates channel gating. Bioorthogonal hydrazide and copper-assisted azide-alkyne cycloaddition conjugation chemistries were employed to generate heterobivalent ligands using polyethylene glycol linkers spanning 40–120 Å. The ligand with an 80 Å linker had the most pronounced bivalent effects, with a significantly slower dissociation rate and 4–24-fold higher potency compared to those of the monovalent peptides for the human Na_V1.4 channel. This study highlights the power of heterobivalent ligand design and expands the repertoire of pharmacological probes for exploring the function of Na_V channels.

Chemical transfection is broadly used to transiently transfect mammalian cells, although often associated with cellular stress and membrane instability, which imposes challenges for most cellular assays, including high-throughput (HT) assays. In the current study, we compared the effectiveness of calcium phosphate, FuGENE and Lipofectamine 3000 to transiently express two key voltage-gated ion channels critical in pain pathways, Ca_V2.2 and Na_V1.7. The expression and function of these channels were validated using two HT platforms, the Fluorescence Imaging Plate Reader FLIPR^Tetra and the automated patch clamp QPatch 16X. We found that all transfection methods tested demonstrated similar effectiveness when applied to FLIPR^Tetra assays. Lipofectamine 3000-mediated transfection produced the largest peak currents for automated patch clamp QPatch assays. However, the FuGENE-mediated transfection was the most effective for QPatch assays as indicated by the superior number of cells displaying GΩ seal formation in whole-cell patch clamp configuration, medium to large peak currents, and higher rates of accomplished assays for both Ca_V2.2 and Na_V1.7 channels. Our findings can facilitate the development of HT automated patch clamp assays for the discovery and characterization of novel analgesics and modulators of pain pathways, as well as assisting studies examining the pharmacology of mutated channels.

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 NaV1.4 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 (NaV) 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 NaV 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 NaV 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 NaV toxins that will provide significant insights into the pharmacology and physiology of NaV. 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.

We report the chemical synthesis of scorpion toxin Cn2, a potent and highly selective activator of the human voltage-gated sodium channel Na_V1.6. In an attempt to decouple channel activation from channel binding, we also synthesized the first analogue of this toxin, Cn2[E15R]. This mutation caused uncoupling of the toxin’s excitatory and depressant activities, effectively resulting in a Na_V1.6 inhibitor. In agreement with the in vitro observations, Cn2[E15R] is antinociceptive in mouse models of Na_V1.6-mediated pain.

T-type calcium channel (Cav3.x) blockers are receiving increasing attention as potential therapeutics for the treatment of pathophysiological disorders and diseases, including absence epilepsy, Parkinson’s disease (PD), hypertension, cardiovascular diseases, cancers, and pain. However, few clinically approved Cav3.x blockers are available, and selective pharmacological tools are needed to further unravel the roles of individual Cav3.x subtypes. In this work, through an efficient synthetic route to the marine fungal product pseudellone C, we obtained bisindole alkaloid analogs of pseudellone C with a modified tryptophan moiety and identified two Cav3.2 (2, IC50 = 18.24 µM; 3, IC50 = 6.59 µM) and Cav3.3 (2, IC50 = 7.71 µM; 3, IC50 = 3.81 µM) selective blockers using a FLIPR cell-based assay measuring Cav3.x window currents. Further characterization by whole-cell patch-clamp revealed a preferential block of Cav3.1 activated current (2, IC50 = 5.60 µM; 3, IC50 = 9.91 µM), suggesting their state-dependent block is subtype specific.

μ-Conotoxins are components of cone snail venom, well-known for their analgesic activity through potent inhibition of voltage-gated sodium channel (Nav) subtypes, including Nav1.7. These small, disulfide-rich peptides are typically stabilized by three disulfide bonds arranged in a ‘native’ CysI-CysIV, CysII-CysV, CysIII-CysVI pattern of disulfide connectivity. However, μ-conotoxin KIIIA, the smallest and most studied μ-conotoxin with inhibitory activity at Nav1.7, forms two distinct disulfide bond isomers during thermodynamic oxidative folding, including Isomer 1 (CysI-CysV, CysII-CysIV, CysIII-CysVI) and Isomer 2 (CysI-CysVI, CysII-CysIV, CysIII-CysV), but not the native μ-conotoxin arrangement. To date, there has been no study on the structure and activity of KIIIA comprising the native μ-conotoxin disulfide bond arrangement. Here, we evaluated the synthesis, potency, sodium channel subtype selectivity, and 3D structure of the three isomers of KIIIA. Using a regioselective disulfide bond-forming strategy, we synthetically produced the three μ-conotoxin KIIIA isomers displaying distinct bioactivity and NaV subtype selectivity across human Nav channel subtypes 1.2, 1.4, and 1.7. We show that Isomer 1 inhibits Nav subtypes with a rank order of potency of Nav1.4 > 1.2 > 1.7 and Isomer 2 in the order of Nav1.4≈1.2 > 1.7, while the native isomer inhibited Nav1.4 > 1.7≈1.2. The three KIIIA isomers were further evaluated by NMR solution structure analysis and molecular docking with hNav1.2. Our study highlights the importance of investigating alternate disulfide isomers, as disulfide connectivity affects not only the overall structure of the peptides but also the potency and subtype selectivity of μ-conotoxins targeting therapeutically relevant Nav subtypes.

