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Animals that specialise in blood feeding have particular challenges in obtaining their meal, whereby they impair blood hemostasis by promoting anticoagulation and vasodilation in order to facilitate feeding. These convergent selection pressures have been studied in a number of lineages, ranging from fleas to leeches. However, the vampire bat ( Desmondus rotundus ) is unstudied in regards to potential vasodilatory mechanisms of their feeding secretions (which are a type of venom). This is despite the intense investigations of their anticoagulant properties which have demonstrated that D. rotundus venom contains strong anticoagulant and proteolytic activities which delay the formation of blood clots and interfere with the blood coagulation cascade. In this study, we identified and tested a compound from D. rotundus venom that is similar in size and amino acid sequence to human calcitonin gene-related peptide (CGRP) which has potent vasodilatory properties. We found that the vampire bat-derived form of CGRP (i.e., vCGRP) selectively caused endothelium-independent relaxation of pre-contracted rat small mesenteric arteries. The vasorelaxant efficacy and potency of vCGRP were similar to that of CGRP, in activating CGRP receptors and Kv channels to relax arteriole smooth muscle, which would facilitate blood meal feeding by promoting continual blood flow. Our results provide, for the first time, a detailed investigation into the identification and function of a vasodilatory peptide found in D. rotundus venom, which provides a basis in understanding the convergent pathways and selectivity of hematophagous venoms. These unique peptides also show excellent drug design and development potential, thus highlighting the social and economic value of venomous animals.

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., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Sea anemone venoms have long been recognized as a rich source of peptides with interesting pharmacological and structural properties, but they still contain many uncharacterized bioactive compounds. Here we report the discovery, three-dimensional structure, activity, tissue localization, and putative function of a novel sea anemone peptide toxin that constitutes a new, sixth type of voltage-gated potassium channel (K V ) toxin from sea anemones. Comprised of just 17 residues, κ-actitoxin-Ate1a (Ate1a) is the shortest sea anemone toxin reported to date, and it adopts a novel three-dimensional structure that we have named the Proline-Hinged Asymmetric β-hairpin (PHAB) fold. Mass spectrometry imaging and bioassays suggest that Ate1a serves a primarily predatory function by immobilising prey, and we show this is achieved through inhibition of Shaker-type K V channels. Ate1a is encoded as a multi-domain precursor protein that yields multiple identical mature peptides, which likely evolved by multiple domain duplication events in an actinioidean ancestor. Despite this ancient evolutionary history, the PHAB-encoding gene family exhibits remarkable sequence conservation in the mature peptide domains. We demonstrate that this conservation is likely due to intra-gene concerted evolution, which has to our knowledge not previously been reported for toxin genes. We propose that the concerted evolution of toxin domains provides a hitherto unrecognised way to circumvent the effects of the costly evolutionary arms race considered to drive toxin gene evolution by ensuring efficient secretion of ecologically important predatory toxins.

Cone snails are a diverse group of predatory marine invertebrates that deploy remarkably complex venoms to rapidly paralyse worm, mollusc or fish prey. ω-Conotoxins are neurotoxic peptides from cone snail venoms that inhibit Ca v 2.2 voltage-gated calcium channel, demonstrating potential for pain management via intrathecal (IT) administration. Here, we isolated and characterized two novel ω-conotoxins, MoVIA and MoVIB from Conus moncuri, the first to be identified in vermivorous (worm-hunting) cone snails. MoVIA and MoVIB potently inhibited human Ca v 2.2 in fluorimetric assays and rat Ca v 2.2 in patch clamp studies, and both potently displaced radiolabeled ω-conotoxin GVIA ( 125 I-GVIA) from human SH-SY5Y cells and fish brain membranes (IC 50 2-9 pM). Intriguingly, an arginine at position 13 in MoVIA and MoVIB replaced the functionally critical tyrosine found in piscivorous ω-conotoxins. To investigate its role, we synthesized MoVIB-[R13Y] and MVIIA-[Y13R]. Interestingly, MVIIA-[Y13R] completely lost Ca v 2.2 activity and MoVIB-[R13Y] had reduced activity, indicating that Arg at position 13 was preferred in these vermivorous ω-conotoxins whereas tyrosine 13 is preferred in piscivorous ω-conotoxins. MoVIB reversed pain behavior in a rat neuropathic pain model, confirming that vermivorous cone snails are a new source of analgesic ω-conotoxins. Given vermivorous cone snails are ancestral to piscivorous species, our findings support the repurposing of defensive venom peptides in the evolution of piscivorous Conidae.

Spider venoms are a rich source of insecticidal peptide toxins. Their development as bioinsecticides has, however, been hampered due to concerns about potential lack of stability and oral bioactivity. We therefore systematically evaluated several synthetic strategies to increase the stability and oral potency of the potent insecticidal spider-venom peptide ω-HXTX-Hv1a (Hv1a). Selective chemical replacement of disulfide bridges with diselenide bonds and N- to C-terminal cyclization were anticipated to improve Hv1a resistance to proteolytic digestion, and thereby its activity when delivered orally. We found that native Hv1a is orally active in blowflies, but 91-fold less potent than when administered by injection. Introduction of a single diselenide bond had no effect on the susceptibility to scrambling or the oral activity of Hv1a. N- to C-terminal cyclization of the peptide backbone did not significantly improve the potency of Hv1a when injected into blowflies and it led to a significant decrease in oral activity. We show that this is likely due to a dramatically reduced rate of translocation of cyclic Hv1a across the insect midgut, highlighting the importance of testing bioavailability in addition to toxin stability.

Consistent with their diverse pharmacology, peptides derived from venomous animals have been developed as drugs to treat disorders as diverse as hypertension, diabetes and chronic pain. Melanoma has a poor prognosis due in part to its metastatic capacity, warranting further development of novel targeted therapies. This prompted us to examine the anti-melanoma activity of the spider peptides gomesin (AgGom) and a gomesin-like homolog (HiGom). AgGom and HiGom dose-dependently reduced the viability and proliferation of melanoma cells whereas it had no deleterious effects on non-transformed neonatal foreskin fibroblasts. Concordantly, gomesin-treated melanoma cells showed a reduced G0/G1 cell population. AgGom and HiGom compromised proliferation of melanoma cells via activation of the p53/p21 cell cycle check-point axis and the Hippo signaling cascade, together with attenuation of the MAP kinase pathway. We show that both gomesin peptides exhibit antitumoral activity in melanoma AVATAR-zebrafish xenograft tumors and that HiGom also reduces tumour progression in a melanoma xenograft mouse model. Taken together, our data highlight the potential of gomesin for development as a novel melanoma-targeted therapy.

