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Abstract: Animal venoms are complex chemical mixtures that typically contain hundreds of proteins and non-proteinaceous compounds, resulting in a potent weapon for prey immobilization and predator deterrence. However, because venoms are protein-rich, they come with a high metabolic price tag. The metabolic cost of venom is sufficiently high to result in secondary loss of venom whenever its use becomes non-essential to survival of the animal. The high metabolic cost of venom leads to the prediction that venomous animals may have evolved strategies for minimizing venom expenditure. Indeed, various behaviors have been identified that appear consistent with frugality of venom use. This has led to formulation of the “venom optimization hypothesis” (Wigger et al. (2002) Toxicon 40, 749–752), also known as “venom metering”, which postulates that venom is metabolically expensive and therefore used frugally through behavioral control. Here, we review the available data concerning economy of venom use by animals with either ancient or more recently evolved venom systems. We conclude that the convergent nature of the evidence in multiple taxa strongly suggests the existence of evolutionary pressures favoring frugal use of venom. However, there remains an unresolved dichotomy between this economy of venom use and the lavish biochemical complexity of venom, which includes a high degree of functional redundancy. We discuss the evidence for biochemical optimization of venom as a means of resolving this conundrum. [Copyright &y& Elsevier]

Abstract: Centipedes are among the oldest extant terrestrial arthropods and are an ecologically important group of soil and leaf litter predators. Despite their abundance and frequent, often painful, encounters with humans, little is known about the venom and venom apparatus of centipedes, although it is apparent that these are both quite different from other venomous lineages. The venom gland can be regarded as an invaginated cuticle and epidermis, consisting of numerous epithelial secretory units each with its own unique valve-like excretory system. The venom contains several different enzymes, but is strikingly different to most other arthropods in that metalloproteases appear to be important. Myotoxic, cardiotoxic, and neurotoxic activities have been described, most of which have been attributed to high molecular weight proteins. Neurotoxic activities are also unusual in that G-protein coupled receptors often seem to be involved, either directly as targets of neurotoxins or indirectly by activating endogenous agonists. These relatively slow responses may be complemented by the rapid effects caused by histamines present in the venom and from endogenous release of histamines induced by venom cytotoxins. The differences probably reflect the ancient and independent evolutionary history of the centipede venom system, although they may also be somewhat exaggerated by the paucity of information available on this largely neglected group. [Copyright &y& Elsevier]

Abstract: Animal toxins are the major class of secreted disulfide-rich proteins, with ∼70% containing two or more disulfide bonds. Incorrect pairing of these disulfide bonds typically leads to a non-native three-dimensional fold accompanied by a loss of protein function. Determination of the native disulfide-bond framework is therefore a key component in the structural characterization of toxins. In this article, we review NMR approaches for elucidation of disulfide-bond connectivities. A major advantage of these NMR approaches is that they are non-invasive, leaving the sample intact at the end of the analysis for use in other studies. [ABSTRACT FROM AUTHOR]

Abstract: Molecular toxinology research was initially driven by an interest in the small subset of animal toxins that are lethal to humans. However, the realization that many venomous creatures possess a complex repertoire of bioactive peptide toxins with potential pharmaceutical and agrochemical applications has led to an explosion in the number of new peptide toxins being discovered and characterized. Unfortunately, this increased awareness of peptide-toxin diversity has not been matched by the development of a generic nomenclature that enables these toxins to be rationally classified, catalogued, and compared. In this article, we introduce a rational nomenclature that can be applied to the naming of peptide toxins from spiders and other venomous animals. [Copyright &y& Elsevier]

Abstract: Insects have a much smaller repertoire of voltage-gated calcium (CaV) channels than vertebrates. Drosophila melanogaster harbors only a single ortholog of each of the vertebrate CaV1, CaV2, and CaV3 subtypes, although its basal inventory is expanded by alternative splicing and editing of CaV channel transcripts. Nevertheless, there appears to be little functional plasticity within this limited panel of insect CaV channels, since severe loss-of-function mutations in genes encoding the pore-forming α 1 subunits in Drosophila are embryonic lethal. Since the primary role of spider venom is to paralyze or kill insect prey, it is not surprising that most, if not all, spider venoms contain peptides that potently modify the activity of these functionally critical insect CaV channels. Unfortunately, it has proven difficult to determine the precise ion channel subtypes recognized by these peptide toxins since insect CaV channels have significantly different pharmacology to their vertebrate counterparts, and cloned insect CaV channels are not available for electrophysiological studies. However, biochemical and genetic studies indicate that some of these spider toxins might ultimately become the defining pharmacology for certain subtypes of insect CaV channels. This review focuses on peptidic spider toxins that specifically target insect CaV channels. In addition to providing novel molecular tools for ion channel characterization, some of these toxins are being used as leads to develop new methods for controlling insect pests. [Copyright &y& Elsevier]

Spiders are remarkable for their complete reliance on predation as a trophic strategy. Their evolutionary success is due in large part to production of a complex venom that is designed to rapidly subdue or kill their prey. This issue provides an overview of the fascinating complexity of these venoms, a realistic account of the danger they pose to humans, and an examination of their immense but largely untapped potential for drug and insecticide discovery. [Copyright &y& Elsevier]

The Janus-faced atracotoxins (J-ACTXs) are a family of insect-specific excitatory toxins isolated from the venom of Australian funnel-web spiders (genera Atrax and Hadronyche). In addition to a classical cystine knot motif, these toxins contain a rare vicinal disulfide bond. While the vicinal disulfide is known to be critical for insecticidal activity, the role of other residues in toxin function remains to be determined. In this study, we probed the role of the structurally disordered N- and C-terminal residues using a panel of recombinant mutants of the prototypic family member J-ACTX-Hv1c. We found that the structurally disordered C-terminal residues (Glu 36 and Pro 37) were dispensable for toxin function. However, whereas deletion of Ala 1 had minimal impact on toxin function, deletion of both Ala 1 and Ile 2 decreased insecticidal activity more than 70-fold. We propose that Ile 2 forms a part of the target binding site of J-ACTX-Hv1c. [Copyright &y& Elsevier]