Chronic pain is a common and often debilitating condition. Existing treatments are either inefficacious or associated with a wide range of side effects. The progress on developing safer and more effective analgesics has been slow, in large part due to our limited understanding of the physiological mechanisms underlying pain in different diseases. Generation and propagation of action potentials is a central component of pain sensation and voltage-gated sodium channels (Na_Vs) play a critical role in this process. In particular, Na_V subtype 1.7, has emerged as a promising universal target for the treatment of pain.

Recently, a spider venom peptide, μ-TRTX-Pn3a, was found to be a highly selective inhibitor of Na_V1.7. Here, we report the first recombinant expression method for Pn3a in a bacterial host, which provides an inexpensive route to production. Furthermore, we have developed a method for bio-conjugation of our recombinantly produced Pn3a using sortase A mediated ligation, providing avenues for further pre-clinical development.

We demonstrate how heterologous expression in bacteria enables facile isotope labelling of Pn3a, which allowed us to study the membrane binding properties of the peptide by high-resolution solution-state nuclear magnetic resonance (NMR) spectroscopy using a recently developed lipid nanodisc system. The heteronuclear NMR data indicate that the C-terminal region of the peptide undergoes a conformational change upon lipid binding. The membrane binding properties of Pn3a are further validated using isothermal titration calorimetry (ITC), which revealed that Pn3a binds to zwitterionic planar lipid bilayers with thermodynamics that are largely driven by enthalpic contributions.

Chronic pain is a common and often debilitating condition. Existing treatments are either inefficacious or associated with a wide range of side effects. The progress on developing safer and more effective analgesics has been slow, in large part due to our limited understanding of the physiological mechanisms underlying pain in different diseases. Generation and propagation of action potentials is a central component of pain sensation and voltage-gated sodium channels (Na_Vs) play a critical role in this process. In particular, the Na_V subtype 1.7, has emerged as a promising universal target for the treatment of pain.

Recently, a spider venom peptide, μ-TRTX-Pn3a, was found to be a highly selective inhibitor of Na_V1.7. Here, we report the first recombinant expression method for Pn3a in a bacterial host, which provides an inexpensive route to production. Furthermore, we have developed a method for bio-conjugation of our recombinantly produced Pn3a via sortase A-mediated ligation, providing avenues for further pre-clinical development.

We demonstrate how heterologous expression in bacteria enables facile isotope labelling of Pn3a, which allowed us to study the membrane-binding properties of the peptide by high-resolution solution-state nuclear magnetic resonance (NMR) spectroscopy using a recently developed lipid nanodisc system. The heteronuclear NMR data indicate that the C-terminal region of the peptide undergoes a conformational change upon lipid binding. The membrane-binding properties of Pn3a are further validated using isothermal titration calorimetry (ITC), which revealed that Pn3a binds to zwitterionic planar lipid bilayers with thermodynamics that are largely driven by enthalpic contributions.

Many spider venom-peptides are known to modulate the activity of the voltage-gated sodium channel Na_V1.7, which has emerged as a promising analgesic target. A class of spider venom-peptides (NaSpTx1) in particular have been found to potently inhibit Na_V1.7 (nanomolar IC50), and shown to produce analgesic effects in animals. However, one member of this family, μ TRTX Hhn2b (Hhn2b), does not inhibit mammalian Na_V channels expressed in dorsal root ganglia at concentrations up to 100 μM. This peptide is classified as a NaSpTx1 member by virtue of its cysteine spacing and sequence conservation over functionally important residues. Here, we have performed detailed structural and functional analyses of Hhn2b leading us to identify two non-pharmacophore residues that contribute to hNa_V1.7 inhibition by non-overlapping mechanisms. These findings allowed us to produce a double mutant of Hhn2b that shows nanomolar inhibition of hNa_V1.7. Traditional structure/function analysis did not provide sufficient resolution to identify the mechanism underlying the observed gain of function. However, by solving the high-resolution structure of both the wild type and mutant peptides using advanced multidimensional NMR experiments, we were able to uncover a previously unknown network of interactions that stabilize the pharmacophore region of this class of venom-peptides. We further monitored the lipid binding properties of the peptides and identified that one of the key amino acid substitutions also selectively modulates the binding of the peptide to anionic lipids. These results will further aid the development of peptide-based analgesics for the treatment of chronic pain.

Venomous animals occupy one of the most successful evolutionary niches and occur on nearly every continent. They deliver venoms via biting and stinging apparatuses with the aim to rapidly incapacitate prey and deter predators. This has led to the evolution of venom components that act at a number of biological targets – including ion channels, G-protein coupled receptors, transporters and enzymes – with exquisite selectivity and potency, making venom-derived components attractive pharmacological tool compounds and drug leads. In recent years, plate-based pharmacological screening approaches have been introduced to accelerate venom-derived drug discovery. A range of assays are amenable to this purpose, including high-throughput electrophysiology, fluorescence-based functional and binding assays. However, despite these technological advances, the traditional activity-guided fractionation approach is time-consuming and resource-intensive. The combination of screening techniques suitable for miniaturization with sequence-based discovery approaches – supported by advanced proteomics, mass spectrometry, chromatography as well as synthesis and expression techniques – promises to further improve venom peptide discovery. Here, we discuss practical aspects of establishing a pipeline for venom peptide drug discovery with a particular emphasis on pharmacology and pharmacological screening approaches.