The Tasmanian devil faces extinction due to devil facial tumour disease (DFTD), a highly transmittable clonal form of cancer without available treatment. In this study, we report the cell-autonomous antiproliferative and cytotoxic activities exhibited by the spider peptide gomesin (AgGom) and gomesin-like homologue (HiGom) in DFTD cells. Mechanistically, both peptides caused a significant reduction at G0/G1 phase, in correlation with an augmented expression of the cell cycle inhibitory proteins p53, p27, p21, necrosis, exacerbated generation of reactive oxygen species and diminished mitochondrial membrane potential, all hallmarks of cellular stress. The screening of a novel panel of AgGom-analogues revealed that, unlike changes in the hydrophobicity and electrostatic surface, the cytotoxic potential of the gomesin analogues in DFTD cells lies on specific arginine substitutions in the eight and nine positions and alanine replacement in three, five and 12 positions. In conclusion, the evidence supports gomesin as a potential antiproliferative compound against DFTD disease., Competing Interests: The authors declare that they have no conflict of interest.

Oxytocin and vasopressin mediate various physiological functions that are important for osmoregulation, reproduction, cardiovascular function, social behavior, memory, and learning through four G protein-coupled receptors that are also implicated in high-profile disorders. Targeting these receptors is challenging because of the difficulty in obtaining ligands that retain selectivity across rodents and humans for translational studies. We identified a selective and more stable oxytocin receptor (OTR) agonist by subtly modifying the pharmacophore framework of human oxytocin and vasopressin. [Se-Se]-oxytocin-OH displayed similar potency to oxytocin but improved selectivity for OTR, an effect that was retained in mice. Centrally infused [Se-Se]-oxytocin-OH potently reversed social fear in mice, confirming that this action was mediated by OTR and not by V1a or V1b vasopressin receptors. In addition, [Se-Se]-oxytocin-OH produced a more regular contraction pattern than did oxytocin in a preclinical labor induction and augmentation model using myometrial strips from cesarean sections. [Se-Se]-oxytocin-OH had no activity in human cardiomyocytes, indicating a potentially improved safety profile and therapeutic window compared to those of clinically used oxytocin. In conclusion, [Se-Se]-oxytocin-OH is a novel probe for validating OTR as a therapeutic target in various biological systems and is a promising new lead for therapeutic development. Our medicinal chemistry approach may also be applicable to other peptidergic signaling systems with similar selectivity issues., (Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Conotoxins are a large family of disulfide-rich peptides that contain unique cysteine frameworks that target a broad range of ion channels and receptors. We recently discovered the 33-residue conotoxin Φ-MiXXVIIA from Conus miles with a novel cysteine framework comprising three consecutive cysteine residues and four disulfide bonds. Regioselective chemical synthesis helped decipher the disulfide bond connectivity and the structure of Φ-MiXXVIIA was determined by NMR spectroscopy. The 3D structure displays a unique topology containing two β-hairpins that resemble the N-terminal domain of granulin. Similar to granulin, Φ-MiXXVIIA promotes cell proliferation (EC 50 17.85 μm) while inhibiting apoptosis (EC 50 2.2 μm). Additional framework XXVII sequences were discovered with homologous signal peptides that define the new conotoxin superfamily G2. The novel structure and biological activity of Φ-MiXXVIIA expands the repertoire of disulfide-rich conotoxins that recognize mammalian receptors., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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 Cav2.2 (IC50 2.6 μM) but not Cav1.3 or Cav3.1 (IC50 > 30 μM) in fluorimetric assays. In patch-clamp electrophysiological assays Cd1a inhibited rat Cav2.2 with similar potency (IC50 3 μM) without influencing the voltage dependence of Cav2.2 activation gating, suggesting that Cd1a doesn't act on Cav2.2 as a classical gating modifier toxin. The Cd1a binding site on Cav2.2 did not overlap with that of the pore blocker ω-conotoxin GVIA, but its activity at Cav2.2-mutant indicated that Cd1a shares some molecular determinants with GVIA and MVIIA, localized near the pore region. Cd1a also inhibited human Nav1.1-1.2 and Nav1.7-1.8 (IC50 0.1-6.9 μM) but not Nav1.3-1.6 (IC50 > 30 μM) in fluorimetric assays. In patch-clamp assays, Cd1a strongly inhibited human Nav1.7 (IC50 16 nM) and produced a 29 mV depolarising shift in Nav1.7 voltage dependence of activation. Cd1a (400 pmol) fully reversed Nav1.7-evoked pain behaviours in mice without producing side effects. In conclusion, Cd1a inhibited two anti-nociceptive targets, appearing to interfere with Cav2.2 inactivation gating, associated with the Cav2.2 α-subunit pore, while altering the activation gating of Nav1.7. Cd1a was inactive at some of the Nav and Cav 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.

Background and Purpose: Naturally occurring dysfunction of voltage-gated sodium (Na V ) channels results in complex disorders such as chronic pain, making these channels an attractive target for new therapies. In the pursuit of novel Na V modulators, we investigated spider venoms for new inhibitors of Na V channels., Experimental Approach: We used high-throughput screens to identify a Na V modulator in venom of the spider Davus fasciatus. Further characterization of this venom peptide was undertaken using fluorescent and electrophysiological assays, molecular modelling and a rodent pain model., Key Results: We identified a potent Na V inhibitor named μ-TRTX-Df1a. This 34-residue peptide fully inhibited responses mediated by Na V 1.7 endogenously expressed in SH-SY5Y cells. Df1a also inhibited voltage-gated calcium (Ca V 3) currents but had no activity against the voltage-gated potassium (K V 2) channel. The modelled structure of Df1a, which contains an inhibitor cystine knot motif, is reminiscent of the Na V channel toxin ProTx-I. Electrophysiology revealed that Df1a inhibits all Na V subtypes tested (hNa V 1.1-1.7). Df1a also slowed fast inactivation of Na V 1.1, Na V 1.3 and Na V 1.5 and modified the voltage-dependence of activation and inactivation of most of the Na V subtypes. Df1a preferentially binds to the domain II voltage-sensor and has additional interactions with the voltage sensors domains III and IV, which probably explains its modulatory features. Df1a was analgesic in vivo, reversing the spontaneous pain behaviours induced by the Na V activator OD1., Conclusion and Implications: μ-TRTX-Df1a shows potential as a new molecule for the development of drugs to treat pain disorders mediated by voltage-gated ion channels., (© 2017 The British Pharmacological Society.)

