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Spider venom-derived cysteine knot peptides are a mega-diverse class of molecules that exhibit unique pharmacological properties to modulate key membrane protein targets. Voltage-gated sodium channels (Na ) are often targeted by these peptides to allosterically promote opening or closing of the channel by binding to structural domains outside the channel pore. These effects can result in modified pain responses, muscle paralysis, cardiac arrest, priapism, and numbness. Although such effects are often deleterious, subtype selective spider venom peptides are showing potential to treat a range of neurological disorders, including chronic pain and epilepsy. This review examines the structure-activity relationships of cysteine knot peptides from spider venoms that modulate Na and discusses their potential as leads to novel therapies for neurological disorders.

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 V 2.2 and Na V 1.7. 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 V 2.2 and Na V 1.7 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.

Voltage-gated sodium channels (NaVs) are a key determinant of neuronal signalling. Neurotoxins from diverse taxa that selectively activate or inhibit NaV channels have helped unravel the role of NaV channels in diseases, including chronic pain. Spider venoms contain the most diverse array of inhibitor cystine knot (ICK) toxins (knottins). This review provides an overview on how spider knottins modulate NaV channels and describes the structural features and molecular determinants that influence their affinity and subtype selectivity. Genetic and functional evidence support a major involvement of NaV subtypes in various chronic pain conditions. The exquisite inhibitory properties of spider knottins over key NaV subtypes make them the best lead molecules for the development of novel analgesics to treat chronic pain.

Snakebite envenoming is an important public health issue responsible for mortality and severe morbidity. Where mortality is mainly caused by venom toxins that induce cardiovascular disturbances, neurotoxicity, and acute kidney injury, morbidity is caused by toxins that directly or indirectly destroy cells and degrade the extracellular matrix. These are referred to as ‘tissue-damaging toxins’ and have previously been classified in various ways, most of which are based on the tissues being affected (e.g., cardiotoxins, myotoxins). This categorisation, however, is primarily phenomenological and not mechanistic. In this review, we propose an alternative way of classifying cytotoxins based on their mechanistic effects rather than using a description that is organ- or tissue-based. The mechanisms of toxin-induced tissue damage and their clinical implications are discussed. This review contributes to our understanding of fundamental biological processes associated with snakebite envenoming, which may pave the way for a knowledge-based search for novel therapeutic options. The snake venom toxins responsible for tissue damage, their mechanisms of action and pathological effects are reviewed, together with the search of novel therapeutic alternatives to abrogate their effects

Zygaenoidea is a superfamily of lepidopterans containing many venomous species, including the Limacodidae (nettle caterpillars) and Megalopygidae (asp caterpillars). Venom proteomes have been recently documented for several species from each of these families, but further data are required to understand the evolution of venom in Zygaenoidea. In this study, we examined the ‘electric’ caterpillar from North-Eastern Australia, a limacodid caterpillar densely covered in venomous spines. We used DNA barcoding to identify this caterpillar as the larva of the moth Comana monomorpha (Turner, 1904). We report the clinical symptoms of C. monomorpha envenomation, which include acute pain, and erythema and oedema lasting for more than a week. Combining transcriptomics of venom spines with proteomics of venom harvested from the spine tips revealed a venom markedly different in composition from previously examined limacodid venoms that are rich in peptides. In contrast, the venom of C. monomorpha is rich in aerolysin-like proteins similar to those found in venoms of asp caterpillars (Megalopygidae). Consistent with this composition, the venom potently permeabilises sensory neurons and human neuroblastoma cells. This study highlights the diversity of venom composition in Limacodidae.

Venom-derived peptides have attracted much attention as potential lead molecules for pharmaceutical development. A well-known example is Huwentoxin-IV (HwTx-IV), a peptide toxin isolated from the venom of the Chinese bird-eating spider Haplopelma schmitdi. HwTx-IV was identified as a potent blocker of a human voltage-gated sodium channel (hNaV1.7), which is a genetically validated analgesic target. The peptide was promising as it showed high potency at NaV1.7 (IC50 ~26 nM) and selectivity over the cardiac NaV subtype (NaV1.5). Mutagenesis studies aimed at optimising the potency of the peptide resulted in the development of a triple-mutant of HwTx-IV (E1G, E4G, Y33W, m3-HwTx-IV) with significantly increased potency against hNaV1.7 (IC50 = 0.4 ± 0.1 nM) without increased potency against hNaV1.5. The activity of m3-HwTx-IV against other NaV subtypes was, however, not investigated. Similarly, the structure of the mutant peptide was not characterised, limiting the interpretation of the observed increase in potency. In this study we produced isotope-labelled recombinant m3-HwTx-IV in E. coli, which enabled us to characterise the atomic-resolution structure and dynamics of the peptide by NMR spectroscopy. The results show that the structure of the peptide is not perturbed by the mutations, whilst the relaxation studies reveal that residues in the active site of the peptide undergo conformational exchange. Additionally, the NaV subtype selectivity of the recombinant peptide was characterised, revealing potent inhibition of neuronal NaV subtypes 1.1, 1.2, 1.3, 1.6 and 1.7. In parallel to the in vitro studies, we investigated NaV1.7 target engagement of the peptide in vivo using a rodent pain model, where m3-HwTx-IV dose-dependently suppressed spontaneous pain induced by the NaV1.7 activator OD1. Thus, our results provide further insight into the structure and dynamics of this class of peptides that may prove useful in guiding the development of inhibitors with improved selectivity for analgesic NaV subtypes.