The human nociceptor-specific voltage-gated sodium channel 1.7 (hNaV1.7) is critical for sensing various types of somatic pain, but it appears not to play a primary role in acute visceral pain. However, its role in chronic visceral pain remains to be determined. We used assay-guided fractionation to isolate a novel hNaV1.7 inhibitor, Tsp1a, from tarantula venom. Tsp1a is 28-residue peptide that potently inhibits hNaV1.7 (IC50 = 10 nM), with greater than 100-fold selectivity over hNaV1.3–hNaV1.6, 45-fold selectivity over hNaV1.1, and 24-fold selectivity over hNaV1.2. Tsp1a is a gating modifier that inhibits NaV1.7 by inducing a hyperpolarizing shift in the voltage-dependence of channel inactivation and slowing recovery from fast inactivation. NMR studies revealed that Tsp1a adopts a classical knottin fold, and like many knottin peptides, it is exceptionally stable in human serum. Remarkably, intracolonic administration of Tsp1a completely reversed chronic visceral hypersensitivity in a mouse model of irritable bowel syndrome. The ability of Tsp1a to reduce visceral hypersensitivity in a model of irritable bowel syndrome suggests that pharmacological inhibition of hNaV1.7 at peripheral sensory nerve endings might be a viable approach for eliciting analgesia in patients suffering from chronic visceral 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_Vsubtypes. 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.

The role of voltage-gated sodium (Na_V) channels in pain perception is indisputable. Of particular interest as targets for the development of pain therapeutics are the tetrodotoxin-resistant isoforms Na_V1.8 and Na_V1.9, based on animal as well as human genetic studies linking these ion channel subtypes to the pathogenesis of pain. However, only a limited number of inhibitors selectively targeting these channels have been reported. HSTX-I is a peptide toxin identified from saliva of the leech Haemadipsa sylvestris. The native 23-residue peptide, stabilised by two disulfide bonds, has been reported to inhibit rat Na_V1.8 and mouse Na_V1.9 with low micromolar activity, and may therefore represent a scaffold for development of novel modulators with activity at human tetrodotoxin-resistant Na_V isoforms. We synthetically produced this hydrophobic peptide in high yield using a one-pot oxidation and single-step purification and determined the three-dimensional solution structure of HSTX-I using NMR solution spectroscopy. However, in our hands, the synthetic HSTX-I displayed only very modest activity at human Na_V1.8 and Na_V1.9 and lacked analgesic efficacy in a murine model of inflammatory pain.

The role of voltage-gated sodium (Na_V) channels in pain perception is indisputable. Of particular interest as targets for the development of pain therapeutics are the tetrodotoxin-resistant isoforms Na_V1.8 and Na_V1.9, based on animal as well as human genetic studies linking these ion channel subtypes to the pathogenesis of pain. However, only a limited number of inhibitors selectively targeting these channels have been reported. HSTX-I is a peptide toxin identified from saliva of the leech Haemadipsa sylvestris. The native 23-residue peptide, stabilised by two disulfide bonds, has been reported to inhibit rat Na_V1.8 and mouse Na_V1.9 with low micromolar activity, and may therefore represent a scaffold for development of novel modulators with activity at human tetrodotoxin-resistant Na_V isoforms. We synthetically produced this hydrophobic peptide in high yield using a one-pot oxidation and single-step purification and determined the three-dimensional solution structure of HSTX-I using NMR solution spectroscopy. However, in our hands, the synthetic HSTX-I displayed only very modest activity at human Na_V1.8 and Na_V1.9, and lacked analgesic efficacy in a murine model of inflammatory pain.

In this work, we investigated the in vitro neurotoxicity of Calliophis intestinalis venom using chick biventer cervicis neuromuscular preparations and electrophysiological analysis of voltage-gated sodium (NaV) channels expressed in HEK293 cells. We found that the indirect twitches of the neuromuscular preparations decreased over time when exposed to venom. However, the responses of these preparations to the agonists acetylcholine, carbachol, and potassium chloride were not changed after incubation with the venom. Our electrophysiological experiments show that C. intestinalis venom acts as a NaV channel antagonist—the first known from a vertebrate venom—by decreasing the peak current of NaV1.4 channels without changing the kinetics of activation or inactivation. Our proteomic results accord with earlier analyses and find that the venom contains three-finger toxins, cysteine-rich secretory proteins, kunitz peptides, phospholipase A2s, snake venom metalloproteases, and vespryns. Some of the three-finger toxins are similar to the δ-elapitoxins from the venom of the closely related Calliophis bivirgatus. However, δ-elapitoxins act as NaV channel agonists in C. bivirgatus whereas C. intestinalis venom contains NaV channel antagonists. The toxins and mechanisms responsible for the neuromuscular symptoms remain unclear as does the identity of the NaV channel antagonists. These aspects of this unusual venom require further study.