Δ-Myrtoxin-Mp1a (Mp1a), a 49-residue heterodimeric peptide from the venom of Myrmecia pilosula, comprises a 26-mer A chain and a 23-mer B chain connected by two disulfide bonds in an antiparallel arrangement. Combination of the individual synthetic chains through aerial oxidation remarkably resulted in the self-assembly of Mp1a as a homogenous product without the need for directed disulfide-bond formation. NMR analysis revealed a well-defined, unique structure containing an antiparallel α-helix pair. Dual polarization interferometry (DPI) analysis showed strong interaction with supported lipid bilayers and insertion within the bilayers. Mp1a caused non-specific Ca 2+ influx in SH-SY5Y cells with a half maximal effective concentration (EC 50 ) of 4.3 μm. Mp1a also displayed broad-spectrum antimicrobial activity, with the highest potency against Gram-negative Acinetobacter baumannii (MIC 25 nm). Intraplantar injection (10 μm) in mice elicited spontaneous pain and mechanical allodynia. Single- and two-chain mimetics of Mp1a revealed functional selectivity., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Voltage-gated sodium (Na V ) channels are essential for the transmission of pain signals in humans making them prime targets for the development of new analgesics. Spider venoms are a rich source of peptide modulators useful to study ion channel structure and function. Here we describe β/δ-TRTX-Pre1a, a 35-residue tarantula peptide that selectively interacts with neuronal Na V channels inhibiting peak current of hNa V 1.1, rNa V 1.2, hNa V 1.6, and hNa V 1.7 while concurrently inhibiting fast inactivation of hNa V 1.1 and rNa V 1.3. The DII and DIV S3-S4 loops of Na V channel voltage sensors are important for the interaction of Pre1a with Na V channels but cannot account for its unique subtype selectivity. Through analysis of the binding regions we ascertained that the variability of the S1-S2 loops between Na V channels contributes substantially to the selectivity profile observed for Pre1a, particularly with regards to fast inactivation. A serine residue on the DIV S2 helix was found to be sufficient to explain Pre1a's potent and selective inhibitory effect on the fast inactivation process of Na V 1.1 and 1.3. This work highlights that interactions with both S1-S2 and S3-S4 of Na V channels may be necessary for functional modulation, and that targeting the diverse S1-S2 region within voltage-sensing domains provides an avenue to develop subtype selective tools.

Nicotinic acetylcholine receptors (nAChR) are therapeutic targets for a range of human diseases. α-Conotoxins are naturally occurring peptide antagonists of nAChRs that have been used as pharmacological probes and investigated as drug leads for nAChR related disorders. However, α-conotoxin interactions have been mostly characterised at the α7 and α3β2 nAChRs, with interactions at other subtypes poorly understood. This study provides novel structural insights into the molecular basis for α-conotoxin activity at α3β4 nAChR, a therapeutic target where subtype specific antagonists have potential to treat nicotine addiction and lung cancer. A co-crystal structure of α-conotoxin LsIA with Lymnaea stagnalis acetylcholine binding protein guided the design and functional characterisations of LsIA analogues that identified the minimum pharmacophore regulating α3β4 antagonism. Interactions of the LsIA R10F with β4 K57 and the conserved -NN- α-conotoxin motif with β4 I77 and I109 conferred α3β4 activity to the otherwise inactive LsIA. Using these structural insights, we designed LsIA analogues with α3β4 activity. This new understanding of the structural basis of protein-protein interactions between α-conotoxins and α3β4 may help rationally guide the development of α3β4 selective antagonists with therapeutic potential.

Characterisation of G protein-coupled receptors (GPCR) relies on the availability of a toolbox of ligands that selectively modulate different functional states of the receptors. To uncover such molecules, we explored a unique strategy for ligand discovery that takes advantage of the evolutionary conservation of the 600-million-year-old oxytocin/vasopressin signalling system. We isolated the insect oxytocin/vasopressin orthologue inotocin from the black garden ant (Lasius niger), identified and cloned its cognate receptor and determined its pharmacological properties on the insect and human oxytocin/vasopressin receptors. Subsequently, we identified a functional dichotomy: inotocin activated the insect inotocin and the human vasopressin V 1b receptors, but inhibited the human V 1a R. Replacement of Arg8 of inotocin by D-Arg8 led to a potent, stable and competitive V 1a R-antagonist ([D-Arg8]-inotocin) with a 3,000-fold binding selectivity for the human V 1a R over the other three subtypes, OTR, V 1b R and V 2 R. The Arg8/D-Arg8 ligand-pair was further investigated to gain novel insights into the oxytocin/vasopressin peptide-receptor interaction, which led to the identification of key residues of the receptors that are important for ligand functionality and selectivity. These observations could play an important role for development of oxytocin/vasopressin receptor modulators that would enable clear distinction of the physiological and pathological responses of the individual receptor subtypes., Competing Interests: The authors declare no competing financial interests.

Human genetic studies have implicated the voltage-gated sodium channel Na V 1.7 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 V 1.7 (IC 50 0.9 nM) with at least 40-1000-fold selectivity over all other Na V subtypes. Despite on-target activity in small-diameter dorsal root ganglia, spinal slices, and in a mouse model of pain induced by Na V 1.7 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 V 1.7 inhibitors PF-04856264 and phlotoxin 1. However, when administered with subtherapeutic doses of opioids or the enkephalinase inhibitor thiorphan, these subtype-selective Na V 1.7 inhibitors produced profound analgesia. Our results suggest that in these inflammatory models, acute administration of peripherally restricted Na V 1.7 inhibitors can only produce analgesia when administered in combination with an opioid.

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., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

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, U1-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 (CaV1)., 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., (Crown Copyright © 2016. Published by Elsevier B.V. All rights reserved.)