Harvester ants (genus Pogonomyrmex) are renowned for their stings which cause intense, long-lasting pain, and other neurotoxic symptoms in vertebrates. Here, we show that harvester ant venoms are relatively simple and composed largely of peptide toxins. One class of peptides is primarily responsible for the long-lasting local pain of envenomation via activation of peripheral sensory neurons. These hydrophobic, cysteine-free peptides potently modulate mammalian voltagegated sodium (NaV) channels, reducing the voltage threshold for activation and inhibiting channel inactivation. These toxins appear to have evolved specifically to deter vertebrates. [ABSTRACT FROM AUTHOR]

The growing world population is placing an increasing demand on food production. In addition, abuse and misuse of chemical insecticides has led to the evolution of resistance in insect pests as well as environmental damage. Together, these developments have created a demand for new insecticidal compounds to facilitate global food production. Arachnid venom peptides provide an environmentally-friendly alternative as potential bioinsecticides given their advantages of being fully biodegradable, highly potent, and phyletically selective. However, the use of arachnid venom peptides as bioinsecticides has been questioned due to their presumed lack of oral toxicity. Thus, the aim of this work was to develop screens for oral insecticidal activity. Based on the high susceptibility of dipterans to venom peptides, fruit flies ( Drosophila melanogaster ) and sheep blowflies ( Lucilia cuprina ) were selected for screening 56 arachnid venoms. 71.4% of these venoms caused 50% or higher mortality in Drosophila , whereas 30.4% were lethal to blowflies at oral doses of 1 or 30 μg/fly, respectively. We used these assays to compare the oral and injection activity of four well-known spider venom peptides (Hv1a, Hv1c, Dc1a and Ta1a). Hv1c and Ta1a only showed weak or no oral activity in both species, while Hv1a and Dc1a showed higher oral activity in blowflies than Drosophila . Overall, we have established screens for oral toxicity in two dipteran insects. Our results indicate that oral insecticidal activity is more widespread in arachnid venoms than expected, and that some arachnid venoms and venom peptides exhibit phyletic differences in oral toxicity. [ABSTRACT FROM AUTHOR]

Disulfide-rich peptides such as defensins play diverse roles in immunity and ion channel modulation, as well as constituting the bioactive components of many animal venoms. We investigated the structure and bioactivity of U-RDTX-Pp19, a peptide previously discovered in venom of the assassin bug Pristhesancus plagipennis. Recombinant Pp19 (rPp19) was found to possess insecticidal activity when injected into Drosophila melanogaster. A bioinformatic search revealed that domains homologous to Pp19 are produced by assassin bugs and diverse other arthropods. rPp19 co-eluted with native Pp19 isolated from P. plagipennis , which we found is more abundant in hemolymph than venom. We solved the three-dimensional structure of rPp19 using 2D 1H NMR spectroscopy, finding that it adopts a disulfide-stabilized structure highly similar to known trans -defensins, with the same cystine connectivity as human α-defensin (I–VI, II–IV, and III–V). The structure of Pp19 is unique among reported structures of arthropod peptides. [Display omitted] • Recombinant expression of Pp19, a disulfide-rich peptide from assassin bug venom • The Pp19 structure was determined by solution NMR spectroscopy • Pp19 adopts a fold similar to human α-defensin with the same disulfide connectivity Walker et al. produced an insecticidal peptide from the venom of an assassin bug using bacterial expression. The structure of this peptide was determined using nuclear magnetic resonance spectroscopy. Pp19 adopts a unique fold previously unknown among defensins produced by arthropods. [ABSTRACT FROM AUTHOR]

In this study, we report the innovative application of whole-cell patch-clamp electrophysiology in assessing broad-spectrum neutralisation by three different antivenoms, of venoms from the medically significant scorpion genus Centruroides. Envenomations by as many as 21 species from the Centruroides genus result in up to 300,000 envenomations per year in Mexico, which poses significant and potentially life-threatening pathophysiology. We first evaluated the in vitro manifestation of envenomation against two human voltage-gated sodium (hNa V) channel subtypes: hNa V 1.4 and hNa V 1.5, which are primarily expressed in skeletal muscles and cardiomyocytes, respectively. The neutralisation of venom activity was then characterised for three different antivenoms using a direct competition model against the more potent target, hNa V 1.4. While broad-spectrum neutralisation was identified, variation in neutralisation arose for Centruroides elegans, C. limpidus , C. noxius and C. suffusus venoms, despite the presence of a number of these venoms within the immunising mixture. This raises questions regarding the truly "broad" neutralisation capacity of the antivenoms. This study not only extends previous validation of the in vitro investigation of antivenom efficacy utilising the whole-cell patch-clamp technique but also underscores the potential of this animal-free model in exploring cross-reactivity, experimental scalability, and most importantly, informing clinical management practices regarding the administration of antivenom in Mexico. [Display omitted] • Novel application of in vitro methodology to assess broad antivenom efficacy. • Mexican scorpion venom shows distinct activity against select human sodium ion channels. • Antivenom efficacy was not universal in treatment across the Centruroides genus. • Antivenom manufacture should consider broader immunising mixtures. [ABSTRACT FROM AUTHOR]

Abstract: Insect-specific neurotoxins that act within the insect hemocoel (body cavity) represent an untapped resource for insect pest management. On the basis of recent advances made in development of appropriate delivery systems for transport of these toxins from the insect gut, across the gut epithelium to their target site, we screened neurotoxins derived from scorpion or spider venom for efficacy against the pea aphid, Acyrthosiphon pisum, and the green peach aphid, Myzus persicae. Toxins were selected to represent different modes of electrophysiological action, including activity on voltage-gated calcium channels (ω-TRTX-Gr1a, ω-agatoxin Aa4a, ω-hexatoxin-Hv1a), calcium- and voltage-activated potassium channels (charybdotoxin, maurotoxin), chloride channels (chlorotoxin) and voltage-gated sodium channels (LqhαIT). The Bacillus thuringiensis-derived toxin Cyt1Aa was also tested as a positive control for toxicity. In per os bioassays with both aphid species, toxicity was only seen for ω-TRTX-Gr1a and Cyt1Aa. On injection into the hemocoel of A. pisum, LD50 values ranged from 1 to 8 ng/mg body weight, with ω-hexatoxin-Hv1a being the most toxic (1.02 ng/mg body weight). All neurotoxins caused rapid paralysis, with charybdotoxin, maurotoxin and chlorotoxin also causing melanization of injected aphids. These data represent the first comprehensive screen of neurotoxins against aphids, and highlight the potential for practical use of the insect-specific toxin ω-hexatoxin-Hv1a in aphid management. [Copyright &y& Elsevier]