Stinging trees from Australasia produce remarkably persistent and painful stings upon contact of their stiff epidermal hairs, called trichomes, with mammalian skin. Dendrocnide-induced acute pain typically lasts for several hours, and intermittent painful flares can persist for days and weeks. Pharmacological activity has been attributed to small-molecule neurotransmitters and inflammatory mediators, but these compounds alone cannot explain the observed sensory effects. We show here that the venoms of Australian Dendrocnide species contain heretofore unknown pain-inducing peptides that potently activate mouse sensory neurons and delay inactivation of voltage-gated sodium channels. These neurotoxins localize specifically to the stinging hairs and are miniproteins of 4 kDa, whose 3D structure is stabilized in an inhibitory cystine knot motif, a characteristic shared with neurotoxins found in spider and cone snail venoms. Our results provide an intriguing example of inter-kingdom convergent evolution of animal and plant venoms with shared modes of delivery, molecular structure, and pharmacology.

Human intoxication with the seafood poison ciguatoxin, a dinoflagellate polyether that activates voltage-gated sodium channels (Na_V), causes ciguatera, a disease characterised by gastrointestinal and neurological disturbances. We assessed the activity of the most potent congener, Pacific ciguatoxin-1 (P-CTX-1), on Na_V1.1–1.9 using imaging and electrophysiological approaches. Although P-CTX-1 is essentially a non-selective NaV toxin and shifted the voltage-dependence of activation to more hyperpolarising potentials at all NaV subtypes, an increase in the inactivation time constant was observed only at Na_V1.8, while the slope factor of the conductance-voltage curves was significantly increased for Na_V1.7 and peak current was significantly increased for Na_V1.6. Accordingly, P-CTX-1-induced visceral and cutaneous pain behaviours were significantly decreased after pharmacological inhibition of Na_V1.8 and the tetrodotoxin-sensitive isoforms Na_V1.7 and Na_V1.6, respectively. The contribution of these isoforms to excitability of peripheral C- and A-fibre sensory neurons, confirmed using murine skin and visceral single-fibre recordings, reflects the expression pattern of Na_V isoforms in peripheral sensory neurons and their contribution to membrane depolarisation, action potential initiation and propagation.

Compelling human genetic studies have identified the voltage-gated sodium channel Na_V1.7 as a promising therapeutic target for the treatment of pain. The analgesic spider-venom-derived peptide μ-theraphotoxin-Pn3a is an exceptionally potent and selective inhibitor of Na_V1.7; however, little is known about the structure-activity relationships or channel interactions that define this activity. We rationally designed 17 Pn3a analogues and determined their activity at hNa_V1.7 using patch-clamp electrophysiology. The positively charged amino acids K22 and K24 were identified as crucial for Pn3a activity, with molecular modeling identifying interactions of these residues with the S3–S4 loop of domain II of hNa_V1.7. Removal of hydrophobic residues Y4, Y27, and W30 led to a loss of potency (>250-fold), while replacement of negatively charged D1 and D8 residues with a positively charged lysine led to increased potencies (>13-fold), likely through alterations in membrane lipid interactions. Mutating D8 to an asparagine led to the greatest improvement in Pn3a potency at Na_V1.7 (20-fold) while maintaining >100-fold selectivity over the major off-targets Na_V1.4, Na_V1.5, and Na_V1.6. The Pn3a[D8N] mutant retained analgesic activity in vivo, significantly attenuating mechanical allodynia in a clinically relevant mouse model of postsurgical pain at doses 3-fold lower than those with wild-type Pn3a, without causing motor-adverse effects. Results from this study will facilitate future rational design of potent and selective peptidic Na_V1.7 inhibitors for the development of more efficacious and safer analgesics as well as to further investigate the involvement of Na_V1.7 in pain.

Huwentoxin-IV (HwTx-IV) is a gating modifier peptide toxin from spiders that has weak affinity for the lipid bilayer. As some gating modifier toxins have affinity for model lipid bilayers, a tripartite relationship among gating modifier toxins, voltage-gated ion channels and the lipid membrane surrounding the channels has been proposed. We previously designed an HwTx-IV analog (gHwTx-IV) with reduced negative charge and increased hydrophobic surface profile, which displays increased lipid bilayer affinity and in vitro activity at the voltage-gated sodium channel subtype 1.7 (Na_V1.7), a channel targeted in pain management. Here, we show that replacements of the positively charged residues that contribute to the activity of the peptide can improve gHwTx-IV’s potency and selectivity for Na_V1.7. Using HwTx-IV, gHwTx- IV,
[R26A]gHwTx-IV, [K27A]gHwTx-IV, and [R29A]gHwTx-IV variants, we examined their potency and selectivity at human Na_V1.7 and their affinity for the lipid bilayer. [R26A]gHwTxIV consistently displayed the most improved potency and selectivity for Na_V1.7, examined alongside off-target Na_Vs, compared with HwTxIV and gHwTx-IV. The lipid affinity of each of the three novel analogs was weaker than that of gHwTx-IV, but stronger than that of HwTx-IV, suggesting a possible relationship between in vitro potency at Na_V1.7 and affinity for lipid bilayers. In a murine Na_V1.7 engagement model, [R26A]gHwTx-IV exhibited an efficacy comparable to that of native HwTx- IV. In summary, the present study reports the development of an HwTx-IV analog with improved in vitro selectivity for the pain target Na_V1.7 and with an in Vivo efficacy similar to that of native HwTx-IV.