Millions of years of evolution have fine-tuned the ability of venom peptides to rapidly incapacitate both prey and potential predators. Toxicofera reptiles are characterized by serous-secreting mandibular or maxillary glands with heightened levels of protein expression. These glands are the core anatomical components of the toxicoferan venom system, which exists in myriad points along an evolutionary continuum. Neofunctionalisation of toxins is facilitated by positive selection at functional hotspots on the ancestral protein and venom proteins have undergone dynamic diversification in helodermatid and varanid lizards as well as advanced snakes. A spectacular point on the venom system continuum is the long-glanded blue coral snake ( Calliophis bivirgatus ), a specialist feeder that preys on fast moving, venomous snakes which have both a high likelihood of prey escape but also represent significant danger to the predator itself. The maxillary venom glands of C. bivirgatus extend one quarter of the snake's body length and nestle within the rib cavity. Despite the snake's notoriety its venom has remained largely unstudied. Here we show that the venom uniquely produces spastic paralysis, in contrast to the flaccid paralysis typically produced by neurotoxic snake venoms. The toxin responsible, which we have called calliotoxin (δ-elapitoxin-Cb1a), is a three-finger toxin (3FTx). Calliotoxin shifts the voltage-dependence of Na V 1.4 activation to more hyperpolarised potentials, inhibits inactivation, and produces large ramp currents, consistent with its profound effects on contractile force in an isolated skeletal muscle preparation. Voltage-gated sodium channels (Na V ) are a particularly attractive pharmacological target as they are involved in almost all physiological processes including action potential generation and conduction. Accordingly, venom peptides that interfere with Na V function provide a key defensive and predatory advantage to a range of invertebrate venomous species including cone snails, scorpions, spiders, and anemones. Enhanced activation or delayed inactivation of sodium channels by toxins is associated with the extremely rapid onset of tetanic/excitatory paralysis in envenomed prey animals. A strong selection pressure exists for the evolution of such toxins where there is a high chance of prey escape. However, despite their prevalence in other venomous species, toxins causing delay of sodium channel inhibition have never previously been described in vertebrate venoms. Here we show that Na V modulators, convergent with those of invertebrates, have evolved in the venom of the long-glanded coral snake. Calliotoxin represents a functionally novel class of 3FTx and a structurally novel class of Na V toxins that will provide significant insights into the pharmacology and physiology of Na V . The toxin represents a remarkable case of functional convergence between invertebrate and vertebrate venom systems in response to similar selection pressures. These results underscore the dynamic evolution of the Toxicofera reptile system and reinforces the value of using evolution as a roadmap for biodiscovery., Competing Interests: The authors declare no conflict of interest and the founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

The μO-conotoxins MrVIA, MrVIB, and MfVIA inhibit the voltage-gated sodium channel NaV1.8, a well described target for the treatment of pain; however, little is known about the residues or structural elements that define this activity. In this study, we determined the three-dimensional structure of MfVIA, examined its membrane binding properties, performed alanine-scanning mutagenesis, and identified residues important for its activity at human NaV1.8. A second round of mutations resulted in (E5K,E8K)MfVIA, a double mutant with greater positive surface charge and greater affinity for lipid membranes compared with MfVIA. This analogue had increased potency at NaV1.8 and was analgesic in the mouse formalin assay., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Peptide dendrimers are a novel class of macromolecules of emerging interest with the potential of delayed renal clearance due to their molecular size and enhanced activity due to the multivalency effect. In this work, an active analogue of the disulfide-rich χ-conotoxin χ-MrIA (χ-MrIA), a norepinephrine reuptake (norepinephrine transporter) inhibitor, was grafted onto a polylysine dendron. Dendron decoration was achieved by employing copper-catalyzed alkyne-azide cycloaddition with azido-PEG chain-modified χ-MrIA analogues, leading to homogenous 4-mer and 8-mer χ-MrIA dendrimers with molecular weights ranging from 8 to 22 kDa. These dendrimers were investigated for their impact on peptide secondary structure, in vitro functional activity, and potential anti-allodynia in vivo. NMR studies showed that the χ-MrIA tertiary structure was maintained in the χ-MrIA dendrimers. In a functional norepinephrine transporter reuptake assay, χ-MrIA dendrimers showed slightly increased potency relative to the azido-PEGylated χ-MrIA analogues with similar potency to the parent peptide. In contrast to χ-MrIA, no anti-allodynic action was observed when the χ-MrIA dendrimers were administered intrathecally in a rat model of neuropathic pain, suggesting that the larger dendrimer structures are unable to diffuse through the spinal column tissue and reach the norepinephrine transporter. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd., (Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.)

Peptide-decorated dendrimers (PDDs) are a class of spherical, regular, branched polymers that are modified by peptides covalently attached to their surface. PDDs have been used as protein mimetics, novel biomaterials, and in a wide range of biomedical applications. Since their design and development in the late eighties, poly-l-lysine has been a preferred core structure for PDDs. However, numerous recent innovations in polymer synthesis and ligation chemistry have re-energized the field and led to the emergence of well-defined peptide dendrimers with more diverse core structures and functions. This Minireview highlights the development of PDDs driven by significantly improved ligation chemistry incorporating structurally well-defined peptides and the emerging use of PDDs in imaging and drug development., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Opioid receptor screening of a conopeptide library led to a novel selective κ-opioid agonist peptide (conorphin T). Intensive medicinal chemistry, guided by potency, selectivity, and stability assays generated a pharmacophore model supporting rational design of highly potent and selective κ-opioid receptor (KOR) agonists (conorphins) with exceptional plasma stability. Conorphins are defined by a hydrophobic benzoprolyl moiety, a double arginine sequence, a spacer amino acid followed by a hydrophobic residue and a C-terminal vicinal disulfide moiety. The pharmacophore model was supported by computational docking studies, revealing receptor-ligand interactions similar to KOR agonist dynorphin A (1-8). A conorphin agonist inhibited colonic nociceptors in a mouse tissue model of chronic visceral hypersensitivity, suggesting the potential of KOR agonists for the treatment of chronic abdominal pain. This new conorphine KOR agonist class and pharmacophore model provide opportunities for future rational drug development and probes for exploring the role of the κ-opioid receptor.

Loss-of-function mutations of Na(V)1.7 lead to congenital insensitivity to pain, a rare condition resulting in individuals who are otherwise normal except for the inability to sense pain, making pharmacological inhibition of Na(V)1.7 a promising therapeutic strategy for the treatment of pain. We characterized a novel mouse model of Na(V)1.7-mediated pain based on intraplantar injection of the scorpion toxin OD1, which is suitable for rapid in vivo profiling of Na(V)1.7 inhibitors. Intraplantar injection of OD1 caused spontaneous pain behaviors, which were reversed by co-injection with Na(V)1.7 inhibitors and significantly reduced in Na(V)1.7(-/-) mice. To validate the use of the model for profiling Na(V)1.7 inhibitors, we determined the Na(V) selectivity and tested the efficacy of the reported Na(V)1.7 inhibitors GpTx-1, PF-04856264 and CNV1014802 (raxatrigine). GpTx-1 selectively inhibited Na(V)1.7 and was effective when co-administered with OD1, but lacked efficacy when delivered systemically. PF-04856264 state-dependently and selectively inhibited Na(V)1.7 and significantly reduced OD1-induced spontaneous pain when delivered locally and systemically. CNV1014802 state-dependently, but non-selectively, inhibited Na(V) channels and was only effective in the OD1 model when delivered systemically. Our novel model of Na(V)1.7-mediated pain based on intraplantar injection of OD1 is thus suitable for the rapid in vivo characterization of the analgesic efficacy of Na(V)1.7 inhibitors.