Sea anemone toxins are predominantly peptide and proteins that act mainly on sodium and potassium channels, as well as in a variety of target cells causing lysis. Over recent years, the number of sea anemone peptide toxins as well as cytolytic pore-forming proteins and phospholipase A 2 sequences submitted to databases has been rapidly increasing due to the developments in DNA sequencing technology and proteomic approaches. However, the lack of a systematic nomenclature has resulted in multiple names being assigned to the same toxins, toxins from unrelated species being designated by the same name, and ambiguous name designations. Therefore, in this work we propose a systematic nomenclature in which we adopted specific criteria, based on order of discovery and phylogenetic analysis, in order to avoid redundant sea anemone toxin names. Implementation of the nomenclature proposed here not only allowed us to rename the already published 191 anemone toxins without ambiguities, but it can be used to unambiguously name newly discovered toxins whether or not they are related to previously published sea anemone sequences. In the new nomenclature each toxin name contains information about the toxin's biological activity, origin and relationship to known isoforms. Ongoing increases in the speed of DNA sequencing will raise significantly the number of sea anemone toxin sequences in the literature. This will represent a constant challenge in their clear identification and logical classification, which could be solved using the proposed nomenclature. [ABSTRACT FROM AUTHOR]

Abstract: cDNA libraries are increasingly being used for high-throughput interrogation of animal venomes. Most previous studies have focused on discovery of new venom toxins, whereas the dynamics of toxin transcription and associated cellular processes have received much less attention. Here we provide, for the first time, an analysis of a transcriptome from the venom gland of a scorpion (Hottentotta judaicus) that is not actively engaged in regenerating its venom. We demonstrate a low abundance of toxin-encoding transcripts coupled with a previously unobserved proliferation of protease sequences. Additionally, we identified several low abundance, toxin-like sequences that may represent decommissioned toxins that are unlikely to be translated. These sequences are not evenly distributed across all toxin families, but rather appear more frequently in transcripts related to α-toxins and β-toxins that are known to target voltage-gated sodium channels. The transcriptomic profile of the replete venom gland is very different to that obtained previously from scorpion venom glands actively engaged in venom regeneration, and it highlights our lack of knowledge as to how the dynamics of transcription changes as the gland progresses from venom regeneration to a “resting” state. This study therefore provides an important foundation for future studies into the dynamics of transcription in the venom glands of scorpions and other venomous animals. [Copyright &y& Elsevier]

Abstract: The complexity of Australian funnel-web spider venoms has been explored via the combined use of MALDI–TOF mass spectrometry coupled with chromatographic separation and the analysis of venom-gland cDNA libraries. The results show that these venoms are far more complex than previously realized. We show that the venoms of Australian funnel-web spiders contain many hundreds of peptides that follow a bimodal distribution, with about 75% of the peptides having a mass of 3000–5000Da. The mass spectral data were validated by matching the experimentally observed masses with those predicted from peptide sequences derived from analysis of venom-gland cDNA libraries. We show that multiple isoforms of these peptides are found in small chromatographic windows, which suggests that the wide distribution of close molecular weights among the chromatographic fractions probably reflects a diversity of structures and physicochemical properties. The combination of all predicted and measured parameters permits the interpretation of three-dimensional ‘venom landscapes’ derived from LC-MALDI analysis. We propose that these venom landscapes might have predictive value for the discovery of various groups of pharmacologically distinct toxins in complex venoms. [Copyright &y& Elsevier]

Endoparasitoid wasps use complex biochemical arsenals to suppress the normal humoral and cellular immune responses of their hosts in order to transform them into a suitable environment for development of their eggs and larvae. Venom injected during oviposition is a key component of this arsenal, but the functions of individual venom toxins are still poorly understood. Furthermore, there has been little investigation of the potential biotechnological use of these venom toxins, for example for control of agricultural pests. The endoparasitoid Cotesia flavipes (Hymenoptera: Braconidae) is a biocontrol agent reared in biofactories and released extensively in Brazil to control the sugarcane borer Diatraea saccharalis (Lepidoptera: Crambidae). The objectives of this work were to reveal venom components produced by C. flavipes and explore the function of a major venom peptide, Cf4. Using a combined proteomic/transcriptomic approach, we identified 38 putative venom toxins including both linear and disulfide-rich peptides, hydrolases, protease inhibitors, apolipophorins, lipid-binding proteins, and proteins of the odorant binding families. Because of its high abundance in the venom, we selected Cf4, a 33-residue peptide with three disulfide bonds, for synthesis and further characterization. We found that synthetic Cf4 reduced the capacity of D. saccharalis hemocytes to encapsulate foreign bodies without any effect on phenoloxidase activity, consistent with a role in disruption of the cellular host immune response. Feeding leaves coated with Cf4 to neonate D. saccharalis resulted in increased mortality and significantly reduced feeding compared to caterpillars fed untreated leaves, indicating that Cf4 is a potential candidate for insect pest control through ingestion. This study adds to our knowledge of endoparasitoid wasp venoms composition, host regulation mechanisms and their biotechnological potential for pest management. [Display omitted] • A combined proteomic/transcriptomic approach was used to reveal 38 components in the venom of Cotesia flavipes. • Disulfide-rich peptides, hydrolases, protease inhibitors, apolipophorins, lipid-binding proteins and others were identified. • Synthetic venom-peptide Cf4 reduces the capacity of Diatraea saccharalis hemocytes to encapsulate foreign bodies. • The ingestion of Cf4 decreases larval viability and leaf consumption by Diatraea saccharalis. [ABSTRACT FROM AUTHOR]