Spider venoms are a rich source of ion channel modulators with therapeutic potential. Given the analgesic potential of subtypeselective inhibitors of voltage-gated sodium (Na_V) channels, we screened spider venoms for inhibitors of human Na_V1.7 (hNa_V1.7) using a high-throughput fluorescent assay. Here, we describe the discovery of a novel Na_V1.7 inhibitor, m-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. N_aV1.7 inhibition was diminished (IC₅₀ 11.5 nM) and the association rate decreased for the C-terminal acid form of Tp1a compared with the native amidated form(IC₅₀ 2.1 nM), suggesting that the peptide C terminus contributes to its interaction with hNaV1.7. Tp1a had no effect on human voltage-gated calcium channels or nicotinic acetylcholine receptors at 5 mM. 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 classic inhibitor cystine knot (ICK) motif. The molecular surface of Tp1a presents a hydrophobic patch surrounded by positively charged residues, with subtle differences from 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.

Double-knotted peptides identified in venoms and synthetic bivalent peptide constructs targeting ion channels are emerging tools for the study of ion channel pharmacology and physiology. These highly complex and disulfide-rich peptides contain two individual cystine knots, each comprising six cysteines and three disulfide bonds. Until now, native double-knotted peptides, such as Hi1a and DkTx, have only been isolated from venom or produced recombinantly, whereas engineered double-knotted peptides have successfully been produced through enzymatic ligation using sortase A to form a seamless amide bond at the ligation site between two knotted toxins, and by alkyne/azide click chemistry, joining two peptide knots via a triazole linkage. To further pursue these double-knotted peptides as pharmacological tools or probes for therapeutically relevant ion channels, we sought to identify a robust methodology resulting in a high yield product that lends itself to rapid production and facile mutational studies. In this study, we evaluated the ligation efficiency of enzymatic (sortase A5°, butelase 1, wild-type OaAEP 1, C247A-OaAEP 1, and peptiligase) and mild chemical approaches (α-ketoacid-hydroxylamine, KAHA) for forming a native amide bond linking the toxins while maintaining the native disulfide connectivity of each pre-folded peptide. We used two Nav1.7 inhibitors: PaurTx3, a spider-derived gating modifier peptide, and KIIIA, a small cone snail-derived pore blocker peptide, which has previously been shown to increase affinity and inhibitory potency on hNav1.7 when ligated together. Correctly folded peptides were successfully ligated in varying yields, without disulfide bond shuffling or reduction, with sortase A5° being the most efficient, resulting in 60% ligation conversion within 15 min. In addition, electrophysiology studies demonstrated that for these two peptides, the amino acid composition of the linker did not affect the activity of the double-knotted peptides. This study demonstrates the powerful application of enzymes in efficiently ligating complex disulfide-rich peptides, paving the way for facile production of double-knotted peptides.

Disulfide-rich animal venom peptides targeting either the voltage-sensing domain or the pore domain of voltage-gated sodium channel 1.7 (Na_V1.7) have been widely studied as drug leads and pharmacological probes for the treatment of chronic pain. However, despite intensive research efforts, the full potential of Na_V1.7 as a therapeutic target is yet to be realized. In this study, using evolved sortase A, we enzymatically ligated two known Na_V1.7 inhibitors–PaurTx3, a spider-derived peptide toxin that modifies the gating mechanism of the channel through interaction with the voltage-sensing domain, and KIIIA, a small cone snail-derived peptide inhibitor of the pore domain–with the aim of creating a bivalent inhibitor which could interact simultaneously with two noncompeting binding sites. Using electrophysiology, we determined the activity at Na_V1.7, and to maximize potency, we systematically evaluated the optimal linker length, which was nine amino acids. Our optimized synthetic bivalent peptide showed improved channel affinity and potency at Na_V1.7 compared to either PaurTx3 or KIIIA individually. This work shows that novel and improved Na_V1.7 inhibitors can be designed by combining a pore blocker toxin and a gating modifier toxin to confer desired pharmacological properties from both the voltage-sensing domain and the pore domain.

Disulfide-rich animal venom peptides targeting either the voltage-sensing domain or the pore domain of voltage-gated sodium channel 1.7 (Na_V1.7) have been widely studied as drug leads and pharmacological probes for the treatment of chronic pain. However, despite intensive research efforts, the full potential of Na_V1.7 as a therapeutic target is yet to be realized. In this study, using evolved sortase A, we enzymatically ligated two known Na_V1.7 inhibitors–PaurTx3, a spider-derived peptide toxin that modifies the gating mechanism of the channel through interaction with the voltage-sensing domain, and KIIIA, a small cone snail-derived peptide inhibitor of the pore domain–with the aim of creating a bivalent inhibitor which could interact simultaneously with two noncompeting binding sites. Using electrophysiology, we determined the activity at Na_V1.7, and to maximize potency, we systematically evaluated the optimal linker length, which was nine amino acids. Our optimized synthetic bivalent peptide showed improved channel affinity and potency at Na_V1.7 compared to either PaurTx3 or KIIIA individually. This work shows that novel and improved Na_V1.7 inhibitors can be designed by combining a pore blocker toxin and a gating modifier toxin to confer desired pharmacological properties from both the voltage-sensing domain and the pore domain.