Australian elapid venom remains an under-investigated resource of novel bioactive peptides. In this study, the venom gland transcriptomes and proteomes of the Australian western brown snakes, Pseudonaja aspidorhyncha and Pseudonaja nuchalis, were compared to Pseudonaja textilis. A deep venomics strategy incorporating high throughput 454 pyrosequencing gave a total of 200,911 raw reads for the three venoms. Subsequent annotation identified 5716 transcripts from 20 different toxin families with inter-specific variation between species observed in eight of the less abundant families. Integration of each venom proteome with the corresponding annotated reads identified 65 isoforms from six toxin families; high sequence coverage highlighted subtle differences between sequences and intra and inter-specific variation between species. High quality MS/MS data identified unusual glycoforms with natriuretic peptides from P. aspidorhyncha and P. nuchaliscontaining O-linked trisaccharides with high homology to the glycosylated region of TNPc. Molecular evolutionary assessments indicated the accelerated evolution of all toxin families with the exception of both natriuretic peptides and P. aspidorhyncha PLA2s that were found to be evolutionarily constrained under purifying selection pressures. This study has revealed a wide range of novel peptide sequences from six bioactive peptide families and highlights the subtle differences between toxins in these closely related species., Biological Significance: Mining Australia's vastly untapped source of toxins from its venomous creatures has been significantly advanced by employing deep venomics methodology. Technological advances in transcriptome analysis using next generation sequencing platforms and proteome analysis by highly sensitive tandem mass spectrometry allowed a more comprehensive interrogation of three underinvestigated brown snake (Pseudonaja) venoms uncovering many novel peptide sequences that are unique to these closely related species. This generic strategy will provide invaluable information when applied to other venomous snakes for a deeper understanding of venom composition, envenomation, venom evolution, as well as identifying research tools and drug leads., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Venoms comprise of complex mixtures of peptides evolved for predation and defensive purposes. Remarkably, some carnivorous cone snails can inject two distinct venoms in response to predatory or defensive stimuli, providing a unique opportunity to study separately how different ecological pressures contribute to toxin diversification. Here, we report the extraordinary defensive strategy of the Rhizoconus subgenus of cone snails. The defensive venom from this worm-hunting subgenus is unusually simple, almost exclusively composed of αD-conotoxins instead of the ubiquitous αA-conotoxins found in the more complex defensive venom of mollusc- and fish-hunting cone snails. A similarly compartmentalized venom gland as those observed in the other dietary groups facilitates the deployment of this defensive venom. Transcriptomic analysis of a Conus vexillum venom gland revealed the αD-conotoxins as the major transcripts, with lower amounts of 15 known and four new conotoxin superfamilies also detected with likely roles in prey capture. Our phylogenetic and molecular evolution analysis of the αD-conotoxins from five subgenera of cone snails suggests they evolved episodically as part of a defensive strategy in the Rhizoconus subgenus. Thus, our results demonstrate an important role for defence in the evolution of conotoxins., (© 2015 John Wiley & Sons Ltd.)

Most venomous predators have evolved complex venom primarily to immobilize their prey and secondarily to defend against predators. In a new paradigm, carnivorous marine gastropods of the genus Conus were shown to rapidly and reversibly switch between two types of venoms in response to predatory or defensive stimulus, suggesting that the defensive use of venom may have a more important role in venom evolution and specialization than previously thought. To further investigate this phenomenon, the defensive repertoire of a vermivorous species, Conus planorbis, was deciphered using second-generation sequencing coupled to high-throughput proteomics. The venom gland transcriptome of C. planorbis revealed 182 unique conotoxin precursors from 25 gene superfamilies, with superfamily T dominating in terms of read and paralog numbers. Analysis of the defense-evoked venom revealed that this vermivorous species uses a similarly complex arsenal to deter aggressors as more recently evolved fish- and mollusk-hunting species, with MS/MS validating 23 conotoxin sequences from six superfamilies. Pharmacological characterization of the defensive venom on human receptors identified the nicotinic acetylcholine receptors as a primary target. This work provides the first insights into the composition and biological activity of specifically evolved defensive venoms in vermivorous cone snails., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Selective activation of peripheral κ opioid receptors (KORs) may overcome the dose-limiting adverse effects of conventional opioid analgesics. We recently developed a vicinal disulfide-stabilized class of peptides with subnanomolar potency at the KOR. The aim of this study was to assess the analgesic effects of one of these peptides, named conorphin-1, in comparison with the prototypical KOR-selective small molecule agonist U-50488, in several rodent pain models. Surprisingly, neither conorphin-1 nor U-50488 were analgesic when delivered peripherally by intraplantar injection at local concentrations expected to fully activate the KOR at cutaneous nerve endings. While U-50488 was analgesic when delivered at high local concentrations, this effect could not be reversed by coadministration with the selective KOR antagonist ML190 or the nonselective opioid antagonist naloxone. Instead, U-50488 likely mediated its peripheral analgesic effect through nonselective inhibition of voltage-gated sodium channels, including peripheral sensory neuron isoforms NaV1.8 and NaV1.7. Our study suggests that targeting the KOR in peripheral sensory nerve endings innervating the skin is not an alternative analgesic approach.

Venomous marine cone snails produce a unique and remarkably diverse range of venom peptides (conotoxins and conopeptides) that have proven to be invaluable as pharmacological probes and leads to new therapies. Conus catus is a hook-and-line fish hunter from clade I, with ∼20 conotoxins identified, including the analgesic ω-conotoxin CVID (AM336). The current study unravels the venom composition of C. catus with tandem mass spectrometry and 454 sequencing data. From the venom gland transcriptome, 104 precursors were recovered from 11 superfamilies, with superfamily A (especially κA-) conotoxins dominating (77%) their venom. Proteomic analysis confirmed that κA-conotoxins dominated the predation-evoked milked venom of each of six C. catus analyzed and revealed remarkable intraspecific variation in both the intensity and type of conotoxins. High-throughput FLIPR assays revealed that the predation-evoked venom contained a range of conotoxins targeting the nAChR, Cav, and Nav ion channels, consistent with α- and ω-conotoxins being used for predation by C. catus. However, the κA-conotoxins did not act at these targets but induced potent and rapid immobilization followed by bursts of activity and finally paralysis when injected intramuscularly in zebrafish. Our venomics approach revealed the complexity of the envenomation strategy used by C. catus, which contains a mix of both excitatory and inhibitory venom peptides.