Snake venom activity exhibits evolutionary patterns within genera and varies according to ecological niche. By examining venoms from species inhabiting distinct environments, niche-specific functional differences can be uncovered. Bothrops , a diverse and medically important Neotropical pit viper genus, shows known clade-specific differences in coagulotoxicity. Here, we expanded this framework by assessing intra-clade variation in cellular activity across twelve Bothrops species using a high-content fluorescence assay that simultaneously measures ion channel responses and membrane cytotoxicity. Most venoms induced rapid membrane damage and cell lysis, whereas arboreal species lacked both activities, suggesting reduced selection for these activities. In contrast, the terrestrial Bothrops mattogrossensis and Bothrops pauloensis, which are sister species inhabiting the Pantanal wetlands and outskirts, respectively, displayed unique intracellular calcium-modulatory effects in the absence of membrane disruption. High throughput venomics revealed candidate toxin families underlying these calcium responses. Our findings demonstrate niche-specific diversification of venom bioactivity and highlight Bothrops venoms as promising sources of pharmacological agents.

Spider-derived venoms are a rich source of cystine knot peptides with immense therapeutic potential. Many of these peptides exert unique biological activities through the modulation of ion channels, including of human voltage-gated sodium (NaV1.1–NaV1.9) channels. NaV channel subtypes have diverse functions determined by their tissue and cellular distribution and biophysical properties, and are pathophysiology mediators in various diseases. Therefore, NaVs are central in studies of human biology. This work investigated the pharmacological properties of venom of the Thai theraphosid Ornithoctonus aureotibialis on NaV channels. We discovered a predominant venom peptide named Oa1a and assessed its pharmacological properties across human NaV channel subtypes. Synthetic forms of the peptide Oa1a showed preferential inhibition of NaV1.1 and NaV1.7, while recombinant Oa1a displayed a preference for inhibiting NaV1.2, NaV1.6, and NaV1.7. Interestingly, all versions of Oa1a peptides exerted dual pharmacological effect by reducing the peak current and slowing fast inactivation of NaV1.3, consistent with Oa1a having more than one binding site on NaV channels. Such complex pharmacology was previously observed for a venom peptide in a Central American and Costa Rican tarantula, suggesting a conserved mechanism of action amongst these geographically distinct species. However, Oa1a lacked activity in the T-type channels observed in the tarantula peptide from Central America. Structure–function relationships investigated using molecular modelling showed that the dual pharmacology is driven by a conserved mechanism utilizing a mix of aromatic and charged residues, while the T-type activity appears to require additional charged residues in loop 2 and fewer positive charges in loop 4. Future structure–activity relationship studies of Oa1a will guide the development of pharmacological tools as well as next-generation drugs to treat NaV channel dysfunction associated with neurological disorders.

The structure-function and optimization studies of Na V -inhibiting spider toxins have focused on developing selective inhibitors for peripheral pain-sensing Na V 1.7. With several Na V subtypes emerging as potential therapeutic targets, structure-function analysis of Na V -inhibiting spider toxins at such subtypes is warranted. Using the recently discovered spider toxin Ssp1a, this study extends the structure-function relationships of Na V -inhibiting spider toxins beyond Na V 1.7 to include the epilepsy target Na V 1.2 and the pain target Na V 1.3. Based on these results and docking studies, we designed analogues for improved potency and/or subtype-selectivity, with S7R-E18K-rSsp1a and N14D-P27R-rSsp1a identified as promising leads. S7R-E18K-rSsp1a increased the rSsp1a potency at these three Na V subtypes, especially at Na V 1.3 (∼10-fold), while N14D-P27R-rSsp1a enhanced Na V 1.2/1.7 selectivity over Na V 1.3. This study highlights the challenge of developing subtype-selective spider toxin inhibitors across multiple Na V subtypes that might offer a more effective therapeutic approach. The findings of this study provide a basis for further rational design of Ssp1a and related NaSpTx1 homologs targeting Na V 1.2, Na V 1.3 and/or Na V 1.7 as research tools and therapeutic leads.

This study presents nanofractionation analytics coupled with in vivo profiling of zebrafish embryo paralysis and lethality in response to toxins in cone snail venoms. The focus of this study is on the development of this approach using venoms of Conus marmoreus, Conus ebraeus, and Conus bandanus. In brief, cone snail venoms were separated using reversed-phase chromatography following high-resolution nanofractionation on microplates with parallel mass spectrometry, enabled via a post-column flow split. All collected fractions were dried overnight, followed by assays on zebrafish embryos. For the paralysis assessment, we monitored swimming behavior and swimming distance and found that exposure to cone snail toxins led to paralysis and decreased movement and swim distance. To correlate the masses of eluted toxins with their paralyzing effects and potency, we compared the fractionation retention time versus normalized swimming distance. This allowed identification of the masses of toxins with paralyzing bioactivity, which were predominantly conopeptides. To assess lethality, zebrafish embryos were exposed to fractionated toxins for 24 h, after which they were inspected. The lethal doses and correlated toxins were identified by comparing retention times of fractionation versus the lethal dose values calculated for each fraction. We found that the most lethal venom was from C. bandanus, displaying the largest number of lethal peptides, followed by C. marmoreus and C. ebraeus. On the other hand, the most paralytic venom was from C. ebraeus, presenting a higher number of peptides with non-lethal paralytic effects, followed by C. bandanus and C. marmoreus. This study provides a pipeline to rapidly identify paralytic and lethal cone snail venom toxins using the zebrafish embryo model.