Gating modifier toxins from spider venom are disulfide-rich peptides that typically comprise a stabilizing inhibitor cystine knot (ICK). These knottin peptides are being pursued as therapeutic leads for a range of conditions linked to transmembrane proteins. Recently, double-knottin peptides discovered in spider venom and produced by recombinant expression have provided insights into the pharmacology of transmembrane channels. Here, we use chemoenzymatic ligation to produce double-knottins to probe the effect of bivalent modulation on the voltage-gated sodium channel subtype 1.7 (NaV1.7), which is implicated in pain signaling. Monovalent knottins were oxidatively folded and then biochemically conjugated using sortase A, to form double-knottins. The structural integrity of the peptides was confirmed using NMR, and fluorescence-based activity assays provided evidence suggesting that coincubated monovalent and bivalent knottins can cooperatively modulate NaV1.7. We anticipate that double-knottins will provide novel tools for enhancing our understanding of, and design strategies for, therapeutically relevant voltage-gated ion channels.

To date, the clinical use of the anti-tubercular therapy bedaqui-line has been somewhat limited due to safety concerns. Recent investigations determined that modification of the Band C-ring units of bedaquiline delivered new diarylquinolines (for example TBAJ-587) with potent anti-tubercular activity yet an improved safety profile due to reduced affinity for the hERG channel. Building on our recent discovery that substitution of the quinoline motif (the A-ring subunit) for C5-aryl pyridine groups within bedaquiline analogues led to retention of anti-tubercular activity, we investigated the concurrent modification of A-, Band C-ring units within bedaquiline variants. This led to the discovery that 4-trifluoromethoxyphenyl and 4-chlorophen-yl pyridyl analogues of TBAJ-587 retained relatively potent anti-tubercular activity and for the 4-chlorophenyl derivative in particular, a significant reduction in hERG inhibition relative to bedaquiline was achieved, demonstrating that modifications of the A-, Band C-ring units within the bedaquiline structure is a viable strategy for the design of effective, yet safer (and less lipophilic) anti-tubercular compounds.

Spider venoms are rich sources of peptidic ion channel modulators with important therapeutical potential. We screened a panel of 60 spider venoms to find modulators of ion channels involved in pain transmission. We isolated, synthesized and pharmacologically characterized Cd1a, a novel peptide from the venom of the spider Ceratogyrus darlingi. Cd1a reversibly paralysed sheep blowflies (PD50 of 1318 pmol/g) and inhibited human Ca_v2.2 (IC₅₀ 2.6 μM) but not Ca_v1.3 or Ca_v3.1 (IC₅₀ > 30 μM) in fluorimetric assays. In patch clamp electrophysiological assays Cd1a inhibited rat Ca_v2.2 with similar potency (IC₅₀ 3 μM) without influencing the voltage dependence of Cav2.2 activation gating, suggesting that Cd1a doesn’t act on Ca_v2.2 as a classical gating modifier toxin. The Cd1a binding site on Ca_v2.2 did not overlap with that of the pore blocker ω-conotoxin GVIA, but its activity at Ca_v2.2-mutant indicated that Cd1a shares some molecular determinants with GVIA and MVIIA, localized near the pore region. Cd1a also inhibited human Na_v1.1–1.2 and Na_v1.7–1.8 (IC₅₀ 0.1–6.9 μM) but not Na_v1.3–1.6 (IC₅₀ > 30 μM) in fluorimetric assays. In patch-clamp assays, Cd1a strongly inhibited human Na_v1.7 (IC₅₀ 16 nM) and produced a 29 mV depolarising shift in Na_v1.7 voltage dependence of activation. Cd1a (400 pmol) fully reversed Na_v1.7-evoked pain behaviours in mice without producing side effects. In conclusion, Cd1a inhibited two anti-nociceptive targets, appearing to interfere with Ca_v2.2 inactivation gating, associated with the Ca_v2.2 α-subunit pore, while altering the activation gating of Na_v1.7. Cd1a was inactive at some of the Na_v and Ca_v channels expressed in skeletal and cardiac muscles and nodes of Ranvier, apparently contributing to the lack of side effects at efficacious doses, and suggesting potential as a lead for development of peripheral pain treatments.