Spider venoms are a rich source of ion channel modulators with therapeutic potential. Given the analgesic potential of subtype-selective inhibitors of voltage-gated sodium (NaV) channels, we screened spider venoms for inhibitors of human NaV1.7 (hNaV1.7) using a high-throughput fluorescent assay. Here, we describe the discovery of a novel NaV1.7 inhibitor, μ-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 hNaV1.7 > hNaV1.6 > hNaV1.2 > hNaV1.1 > hNaV1.3 channels in fluorescent assays. NaV1.7 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 μM. Unlike most spider toxins that modulate NaV channels, Tp1a inhibited hNaV1.7 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 hNaV1.7. 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 hNaV1.7 inhibitors for treatment of chronic pain., (Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.)

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., (© 2015 The Author(s) Published by the Royal Society. All rights reserved.)

Cone snails are predatory marine gastropods characterized by a sophisticated venom apparatus responsible for the biosynthesis and delivery of complex mixtures of cysteine-rich toxin peptides. These conotoxins fold into small highly structured frameworks, allowing them to potently and selectively interact with heterologous ion channels and receptors. Approximately 2,000 toxins from an estimated number of >70,000 bioactive peptides have been identified in the genus Conus to date. Here, we describe a high-resolution interrogation of the transcriptomes (available at www.ddbj.nig.ac.jp) and proteomes of the diverse compartments of the Conus episcopatus venom apparatus. Using biochemical and bioinformatic tools, we found the highest number of conopeptides yet discovered in a single Conus specimen, with 3,305 novel precursor toxin sequences classified into 9 known superfamilies (A, I1, I2, M, O1, O2, S, T, Z), and identified 16 new superfamilies showing unique signal peptide signatures. We were also able to depict the largest population of venom peptides containing the pharmacologically active C-C-CC-C-C inhibitor cystine knot and CC-C-C motifs (168 and 44 toxins, respectively), as well as 208 new conotoxins displaying odd numbers of cysteine residues derived from known conotoxin motifs. Importantly, six novel cysteine-rich frameworks were revealed which may have novel pharmacology. Finally, analyses of codon usage bias and RNA-editing processes of the conotoxin transcripts demonstrate a specific conservation of the cysteine skeleton at the nucleic acid level and provide new insights about the origin of sequence hypervariablity in mature toxin regions.

Cnidarians are the oldest extant lineage of venomous animals. Despite their simple anatomy, they are capable of subduing or repelling prey and predator species that are far more complex and recently evolved. Utilizing specialized penetrating nematocysts, cnidarians inject the nematocyst content or "venom" that initiates toxic and immunological reactions in the envenomated organism. These venoms contain enzymes, potent pore forming toxins, and neurotoxins. Enzymes include lipolytic and proteolytic proteins that catabolize prey tissues. Cnidarian pore forming toxins self-assemble to form robust membrane pores that can cause cell death via osmotic lysis. Neurotoxins exhibit rapid ion channel specific activities. In addition, certain cnidarian venoms contain or induce the release of host vasodilatory biogenic amines such as serotonin, histamine, bunodosine and caissarone accelerating the pathogenic effects of other venom enzymes and porins. The cnidarian attacking/defending mechanism is fast and efficient, and massive envenomation of humans may result in death, in some cases within a few minutes to an hour after sting. The complexity of venom components represents a unique therapeutic challenge and probably reflects the ancient evolutionary history of the cnidarian venom system. Thus, they are invaluable as a therapeutic target for sting treatment or as lead compounds for drug design.

Despite Cnidaria (sea anemones, corals, jellyfish, and hydroids) being the oldest venomous animal lineage, structure-function relationships, phyletic distributions, and the molecular evolutionary regimes of toxins encoded by these intriguing animals are poorly understood. Hence, we have comprehensively elucidated the phylogenetic and molecular evolutionary histories of pharmacologically characterized cnidarian toxin families, including peptide neurotoxins (voltage-gated Na(+) and K(+) channel-targeting toxins: NaTxs and KTxs, respectively), pore-forming toxins (actinoporins, aerolysin-related toxins, and jellyfish toxins), and the newly discovered small cysteine-rich peptides (SCRiPs). We show that despite long evolutionary histories, most cnidarian toxins remain conserved under the strong influence of negative selection-a finding that is in striking contrast to the rapid evolution of toxin families in evolutionarily younger lineages, such as cone snails and advanced snakes. In contrast to the previous suggestions that implicated SCRiPs in the biomineralization process in corals, we demonstrate that they are potent neurotoxins that are likely involved in the envenoming function, and thus represent the first family of neurotoxins from corals. We also demonstrate the common evolutionary origin of type III KTxs and NaTxs in sea anemones. We show that type III KTxs have evolved from NaTxs under the regime of positive selection, and likely represent a unique evolutionary innovation of the Actinioidea lineage. We report a correlation between the accumulation of episodically adaptive sites and the emergence of novel pharmacological activities in this rapidly evolving neurotoxic clade., (© The Author 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Venom as a form of chemical prey capture is a key innovation that has underpinned the explosive radiation of the advanced snakes (Caenophidia). Small venom proteins are often rich in disulfide bonds thus facilitating stable molecular scaffolds that present key functional residues on the protein surface. New toxin types are initially developed through the venom gland over-expression of normal body proteins, their subsequent gene duplication and diversification that leads to neofunctionalisation as random mutations modify their structure and function. This process has led to preferentially selected (privileged) cysteine-rich scaffolds that enable the snake to build arrays of toxins many of which may lead to therapeutic products and research tools. This review focuses on cysteine-rich small proteins and peptides found in snake venoms spanning natriuretic peptides to phospholipase enzymes, while highlighting their three-dimensional structures and biological functions as well as their potential as therapeutic agents or research tools.