• We examined the in vitro anthelmintic activity of diarylamidine compounds. • Pentamidine and diminazene inhibited Haemonchus contortus larval development. • Highlights anthelmintic potential of this drug class. • Raises possibility of acid-sensing ion channels as anthelmintic targets. Parasitic nematodes pose a major threat to livestock production worldwide. The blood-feeding parasite Haemonchus contortus is a key small-ruminant pathogen that causes anaemia, and thereby seriously impacts animal health and production. Control of this parasite relies largely upon broad-spectrum anthelmintics, but new drugs are urgently needed to combat the threat of widespread multidrug resistance. Repurposing drugs can accelerate the development pipeline by reducing costs and risks, and can be an effective way of quickly bringing new antiparasitic drugs to market. Diarylamidine compounds such as pentamidine and diminazene have been employed in the treatment of trypanosomiasis and leishmaniasis in both human and veterinary settings, but their activity against parasitic worms has not yet been reported. We screened a small panel of diarylamidine compounds against H. contortus to assess their potential to be repurposed as anthelmintic drugs. Pentamidine and diminazene inhibited H. contortus larval development at low micromolar concentrations (IC 50 4.9 μM and 16.1 μM, respectively, in a drug-susceptible isolate) with no existing cross-resistance in two multidrug resistant isolates and a monepantel-resistant isolate. Combinations of pentamidine with commercial anthelmintics showed additive activity, with no significant synergism detected. Pentamidine and diminazene showed different life-stage patterns of activity; both were active against early stage larvae in development assays, but only diminazene was active against the infective L3 stage in migration assays. This suggests some differences in uptake of the two drugs across the nematode cuticle, or differences in the nature and expression patterns of their molecular targets. As pentamidine and diminazene have been reported to be potent inhibitors of mammalian acid-sensing ion channels (ASIC), we tested the activity of known ASIC inhibitors against H. contortus to probe whether these channels may represent potential anthelmintic targets in nematodes. Remarkably, the spider-venom peptide Hi1a, a potent inhibitor of ASIC1a, inhibited H. contortus larval development with an IC 50 of 22.9 ± 1.9 μM. This study highlights the potential use of diarylamidines as anthelmintics, although their activity needs to be confirmed in vivo. In addition, our demonstration that ASIC inhibitors have anthelmintic activity raises the possibility that this family of ion channels may represent a novel anthelmintic target. [ABSTRACT FROM AUTHOR]

Graphical abstract Abstract Acid-sensing ion channels (ASICs) are primary acid sensors in the mammalian nervous system that are activated by protons under conditions of local acidosis. They have been implicated in a range of pathologies including ischemic stroke (ASIC1a subtype) and peripheral pain (ASIC1b and ASIC3). Although the spider venom peptide PcTx1 is the best-studied ASIC modulator and is neuroprotective in rodent models of ischemic stroke, little experimental work has been done to examine its molecular interaction with human ASIC1a or the off-target ASIC1b. The complementary face of the acidic pocket binding site of PcTx1 is where these channels differ in sequence. We show here that although PcTx1 is 10-fold less potent at human ASIC1a than the rat channel, the apparent affinity for the two channels is comparable. We examined the pharmacophore of PcTx1 for human ASIC1a and rat ASIC1b, and show that inhibitory and stimulatory effects at each ASIC1 variant is driven mostly by a shared set of core peptide pharmacophore residues that bind to the thumb domain, while peptide residues that interact with the complementary face of the biding site underlie species and subtype-dependent differences in activity that may allow manipulation of ASIC1 variant selectivity. Finally, the stimulatory effect of PcTx1 on rat ASIC1a when applied under mildly alkaline pH correlates with low receptor occupancy. These new insights into the interactions between PcTx1 with ASIC1 subtypes demonstrates the complexity of its mechanism of action, and highlights important implications to consider when using PcTx1 as a pharmacological tool to study ASIC function. [ABSTRACT FROM AUTHOR]

Graphical abstract Abstract Recently, we and other groups revealed that gain-of-function mutations in the human ether à go-go voltage-gated potassium channel hEAG1 (K v 10.1) lead to developmental disorders with associated infantile-onset epilepsy. However, the physiological role of hEAG1 in the central nervous system remains elusive. Potent and selective antagonists of hEAG1 are therefore much sought after, both as pharmacological tools for studying the (patho)physiological functions of this enigmatic channel and as potential leads for development of anti-epileptic drugs. Since animal venoms are a rich source of potent ion channel modifiers that have been finely tuned by millions of year of evolution, we screened 108 arachnid venoms for hEAG1 inhibitors using electrophysiology. Two hit peptides (Aa1a and Ap1a) were isolated, sequenced, and chemically synthesised for structure-function studies. Both of these hEAG1 inhibitors are C-terminally amidated peptides containing an inhibitor cystine knot motif, which provides them with exceptional stability in both plasma and cerebrospinal fluid. Aa1a and Ap1a are the most potent peptidic inhibitors of hEAG1 reported to date, and they present a novel mode of action by targeting both the activation and inactivation gating of the channel. These peptides should be useful pharmacological tools for probing hEAG1 function as well as informative leads for the development of novel anti-epileptic drugs. [ABSTRACT FROM AUTHOR]

Abstract The insects are a hyperdiverse class containing more species than all other animal groups combined—and many employ venom to capture prey, deter predators and micro-organisms, or facilitate parasitism or extra-oral digestion. However, with the exception of those made by Hymenoptera (wasps, ants and bees), little is known about insect venoms. Here, we review the current literature on insects that use venom for prey capture and predator deterrence, finding evidence for fourteen independent origins of venom usage among insects, mostly among the hyperdiverse holometabolan orders. Many lineages, including the True Bugs (Heteroptera), robber flies (Asilidae), and larvae of many Neuroptera, Coleoptera and Diptera, use mouthpart-associated venoms to paralyse and pre-digest prey during hunting. In contrast, some Hymenoptera and larval Lepidoptera, and one species of beetle, use non-mouthpart structures to inject venom in order to cause pain to deter potential predators. Several recently published insect venom proteomes indicate molecular convergence between insects and other venomous animal groups, with all insect venoms studied so far being potently bioactive cocktails containing both peptides and larger proteins, including novel peptide and protein families. This review summarises the current state of the field of entomo-venomics. Highlights • Literature review suggests that venom use has evolved 14 times among the insects, the most diverse class of animals. • Convergent evolution in the molecular and anatomical basis of venom use between distinct groups of venomous insects. • The insects represent an enormous and mostly untapped source of new toxins, including peptides, alkaloids, and others. [ABSTRACT FROM AUTHOR]