The natural product indole-3-carbinol (I3C) and its major digestive product 3,3′-diindolylmethane (DIM) have shown clinical promise in multiple forms of cancer including breast cancer. In this study, we explored the calcium channel activity of DIM, its synthetic derivative 3,3′-Diindolylmethanone (DIM-one) and related I3C and DIM-one analogs. For the first time, DIM, DIM-one and analog IX were identified as selective blockers for T-type CaV3.3 (IC50s DIM 2.09 µM; DIM-one 9.07 µM) while compound IX inhibited both CaV3.2 (6.68 µM) and CaV3.3 (IC50 = 3.05 µM) using a FLIPR cell-based assay to measure inhibition of T-type calcium channel window current. Further characterization of DIM by electrophysiology revealed it inhibited inward Ca2+ current through CaV3.1 (IC50 = 8.32 µM) and CaV3.3 (IC50 = 9.63 µM), while IX partially blocked CaV3.2 and CaV3.3 inward Ca2+ current. In contrast, DIM-one preferentially blocked CaV3.1 inward Ca2+ current (IC50 = 1.53 µM). The anti-proliferative activities of these compounds revealed that oxidation of the methylene group of DIM shifted the selectivity of DIMs from breast cancer cell line MCF-7 to colon cancer cell line HT-29.

Voltage-gated sodium channels (Na_Vs) are key therapeutic targets for pain, epilepsy and cardiac arrhythmias. Here we describe the development of a no-wash fluorescent sodium
influx assay suitable for high-throughput screening and characterization of novel drug leads. Addition of red-violet food dyes (peak absorbance range 495–575 nm) to assays in HEK293 cells heterologously expressing hNa_V1.1–1.8 effectively quenched background fluorescence of the sodium indicator dye Asante NaTRIUM Green-2 (ANG-2; peak emission 540 nm), negating the need for a wash step. Ponceau 4R (1 mM) was identified as a suitable quencher, which had no direct effect on NaV channels as assessed by patch-clamp experiments, and did not alter the pharmacology of the Na_V1.1–1.7 activator veratridine (EC50 10–29 μM) or the Na_V1.1–1.8 inhibitor tetracaine (IC50’s 6–66 μM). In addition, we also identified that the food dyes Ponceau 4R, Brilliant Black BN, Allura Red and Amaranth are effective at quenching the background fluorescence of the calcium indicator dyes fluo-4, fura-2 and fura-5F, identifying them as potential inexpensive alternatives to no-wash calcium ion indicator kits. In summary, we have developed a no-wash fluorescent sodium influx assay suitable for high-throughput screening based on the sodium indicator dye ANG-2 and the quencher Ponceau 4R.

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, as been reported to selectively activate NaV1.3. 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 voltagedependence 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 chimaera, where Tf2 (1 µM) shifted the voltage dependence of activation by −6.3 mV. In an attempt to convert Tf2 into a Na_V1.3 inhibitor, we synthesized 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 NaV1.9 and other yet to be identified target(s), the use of Tf2 as a selective pharmacological probe may be limited.

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.

The 27-amino acid (aa)-long δ-conotoxin TxVIA, originally isolated from the mollusc-hunting cone snail Conus textile, slows voltage-gated sodium (Na_V) channel inactivation in molluscan neurons, but its mammalian ion channel targets remain undetermined. In this study, we confirmed that TxVIA
was inactive on mammalian Na_V1.2 and Na_V1.7 even at high concentrations (10 µM). Given the fact that invertebrate NaV channel and T-type calcium channels (Ca_V3.x) are volutionarily related, we examined the possibility that TxVIA may act on Ca_V3.x. Electrophysiological characterisation of the native TxVIA on Ca_V3.1, 3.2 and 3.3 revealed that TxVIA preferentially inhibits Ca_V3.2 current (IC₅₀ = 0.24 µM) and enhances Ca_V3.1 current at higher concentrations. In fish bioassays, TxVIA showed little effect on zebrafish behaviours when injected intramuscularly at 250 ng/100 mg fish. The binding sites for TxVIA at Na_V1.7 and Ca_V3.1 revealed that their channel binding sites contained a common epitope.

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 NaV1.7 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_vV1.7 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 NaV1.7 inhibitors GpTx-1, PF-04856264 and CNV1014802 (raxatrigine). GpTx-1 selectively inhibited NaV1.7 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 NaV 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.

Chronic pain is a serious debilitating condition that affects ∼20% of the world’s population. Currently available drugs fail to produce effective pain relief in many patients and have dose-limiting side effects. Several voltage-gated sodium (Na_V) and calcium (Ca_V) channels are implicated in the etiology of chronic pain, particularly Na_V1.1, Na_V1.3, Na_V1.7–Na_V1.9, Ca_V2.2, and Ca_V3.2. Numerous Na_V and Ca_V modulators have been described, but with few exceptions, they display poor potency and/or selectivity for pain-related channel subtypes. Here, we report the discovery and characterization of 2 novel tarantula-venom peptides (Tap1a and Tap2a) isolated from Theraphosa apophysis venom that modulate the activity of both Na_V and Ca_V3 channels. Tap1a and Tap2a inhibited on-target Na_V and Ca_V3 channels at nanomolar to micromolar concentrations and displayed moderate off-target selectivity for Na_V1.6 and weak affinity for Na_V1.4 and Na_V1.5. The most potent inhibitor, Tap1a, nearly ablated neuronal mechanosensitivity in afferent fibers innervating the colon and the bladder, with in vivo intracolonic administration reversing colonic mechanical hypersensitivity in a mouse model of irritable bowel syndrome. These findings suggest that targeting a specific combination of Na_V and Ca_V3 subtypes provides a novel route for treatment of chronic visceral pain.