Natriuretic peptides (NP) play important roles in human cardiac physiology through their guanylyl cyclase receptors NPR-A and NPR-B. Described herein is a bifunctional O-glycosylated natriuretic peptide, TcNPa, from Tropidechis carinatus venom and it unusually targets both NPR-A and NPR-B. Characterization using specific glycosidases and ETD-MS identified the glycan as galactosyl-β(1-3)-N-acetylgalactosamine (Gal-GalNAc) and was α-linked to the C-terminal threonine residue. TcNPa contains the characteristic NP 17-membered disulfide ring with conserved phenylalanine and arginine residues. Both glycosylated and nonglycosylated forms were synthesized by Fmoc solid-phase peptide synthesis and NMR analysis identified an α-helix within the disulfide ring containing the putative pharmacophore for NPR-A. Surprisingly, both forms activated NPR-A and NPR-B and were relatively resistant towards proteolytic degradation in plasma. This work will underpin the future development of bifunctional NP peptide mimetics., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

MrIC is a recently described selective agonist of endogenously expressed α7 nAChR. In this study, we further characterize the pharmacological activity of MrIC using Ca(2+) imaging approaches in SH-SY5Y cells endogenously expressing α7 nAChR and demonstrate that MrIC exclusively activates α7 nAChR modulated by type II positive allosteric modulators, including PNU120596. MrIC was a full agonist at PNU120596-modulated α7 nAChR compared with choline, albeit with slower kinetics, but failed to elicit a Ca(2+) response in the absence of PNU120596. Interestingly, the NMR structure of MrIC showed a typical 4/7 α-conotoxin fold, indicating that its unusual pharmacological activity is likely sequence-dependent. Overall, our results suggest that MrIC acts as a biased agonist that can only activate α7 nAChR modified by type II positive allosteric modulators, and thus represents a valuable tool to probe the pharmacological properties of this important ion channel., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Covalently attached peptide dendrimers can enhance binding affinity and functional activity. Homogenous di- and tetravalent dendrimers incorporating the α7-nicotinic receptor blocker α-conotoxin ImI (α-ImI) with polyethylene glycol spacers were designed and synthesized via a copper-catalyzed azide-alkyne cycloaddition of azide-modified α-ImI to an alkyne-modified polylysine dendron. NMR and CD structural analysis confirmed that each α-ImI moiety in the dendrimers had the same 3D structure as native α-ImI. The binding of the α-ImI dendrimers to binding protein Ac-AChBP was measured by surface plasmon resonance and revealed enhanced affinity. Quantitative electrophysiology showed that α-ImI dendrimers had ∼100-fold enhanced potency at hα7 nAChRs (IC50 = 4 nM) compared to native α-ImI (IC50 = 440 nM). In contrast, no significant potency enhancement was observed at heteromeric hα3β2 and hα9α10 nAChRs. These findings indicate that multimeric ligands can significantly enhance conotoxin potency and selectivity at homomeric nicotinic ion channels.

Changes in ion channel function and expression are characteristic of neuropathic pain. Voltage-gated calcium channels (VGCCs) are integral for neurotransmission and membrane excitability, but relatively little is known about changes in their expression after nerve injury. In this study, we investigate whether peripheral nerve ligation is followed by changes in the density and proportion of high-voltage-activated (HVA) VGCC current subtypes in dorsal root ganglion (DRG) neurons, the contribution of presynaptic N-type calcium channels in evoked excitatory postsynaptic currents (EPSCs) recorded from dorsal horn neurons in the spinal cord, and the changes in expression of mRNA encoding VGCC subunits in DRG neurons. Using C57BL/6 mice [8- to 11-wk-old males (n = 91)] for partial sciatic nerve ligation or sham surgery, we performed whole cell patch-clamp recordings on isolated DRG neurons and dorsal horn neurons and measured the expression of all VGCC subunits with RT-PCR in DRG neurons. After nerve injury, the density of P/Q-type current was reduced overall in DRG neurons. There was an increase in the percentage of N-type and a decrease in that of P/Q-type current in medium- to large-diameter neurons. No changes were found in the contribution of presynaptic N-type calcium channels in evoked EPSCs recorded from dorsal horn neurons. The α2δ-1 subunit was upregulated by 1.7-fold and γ-3, γ-2, and β-4 subunits were all downregulated 1.7-fold in injured neurons compared with sham-operated neurons. This comprehensive characterization of HVA VGCC subtypes in mouse DRG neurons after nerve injury revealed changes in N- and P/Q-type current proportions only in medium- to large-diameter neurons., (Copyright © 2015 the American Physiological Society.)

The design of disulfide bond mimetics is an important strategy for optimising cysteine-rich peptides in drug development. Mimetics of the drug lead conotoxin MrIA, in which one disulfide bond is selectively replaced of by a 1,4-disubstituted-1,2,3-triazole bridge, are described. Sequential copper-catalyzed azide-alkyne cycloaddition (CuAAC; click reaction) followed by disulfide formation resulted in the regioselective syntheses of triazole-disulfide hybrid MrIA analogues. Mimetics with a triazole replacing the Cys4-Cys13 disulfide bond retained tertiary structure and full in vitro and in vivo activity as norepinephrine reuptake inhibitors. Importantly, these mimetics are resistant to reduction in the presence of glutathione, thus resulting in improved plasma stability and increased suitability for drug development., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Conus geographus is the most dangerous cone snail species known, with reported human fatality rates as high as 65%. Crude venom gland extracts have been used to determine animal LD50 and to aid the isolation of several potent paralytic toxins. However, not only is the composition of injected venoms known to differ significantly from that in dissected venom glands, but also to vary according to predatory or defensive stimuli. Therefore, to study the venom that is directly relevant to human envenomation, the defense-evoked venom of several specimens of C. geographus was collected and analyzed by standard LC-MS methods. The molecular composition of individual defense-evoked venom showed significant intraspecific variations, but a core of paralytic conotoxins including α-GI, α-GII, μ-GIIIA, ω-GVIA and ω-GVIIA was always present in large amounts, consistent with the symptomology and high fatality rate in humans. Differences between injected and dissected venoms obtained from the same specimen were also evident. Interestingly, an apparent linear correlation between the dry weight/volume of injected venom and the size of the shell allowed extrapolation to a human lethal dose (0.038-0.029 mg/kg) from an historic fatal case of C. geographus envenomation, which may help in the management of future victims., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The sea anemone peptide APETx2 is a potent and selective blocker of acid-sensing ion channel 3 (ASIC3). APETx2 is analgesic in a variety of rodent pain models, but the lack of knowledge of its pharmacophore and binding site on ASIC3 has impeded development of improved analogues. Here we present a detailed structure-activity relationship study of APETx2. Determination of a high-resolution structure of APETx2 combined with scanning mutagenesis revealed a cluster of aromatic and basic residues that mediate its interaction with ASIC3. We show that APETx2 also inhibits the off-target hERG channel by reducing the maximal current amplitude and shifting the voltage dependence of activation to more positive potentials. Electrophysiological screening of selected APETx2 mutants revealed partial overlap between the surfaces on APETx2 that mediate its interaction with ASIC3 and hERG. Characterization of the molecular basis of these interactions is an important first step toward the rational design of more selective APETx2 analogues.