Gating modifier toxins (GMTs) are venom-derived peptides isolated from spiders and other venomous creatures and modulate activity of disease-relevant voltage-gated ion channels and are therefore being pursued as therapeutic leads. The amphipathic surface profile of GMTs has prompted the proposal that some GMTs simultaneously bind to the cell membrane and voltage-gated ion channels in a trimolecular complex. Here, we examined whether there is a relationship among spider GMT amphipathicity, membrane binding, and potency or selectivity for voltage-gated sodium (NaV) channels. We used NMR spectroscopy and in silico calculations to examine the structures and physicochemical properties of a panel of nine GMTs and deployed surface plasmon resonance to measure GMT affinity for lipids putatively found in proximity to NaV channels. Electrophysiology was used to quantify GMT activity on NaV1.7, an ion channel linked to chronic pain. Selectivity of the peptides was further examined against a panel of NaV channel subtypes. We show that GMTs adsorb to the outer leaflet of anionic lipid bilayers through electrostatic interactions. We did not observe a direct correlation between GMT amphipathicity and affinity for lipid bilayers. Furthermore, GMT-lipid bilayer interactions did not correlate with potency or selectivity for NaVs. We therefore propose that increased membrane binding is unlikely to improve subtype selectivity and that the conserved amphipathic GMT surface profile is an adaptation that facilitates simultaneous modulation of multiple NaVs. [ABSTRACT FROM AUTHOR]

[Display omitted] Limacodidae is a family of lepidopteran insects comprising >1500 species. More than half of these species produce pain-inducing defensive venoms in the larval stage, but little is known about their venom toxins. Recently, we characterised proteinaceous toxins from the Australian limacodid caterpillar Doratifera vulnerans , but it is unknown if the venom of this species is typical of other Limacodidae. Here, we use single animal transcriptomics and venom proteomics to investigate the venom of an iconic limacodid, the North American saddleback caterpillar Acharia stimulea. We identified 65 venom polypeptides, grouped into 31 different families. Neurohormones, knottins, and homologues of the immune signaller Diedel make up the majority of A. stimulea venom, indicating strong similarities to D. vulnerans venom, despite the large geographic separation of these caterpillars. One notable difference is the presence of RF-amide peptide toxins in A. stimulea venom. Synthetic versions of one of these RF-amide toxins potently activated the human neuropeptide FF1 receptor, displayed insecticidal activity when injected into Drosophila melanogaster , and moderately inhibited larval development of the parasitic nematode Haemonchus contortus. This study provides insights into the evolution and activity of venom toxins in Limacodidae, and provides a platform for future structure-function characterisation of A. stimulea peptide toxins. [ABSTRACT FROM AUTHOR]

Sheep flystrike is caused by parasitic flies laying eggs on soiled wool or open wounds, after which the hatched maggots feed on the sheep flesh and often cause large lesions. It is a significant economic problem for the livestock industry as infestations are difficult to control due to ongoing cycles of larval development into flies followed by further egg laying. We therefore screened venom fractions from the Australian theraphosid spider Coremiocnemis tropix to identify toxins active against the sheep blowfly Lucilia cuprina , which is the primary cause of flystrike in Australia. This screen led to isolation of two insecticidal peptides, Ct1a and Ct1b, that are lethal to blowflies within 24 h of injection. The primary structure of these peptides was determined using a combination of Edman degradation and sequencing of a C. tropix venom-gland transcriptome. Ct1a and Ct1b contain 39 and 38 amino acid residues, respectively, including six cysteine residues that form three disulfide bonds. Recombinant production in bacteria ( Escherichia coli ) resulted in low yields of Ct1a whereas solid-phase peptide synthesis using native chemical ligation produced sufficient quantities of Ct1a for functional analyses. Synthetic Ct1a had no effect on voltage-gated sodium channels from the American cockroach Periplanata americana or the German cockroach Blattella germanica , but it was lethal to sheep blowflies with an LD 50 of 1687 pmol/g. [ABSTRACT FROM AUTHOR]

Background Most ant venoms consist predominantly of small linear peptides, although some contain disulfide-linked peptides as minor components. However, in striking contrast to other ant species, some Anochetus venoms are composed primarily of disulfide-rich peptides. In this study, we investigated the venom of the ant Anochetus emarginatus with the aim of exploring these novel disulfide-rich peptides. Methods The venom peptidome was initially investigated using a combination of reversed-phase HPLC and mass spectrometry, then the amino acid sequences of the major peptides were determined using a combination of Edman degradation and de novo MS/MS sequencing. We focused on one of these peptides, U 1 -PONTX-Ae1a (Ae1a), because of its novel sequence, which we predicted would form a novel 3D fold. Ae1a was chemically synthesized using Fmoc chemistry and its 3D structure was elucidated using NMR spectroscopy. The peptide was then tested for insecticidal activity and its effect on a range of human ion channels. Results Seven peptides named poneritoxins (PONTXs) were isolated and sequenced. The three-dimensional structure of synthetic Ae1a revealed a novel, compact scaffold in which a C-terminal β-hairpin is connected to the N-terminal region via two disulfide bonds. Synthetic Ae1a reversibly paralyzed blowflies and inhibited human L-type voltage-gated calcium channels (Ca V 1). Conclusions Poneritoxins from Anochetus emarginatus venom are a novel class of toxins that are structurally unique among animal venoms. General significance This study demonstrates that Anochetus ant venoms are a rich source of novel ion channel modulating peptides, some of which might be useful leads for the development of biopesticides. [ABSTRACT FROM AUTHOR]