Dravet syndrome (DS) is a catastrophic epileptic encephalopathy characterised by childhood-onset polymorphic seizures, multiple neuropsychiatric comorbidities, and increased risk of sudden death. Heterozygous loss-of-function mutations in one allele of SCN1A, the gene encoding the voltage-gated sodium channel 1.1 (Na_V1.1), lead to DS. Na_V1.1 is primarily found in the axon initial segment of fast-spiking GABAergic inhibitory interneurons in the brain, and the principal mechanism proposed to underlie seizure genesis in DS is loss of inhibitory input due to dysfunctional firing of GABAergic interneurons. We hypothesised that DS symptoms could be ameliorated by a drug that activates the reduced population of functional Na_V1.1 channels in DS interneurons. We recently identified two homologous disulfide-rich spider-venom peptides (Hm1a and Hm1b) that selectively potentiate Na_V1.1, and showed that selective activation of Na_V1.1 by Hm1a restores the function of inhibitory interneurons in a mouse model of DS. Here we produced recombinant Hm1b (rHm1b) using an E. coli periplasmic expression system and examined its selectivity against a panel of human NaV subtypes using whole-cell patch-clamp recordings. rHm1b is a potent and highly selective agonist of Na_V1.1 and Na_V1.3 (EC50 ~12 nM for both). rHm1b is a gating modifier that shifts the voltage dependence of channel activation and inactivation to hyperpolarised and depolarised potentials respectively, presumably by interacting with the channel’s voltage-sensor domains. Like Hm1a, the structure of rHm1b determined by using NMR revealed a classical inhibitor cystine knot (ICK) motif. However, we show that rHm1b is an order of magnitude more stable than Hm1a in human cerebrospinal fluid. Overall, our data suggest that rHm1b is an exciting lead for a precision therapeutic targeted against DS.

Dravet syndrome (DS) is a catastrophic epileptic encephalopathy characterised by childhood-onset polymorphic seizures, multiple neuropsychiatric comorbidities, and increased risk of sudden death. Heterozygous loss-of-function mutations in one allele of SCN1A, the gene encoding the voltage-gated sodium channel 1.1 (Na_V1.1), lead to DS. Na_V1.1 is primarily found in the axon initial segment of fast-spiking GABAergic inhibitory interneurons in the brain, and the principle mechanism proposed to underlie seizure genesis in DS is loss of inhibitory input due to dysfunctional firing of GABAergic interneurons. We hypothesised that DS symptoms could be ameliorated by a drug that activates the reduced population of functional Na_V1.1 channels in DS interneurons. We recently identified two homologous disulfide-rich spider-venom peptides (Hm1a and Hm1b) that selectively potentiate Na_V1.1, and showed that selective activation of Na_V1.1 by Hm1a restores the function of inhibitory interneurons in a mouse model of DS. Here we produced recombinant Hm1b (rHm1b) using an E. coli periplasmic expression system and examined its selectivity against a panel of human NaV subtypes using whole-cell patch-clamp recordings. rHm1b is a potent and highly selective agonist of Na_V1.1 and Na_V1.3 (EC₅₀ ~12 nM for both). rHm1b is a gating modifier that shifts the voltage dependence of channel activation and inactivation to hyperpolarised and depolarised potentials respectively, presumably by interacting with the channel’s voltage-sensor domains. Like Hm1a, the structure of rHm1b determined by using NMR revealed a classical inhibitor cystine knot (ICK) motif. However, we show that rHm1b is an order of magnitude more stable than Hm1a in human cerebrospinal fluid. Overall, our data suggest that rHm1b is an exciting lead for a precision therapeutic targeted against DS.

Ants (Hymenoptera: Formicidae) are diverse and ubiquitous, and their ability to sting is familiar to many of us. However, their venoms remain largely unstudied. We provide the first comprehensive characterization of a polypeptidic ant venom, that of the giant red bull ant, Myrmecia gulosa. We reveal a suite of novel peptides with a range of posttranslational modifications, including disulfide bond formation, dimerization, and glycosylation. One venom peptide has sequence features consistent with an epidermal growth factor fold, while the remaining peptides have features suggestive of a capacity to form amphipathic helices. We show that these peptides are derived from what appears to be a single, pharmacologically diverse, gene superfamily (aculeatoxins) that includes most venom peptides previously reported from the aculeate Hymenoptera. Two aculeatoxins purified from the venom were found to be capable of activating mammalian sensory neurons, consistent with the capacity to produce pain but via distinct mechanisms of action. Further investigation of the major venom peptide MIITX₁-Mg1a revealed that it can also incapacitate arthropods, indicative of dual utility in both defense and predation. MIITX₁-Mg1a accomplishes these functions by generating a leak in membrane ion conductance, which alters membrane potential and triggers neuronal depolarization. Our results provide the first insights into the evolution of the major toxin gene superfamily of the aculeate Hymenoptera and provide a new paradigm in the functional evolution of toxins from animal venoms.