In the past 100 years minimal venom research has focused on ticks despite several species possessing a highly paralytic and lethal venom cocktail of proteinaceous molecules. The saliva of the Australian paralysis tick, Ixodes holocyclus, has been responsible for 20 human fatalities from 1900 to 1945, and up to 100,000 domestic animal fatalities annually. In the last 50 years, research on this tick has focused on identifying the neurotoxins present in the saliva and in the last ten years the sequence of a potential neurotoxin, HT-1, has been determined. In this study we chemically synthesised HT-1 using Boc-chemistry in combination with native chemical ligation. Following successful oxidative folding, we determined the three-dimensional structure of HT-1 by NMR spectroscopy and found a novel structural fold with three of the four disulfide bonds comprising the inhibitory cystine knot (ICK) motif. The fourth disulfide bond connects the second loop to the N-terminal, which decreases the flexibility of the structure., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Neuronal nicotinic acetylcholine receptors (nAChRs) are a diverse class of ligand-gated ion channels involved in neurological conditions such as neuropathic pain and Alzheimer's disease. α-Conotoxin [A10L]PnIA is a potent and selective antagonist of the mammalian α7 nAChR with a key binding interaction at position 10. We now describe a molecular analysis of the receptor-ligand interactions that determine the role of position 10 in determining potency and selectivity for the α7 and α3β2 nAChR subtypes. Using electrophysiological and radioligand binding methods on a suite of [A10L]PnIA analogs we observed that hydrophobic residues in position 10 maintained potency at both subtypes whereas charged or polar residues abolished α7 binding. Molecular docking revealed dominant hydrophobic interactions with several α7 and α3β2 receptor residues via a hydrophobic funnel. Incorporation of norleucine (Nle) caused the largest (8-fold) increase in affinity for the α7 subtype (Ki=44nM) though selectivity reverted to α3β2 (IC50=0.7nM). It appears that the placement of a single methyl group determines selectivity between α7 and α3β2 nAChRs via different molecular determinants., (Crown Copyright © 2014. Published by Elsevier Inc. All rights reserved.)

Peptide neurotoxins from cone snails called conotoxins are renowned for their therapeutic potential to treat pain and several neurodegenerative diseases. Inefficient assay-guided discovery methods have been replaced by high-throughput bioassays integrated with advanced MS and next-generation sequencing, ushering in the era of 'venomics'. In this review, we focus on the impact of venomics on the understanding of cone snail biology as well as the application of venomics to accelerate the discovery of new conotoxins. We also discuss the continued importance of medicinal chemistry approaches to optimize conotoxins for clinical use, with a descriptive case study of MrIA featured.

Background: Peripheral neuropathy is the major dose-limiting side effect of cisplatin and oxaliplatin, and there are currently no effective treatments available. The aim of this study was to assess the pharmacological mechanisms underlying chemotherapy-induced neuropathy in novel animal models based on intraplantar administration of cisplatin and oxaliplatin and to systematically evaluate the analgesic efficacy of a range of therapeutics., Methods: Neuropathy was induced by a single intraplantar injection of cisplatin or oxaliplatin in C57BL/6J mice and assessed by quantification of mechanical and thermal allodynia. The pharmacological basis of cisplatin-induced neuropathy was characterized using a range of selective pharmacological inhibitors. The analgesic effects of phenytoin, amitriptyline, oxcarbazepine, mexiletine, topiramate, retigabine, gabapentin, fentanyl, and Ca(2+/)Mg(2+) were assessed 24 hours after induction of neuropathy., Results: Intraplantar administration of cisplatin led to the development of mechanical allodynia, mediated through Nav1.6-expressing sensory neurons. Unlike intraplantar injection of oxaliplatin, cold allodynia was not observed with cisplatin, consistent with clinical observations. Surprisingly, only fentanyl was effective at alleviating cisplatin-induced mechanical allodynia despite a lack of efficacy in oxaliplatin-induced cold allodynia. Conversely, lamotrigine, phenytoin, retigabine, and gabapentin were effective at reversing oxaliplatin-induced cold allodynia but had no effect on cisplatin-induced mechanical allodynia. Oxcarbazepine, amitriptyline, mexiletine, and topiramate lacked efficacy in both models of acute chemotherapy-induced neuropathy., Conclusion: This study established a novel animal model of cisplatin-induced mechanical allodynia consistent with the A-fiber neuropathy seen clinically. Systematic assessment of a range of therapeutics identified several candidates that warrant further clinical investigation., (© The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background and Purpose: ω-Conotoxins CVIE and CVIF (CVIE&F) selectively inhibit Cav2.2 channels and are lead molecules in the development of novel analgesics. At physiological membrane potentials, CVIE&F block of Cav2.2 channels is weakly reversible. To improve reversibility, we designed and synthesized arginine CVIE&F analogues in which arginine was substituted for lysine at position 10 ([R10K]CVIE&F), and investigated their serum stability and pharmacological actions on voltage-gated calcium channels (VGCCs)., Experimental Approach: Changes in peptide structure due to R10K substitution were assessed by NMR. Peptide stability in human serum was analysed by reversed-phase HPLC and MS over a 24 h period. Two-electrode voltage-clamp and whole-cell patch clamp techniques were used to study [R10K]CVIE&F effects on VGCC currents in Xenopus oocytes and rat dorsal root ganglion neurons respectively., Key Results: R10K substitution did not change the conserved ω-conotoxin backbone conformations of CVIE&F nor the ω-conotoxin selectivity for recombinant or native Cav2.2 channels, although the inhibitory potency of [R10K]CVIF was better than that of CVIF. At -80 mV, the R10K chemical modification significantly affected ω-conotoxin-channel interaction, resulting in faster onset kinetics than those of CVIE&F. Heterologous and native Cav2.2 channels recovered better from [R10K]CVIE&F block than CVIE&F. In human serum, the ω-conotoxin half-lives were 6-10 h. CVIE&F and [R10K]CVIE&F were more stable than CVID., Conclusions and Implications: R10K substitution in CVIE&F significantly alters the kinetics of ω-conotoxin action and improves reversibility without diminishing conotoxin potency and specificity for the Cav2.2 channel and without diminishing the serum stability. These results may help generate ω-conotoxins with optimized kinetic profiles for target binding., (© 2014 The British Pharmacological Society.)