ProTx-II is a disulfide-rich peptide toxin from tarantula venom able to inhibit the human voltage-gated sodium channel 1.7 (hNav1.7), a channel reported to be involved in nociception, and thus it might have potential as a pain therapeutic. ProTx-II acts by binding to the membrane-embedded voltage sensor domain of hNav1.7, but the precise peptide channel-binding site and the importance of membrane binding on the inhibitory activity of ProTx-II remain unknown. In this study, we examined the structure and membrane-binding properties of ProTx-II and several analogues using NMR spectroscopy, surface plasmon resonance, fluorescence spectroscopy, and molecular dynamics simulations. Our results show a direct correlation between ProTx-II membrane binding affinity and its potency as an hNav1.7 channel inhibitor. The data support a model whereby a hydrophobic patch on the ProTx-II surface anchors the molecule at the cell surface in a position that optimizes interaction of the peptide with the binding site on the voltage sensor domain. This is the first study to demonstrate that binding of ProTx-II to the lipid membrane is directly linked to its potency as an hNav1.7 channel inhibitor. [ABSTRACT FROM AUTHOR]

Many venom peptides are potent and selective inhibitors of voltage-gated ion channels, including channels that are validated therapeutic targets for treatment of a wide range of human diseases. However, the development of novel venom-peptide-based therapeutics requires an understanding of their mechanism of action. In the case of voltage-gated ion channels, venom peptides act either as pore blockers that bind to the extracellular side of the channel pore or gating modifiers that bind to one or more of the membrane-embedded voltage sensor domains. In the case of gating modifiers, it has been debated whether the peptide must partition into the membrane to reach its binding site. In this study, we used surface plasmon resonance, fluorescence spectroscopy and molecular dynamics to directly compare the lipid-binding properties of two gating modifiers (μ-TRTX-Hd1a and ProTx-I) and two pore blockers (ShK and KIIIA). Only ProTx-I was found to bind to model membranes. Our results provide further evidence that the ability to insert into the lipid bilayer is not a requirement to be a gating modifier. In addition, we characterised the surface of ProTx-I that mediates its interaction with neutral and anionic phospholipid membranes and show that it preferentially interacts with anionic lipids. [ABSTRACT FROM AUTHOR]

Acid-sensing ion channel 1a (ASIC1a) is the primary acid sensor in mammalian brain and plays a major role in neuronal injury following cerebral ischemia. Evidence that inhibition of ASIC1a might be neuroprotective following stroke was previously obtained using “PcTx1 venom” from the tarantula Psalmopeous cambridgei . We show here that the ASIC1a-selective blocker PcTx1 is present at only 0.4% abundance in this venom, leading to uncertainty as to whether the observed neuroprotective effects were due to PcTx1 blockade of ASIC1a or inhibition of other ion channels and receptors by the hundreds of peptides and small molecules present in the venom. We therefore examined whether pure PcTx1 is neuroprotective in a conscious model of stroke via direct inhibition of ASIC1a. A focal reperfusion model of stroke was induced in conscious spontaneously hypertensive rats (SHR) by administering endothelin-1 to the middle cerebral artery via a surgically implanted cannula. Two hours later, SHR were treated with a single intracerebroventricular (i.c.v.) dose of PcTx1 (1 ng/kg), an ASIC1a-inactive mutant of PcTx1 (1 ng/kg), or saline, and ledged beam and neurological tests were used to assess the severity of symptomatic changes. PcTx1 markedly reduced cortical and striatal infarct volumes measured 72 h post-stroke, which correlated with improvements in neurological score, motor function and preservation of neuronal architecture. In contrast, the inactive PcTx1 analogue had no effect on stroke outcome. This is the first demonstration that selective pharmacological inhibition of ASIC1a is neuroprotective in conscious SHRs, thus validating inhibition of ASIC1a as a potential treatment for stroke. [ABSTRACT FROM AUTHOR]

Summary Arthropod venoms consist primarily of peptide toxins that are injected into their prey with devastating consequences. Venom proteins are thought to be recruited from endogenous body proteins and mutated to yield neofunctionalized toxins with remarkable affinity for specific subtypes of ion channels and receptors. However, the evolutionary history of venom peptides remains poorly understood. Here we show that a neuropeptide hormone has been convergently recruited into the venom of spiders and centipedes and evolved into a highly stable toxin through divergent modification of the ancestral gene. High-resolution structures of representative hormone-derived toxins revealed they possess a unique structure and disulfide framework and that the key structural adaptation in weaponization of the ancestral hormone was loss of a C-terminal α helix, an adaptation that occurred independently in spiders and centipedes. Our results raise a new paradigm for toxin evolution and highlight the value of structural information in providing insight into protein evolution. [ABSTRACT FROM AUTHOR]

[Display omitted] • Cotesia flavipes disrupts cellular and humoral immune response of its host. • Proteotranscriptomics revealed 57 components released by Cotesia flavipes teratocytes. • A major peptide expressed by C. flavipes teratocytes was annotated as viral. • Teratocytes release antimicrobial-like peptides mostly in the final days of parasitism. Parasitoid wasps have evolved sophisticated mechanisms of host regulation that establish a favorable environment for the development of immature parasitoids. While maternal venom and symbiotic virus-like particles are well-known mechanisms of host regulation, another less-studied mechanism is the secretion of host regulation factors by cells called teratocytes, extra-embryonic cells released during parasitoid larval eclosion. Consequently, identification and characterization of teratocyte secretory products has not been reported in detail for any parasitoid wasp. We aimed to analyze teratocyte secretory products released into hemolymph of the larval sugarcane borer Diatraea saccharalis (Fabricius, 1794) (Lepidoptera: Crambidae) by its biological control agent, the koinobiont endoparasitoid wasp Cotesia flavipes Cameron, 1891 (Hymenoptera: Braconidae). Teratocytes were released upon eclosion of parasitoid larvae four days after parasitization (DAP) and increased in number and size until six DAP. Total D. saccharalis hemocyte viability was reduced immediately after parasitization until DAP 2, while total hemocyte count was lower from the third DAP, and phenoloxidase and lysozyme activity were disrupted compared to non-parasitized controls. To examine the secretory products of teratocytes, we generated a teratocyte transcriptome and compared its in silico translated open reading frames to mass spectra obtained from hemolymph from parasitized and unparasitized hosts. This led to the identification of 57 polypeptides secreted by teratocytes, the abundance of which we tracked over 0–10 DAP. Abundant teratocyte products included proteins similar to bracovirus proteins and multiple disulfide-rich peptides. Most teratocyte products accumulated in hemolymph, reaching their highest concentrations immediately before parasitoid pupation. Our results provide insights into host regulation by teratocytes and reveal molecules that may be useful in biotechnology. [ABSTRACT FROM AUTHOR]

Abstract: Spider venoms are replete with peptidic ion channel modulators, often with novel subtype selectivity, making them a rich source of pharmacological tools and drug leads. In a search for subtype-selective blockers of voltage-gated calcium (CaV) channels, we isolated and characterized a novel 39-residue peptide, ω-TRTX-Cc1a (Cc1a), from the venom of the tarantula Citharischius crawshayi (now Pelinobius muticus). Cc1a is 67% identical to the spider toxin ω-TRTX-Hg1a, an inhibitor of CaV2.3 channels. We assembled Cc1a using a combination of Boc solid-phase peptide synthesis and native chemical ligation. Oxidative folding yielded two stable, slowly interconverting isomers. Cc1a preferentially inhibited Ba2+ currents (I Ba) mediated by L-type (CaV1.2 and CaV1.3) CaV channels heterologously expressed in Xenopus oocytes, with half-maximal inhibitory concentration (IC50) values of 825nM and 2.24μM, respectively. In rat dorsal root ganglion neurons, Cc1a inhibited I Ba mediated by high voltage-activated CaV channels but did not affect low voltage-activated T-type CaV channels. Cc1a exhibited weak activity at NaV1.5 and NaV1.7 voltage-gated sodium (NaV) channels stably expressed in mammalian HEK or CHO cells, respectively. Experiments with modified Cc1a peptides, truncated at the N-terminus (ΔG1–E5) or C-terminus (ΔW35–V39), demonstrated that the N- and C-termini are important for voltage-gated ion channel modulation. We conclude that Cc1a represents a novel pharmacological tool for probing the structure and function of L-type CaV channels. [Copyright &y& Elsevier]

Highlights: [•] Octopus venoms contain tachykinin peptides. [•] These peptides are more similar to vertebrate non-venom types than invertebrate. [•] These peptides are active on both invertebrate and vertebrate receptors. [Copyright &y& Elsevier]

Abstract: One of the most potent insecticidal venom peptides described to date is Aps III from the venom of the trapdoor spider Apomastus schlingeri. Aps III is highly neurotoxic to lepidopteran crop pests, making it a promising candidate for bioinsecticide development. However, its disulfide-connectivity, three-dimensional structure, and mode of action have not been determined. Here we show that recombinant Aps III (rAps III) is an atypical knottin peptide; three of the disulfide bridges form a classical inhibitor cystine knot motif while the fourth disulfide acts as a molecular staple that restricts the flexibility of an unusually large β hairpin loop that often houses the pharmacophore in this class of toxins. We demonstrate that the irreversible paralysis induced in insects by rAps III results from a potent block of insect voltage-gated sodium channels. Channel block by rAps III is voltage-independent insofar as it occurs without significant alteration in the voltage-dependence of channel activation or steady-state inactivation. Thus, rAps III appears to be a pore blocker that plugs the outer vestibule of insect voltage-gated sodium channels. This mechanism of action contrasts strikingly with virtually all other sodium channel modulators isolated from spider venoms that act as gating modifiers by interacting with one or more of the four voltage-sensing domains of the channel. [Copyright &y& Elsevier]

Voltage-gated sodium (Na V ) channels play a central role in the propagation of action potentials in excitable cells in both humans and insects. Many venomous animals have therefore evolved toxins that modulate the activity of Na V channels in order to subdue their prey and deter predators. Spider venoms in particular are rich in Na V channel modulators, with one-third of all known ion channel toxins from spider venoms acting on Na V channels. Here we review the landscape of spider-venom peptides that have so far been described to target vertebrate or invertebrate Na V channels. These peptides fall into 12 distinct families based on their primary structure and cysteine scaffold. Some of these peptides have become useful pharmacological tools, while others have potential as therapeutic leads because they target specific Na V channel subtypes that are considered to be important analgesic targets. Spider venoms are conservatively predicted to contain more than 10 million bioactive peptides and so far only 0.01% of this diversity been characterised. Thus, it is likely that future research will reveal additional structural classes of spider-venom peptides that target Na V channels. [ABSTRACT FROM AUTHOR]

Abstract: The insulin-like growth factor binding proteins are a family of six proteins (IGFBP-1 to -6) that bind insulin-like growth factors-I and -II (IGF-I/II) with high affinity. In addition to regulating IGF actions, IGFBPs have IGF-independent functions. IGFBP-2, the largest member of this family, is over-expressed in many cancers and has been proposed as a possible target for the development of novel anti-cancer therapeutics. The IGFBPs have a common architecture consisting of conserved N- and C-terminal domains joined by a variable linker domain. The solution structure and dynamics of the C-terminal domain of human IGFBP-2 have been reported (Kuang Z. et al. J. Mol. Biol. 364, 690–704, 2006) but neither the N-domain (N-BP-2) nor the linker domain have been characterised. Here we present NMR resonance assignments for human N-BP-2, achieved by recording spectra at low protein concentration using non-uniform sampling and maximum entropy reconstruction. Analysis of secondary chemical shifts shows that N-BP-2 possesses a secondary structure similar to that of other IGFBPs. Although aggregation hampered determination of the solution structure for N-BP-2, a homology model was generated based on the high degree of sequence and structure homology exhibited by the IGFBPs. This model was consistent with experimental NMR and SAXS data and displayed some unique features such as a Pro/Ala-rich non-polar insert, which formed a flexible solvent-exposed loop on the surface of the protein opposite to the IGF-binding interface. NMR data indicated that this loop could adopt either of two alternate conformations in solution – an entirely flexible conformation and one containing nascent helical structure. This loop and an adjacent poly-proline sequence may comprise a potential SH3 domain interaction site for binding to other proteins. [Copyright &y& Elsevier]

Abstract: The venom of the European black widow spider Latrodectus tredecimguttatus (Theridiidae) contains several high molecular mass (110–140kDa) neurotoxins that induce neurotransmitter exocytosis. These include a vertebrate-specific α-latrotoxin (α-LTX-Lt1a) responsible for the clinical symptoms of latrodectism and numerous insect-specific latroinsectoxins (LITs). In contrast, little is known about the expression of these toxins in other Latrodectus species despite the fact that envenomation by these spiders induces a similar clinical syndrome. Here we report highly conserved α-LTX, α-LIT and δ-LIT sequence tags in Latrodectus mactans, Latrodectus hesperus and Latrodectus hasselti venoms using tandem mass spectrometry, following bioassay-guided separation of venoms by liquid chromatography. Despite this sequence similarity, we show that the anti-α-LTX monoclonal antibody 4C4.1, raised against α-LTX-Lt1a, fails to neutralize the neurotoxicity of all other Latrodectus venoms tested in an isolated chick biventer cervicis nerve–muscle bioassay. This suggests that there are important structural differences between α-LTXs in theridiid spider venoms. We therefore cloned and sequenced the α-LTX from the Australian red-back spider L. hasselti (α-LTX-Lh1a). The deduced amino acid sequence of the mature α-LTX-Lh1a comprises 1180 residues (∼132kDa) with ∼93% sequence identity with α-LTX-Lt1a. α-LTX-Lh1a is composed of an N-terminal domain and a central region containing 22 ankyrin-like repeats. The presence of two furin cleavage sites, conserved with α-LTX-Lt1a, indicates that α-LTX-Lh1a is derived from the proteolytic cleavage of an N-terminal signal peptide and C-terminal propeptide region. However, we show that α-LTX-Lh1a has key substitutions in the 4C4.1 epitope that explains the lack of binding of the monoclonal antibody. [Copyright &y& Elsevier]

Abstract: Acid sensing ion channels (ASICs) are pH-sensitive channels that are distributed in the central and peripheral nervous system and which are believed to play a key role in pain perception. APETx2, a 42-residue peptide toxin isolated from the sea anemone Anthopleura elegantissima, is the only known selective inhibitor of ASIC3 channels. Here we describe the total chemical synthesis of APETx2 by solid-phase peptide synthesis and native chemical ligation. The folded synthetic toxin had an IC50 of 57nM for inhibition of rat ASIC3 channels expressed in Xenopus oocytes, in agreement with the IC50 reported for the native toxin (63nM). The native chemical ligation approach should provide an efficient route for synthesis of other pharmacologically useful disulfide-rich toxins from venomous animals. [Copyright &y& Elsevier]

Abstract: We screened a panel of theraphosid venoms in two orders of insect in order to determine whether these bioassays would help in the selection of candidate venoms for future discovery of insecticidal toxins. Venoms from six different theraphosid genera were compared with venom from the Australian funnel-web spider Hadronyche infensa (Hexathelidae). The tarantulas included were Coremiocnemis tropix, Selenocosmia crassipes, and Selenotholus foelschei from Australia and Brachypelma albiceps and Brachypelma hamorii from Mexico. The insects assayed, Tenebrio molitor (Coleoptera: Tenebrionidae) and Acheta domesticus (Orthoptera: Gryllidae), were selected because of their relevance as model holometabolous and hemimetabolous insects, respectively, as well as their taxonomic relationship to economically important pest insects. Despite significant differences in their peptide/protein profiles as determined using SDS-PAGE, HPLC, and mass spectrometry, all of the theraphosid venoms exhibited remarkably similar LD50 values of 46–126μg/g for crickets and 0.5–4.0μg/g for mealworms. Notably, mealworms were on average 50-fold more susceptible than crickets to each of the crude theraphosid venoms and consequently they provide an excellent bioassay system when venom supply is limited. This study indicates that even closely related spiders have evolved quite different toxin repertoires that nevertheless have comparable efficiency with respect to killing their primary prey, namely insects. [Copyright &y& Elsevier]

Abstract: The ω-atracotoxins (ω-ACTX) are a family of arthropod-selective peptide neurotoxins from Australian funnel-web spider venoms (Hexathelidae: Atracinae) that are candidates for development as biopesticides. We isolated a 37-residue insect-selective neurotoxin, ω-ACTX-Ar1a, from the venom of the Sydney funnel-web spider Atrax robustus, with high homology to several previously characterized members of the ω-ACTX-1 family. The peptide induced potent excitatory symptoms, followed by flaccid paralysis leading to death, in acute toxicity tests in house crickets. Using isolated smooth and skeletal nerve-muscle preparations, the toxin was shown to lack overt vertebrate toxicity at concentrations up to 1μM. To further characterize the target of the ω-ACTXs, voltage-clamp analysis using the whole-cell patch-clamp technique was undertaken using cockroach dorsal unpaired median neurons. It is shown here for the first time that ω-ACTX-Ar1a, and its homolog ω-ACTX-Hv1a from Hadronyche versuta, reversibly block both mid–low- (M-LVA) and high-voltage-activated (HVA) insect calcium channel (Cav) currents. This block occurred in the absence of alterations in the voltage-dependence of Cav channel activation, and was voltage-independent, suggesting that ω-ACTX-1 family toxins are pore blockers rather than gating modifiers. At a concentration of 1μM ω-ACTX-Ar1a failed to significantly affect global Kv channel currents. However, 1μM ω-ACTX-Ar1a caused a modest 18% block of insect Nav channel currents, similar to the minor block of Nav channels reported for other insect Cav channel blockers such as ω-agatoxin IVA. These findings validate both M-LVA and HVA Cav channels as potential targets for insecticides. [Copyright &y& Elsevier]