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Venom peptides have evolved to target a wide range of membrane proteins through diverse mechanisms of action and structures, providing promising therapeutic leads for diseases, including pain, epilepsy, and cancer, as well as unique probes of ion channel structure-function. In this work, a high-throughput FLIPR window current screening assay on T-type Ca V 3.2 guided the isolation of a novel peptide named ω-Buthitoxin-Hf1a from scorpion Hottentotta franzwerneri crude venom. At only 10 amino acid residues with one disulfide bond, it is not only the smallest venom peptide known to target T-type Ca V s but also the smallest structured scorpion venom peptide yet discovered. Synthetic Hf1a peptides were prepared with C-terminal amidation (Hf1a-NH 2 ) or a free C-terminus (Hf1a-OH). Electrophysiological characterization revealed Hf1a-NH 2 to be a concentration-dependent partial inhibitor of Ca V 3.2 (IC 50 = 1.18 μM) and Ca V 3.3 (IC 50 = 0.49 μM) depolarized currents but was ineffective at Ca V 3.1. Hf1a-OH did not show activity against any of the three T-type subtypes. Additionally, neither form showed activity against N-type Ca V 2.2 or L-type calcium channels. The three-dimensional structure of Hf1a-NH 2 was determined using NMR spectroscopy and used in docking studies to predict its binding site at Ca V 3.2 and Ca V 3.3. As both Ca V 3.2 and Ca V 3.3 have been implicated in peripheral pain signaling, the analgesic potential of Hf1a-NH 2 was explored in vivo in a mouse model of incision-induced acute post-surgical pain. Consistent with this role, Hf1a-NH 2 produced antiallodynia in both mechanical and thermal pain.

Inhibition of T-type calcium channels (Ca V 3) prevents development of diseases related to cardiovascular and nerve systems. Further, knockout animal studies have revealed that some diseases are mediated by specific subtypes of Ca V 3. However, subtype-specific Ca V 3 inhibitors for therapeutic purposes or for studying the physiological roles of Ca V 3 subtypes are missing. To bridge this gap, we employed our spider venom library and uncovered that Avicularia spec. ("Amazonas Purple", Peru) tarantula venom inhibited specific T-type Ca V channel subtypes. By using chromatographic and mass-spectrometric techniques, we isolated and sequenced the active toxin ω-Avsp1a, a C-terminally amidated 36 residue peptide with a molecular weight of 4224.91 Da, which comprised the major peak in the venom. Both native (4.1 μM) and synthetic ω-Avsp1a (10 μM) inhibited 90% of Ca V 3.1 and Ca V 3.3, but only 25% of Ca V 3.2 currents. In order to investigate the toxin binding site, we generated a range of chimeric channels from the less sensitive Ca V 3.2 and more sensitive Ca V 3.3. Our results suggest that domain-1 of Ca V 3.3 is important for the inhibitory effect of ω-Avsp1a on T-type calcium channels. Further studies revealed that a leucine of T-type calcium channels is crucial for the inhibitory effect of ω-Avsp1a.

α-Conotoxins that target muscle nicotinic acetylcholine receptors (nAChRs) commonly fall into two structural classes, frameworks I and II containing two and three disulfide bonds, respectively. Conotoxin SII is the sole member of the cysteine-rich framework II with ill-defined interactions at the nAChRs. Following directed synthesis of α-SII, NMR analysis revealed a well-defined structure containing a 3 10 -helix frequently employed by framework I α-conotoxins; α-SII acted at the muscle nAChR with half-maximal inhibitory concentrations (IC 50 ) of 120 nM (adult) and 370 nM (fetal) though weakly at neuronal nAChRs. Truncation of α-SII to a two disulfide bond amidated peptide with framework I disulfide connectivity led to similar activity. Surprisingly, the more constrained α-SII was less stable under mild reducing conditions and displayed a unique docking mode at the nAChR.

The King Baboon spider, Pelinobius muticus , is a burrowing African tarantula. Its impressive size and appealing coloration are tempered by reports describing severe localized pain, swelling, itchiness, and muscle cramping after accidental envenomation. Hyperalgesia is the most prominent symptom after bites from P. muticus , but the molecular basis by which the venom induces pain is unknown. Proteotranscriptomic analysis of P. muticus venom uncovered a cysteine-rich peptide, δ/κ-theraphotoxin-Pm1a (δ/κ-TRTX-Pm1a), that elicited nocifensive behavior when injected into mice. In small dorsal root ganglion neurons, synthetic δ/κ-TRTX-Pm1a (sPm1a) induced hyperexcitability by enhancing tetrodotoxin-resistant sodium currents, impairing repolarization and lowering the threshold of action potential firing, consistent with the severe pain associated with envenomation. The molecular mechanism of nociceptor sensitization by sPm1a involves multimodal actions over several ion channel targets, including Na V 1.8, K V 2.1, and tetrodotoxin-sensitive Na V channels. The promiscuous targeting of peptides like δ/κ-TRTX-Pm1a may be an evolutionary adaptation in pain-inducing defensive venoms., Competing Interests: The authors declare no competing interest., (Copyright © 2022 the Author(s). Published by PNAS.)

Venoms from cone snails and arachnids are a rich source of peptide modulators of voltage-gated sodium (Na V ) channels, however relatively few venom-derived peptides with activity at the mammalian Na V 1.8 subtype have been isolated. Here, we describe the discovery and functional characterisation of β-theraphotoxin-Eo1a, a peptide from the venom of the Tanzanian black and olive baboon tarantula Encyocratella olivacea that modulates Na V 1.8. Eo1a is a 37-residue peptide that increases Na V 1.8 peak current (EC 50 894 ± 146 nM) and causes a large hyperpolarising shift in both the voltage-dependence of activation (ΔV 50 -20.5 ± 1.2 mV) and steady-state fast inactivation (ΔV 50 -15.5 ± 1.8 mV). At a concentration of 10 μM, Eo1a has varying effects on the peak current and channel gating of Na V 1.1-Na V 1.7, although its activity is most pronounced at Na V 1.8. Investigations into the binding site of Eo1a using Na V 1.7/Na V 1.8 chimeras revealed a critical contribution of the DII S3-S4 extracellular loop of Na V 1.8 to toxin activity. Results from this work may form the basis for future studies that lead to the rational design of spider venom-derived peptides with improved potency and selectivity at Na V 1.8., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Deuis, Ragnarsson, Robinson, Dekan, Chan, Jin, Tran, McMahon, Li, Wood, Cox, King, Herzig and Vetter.)

The olfactory system is a driver of feeding behavior, whereby olfactory acuity is modulated by the metabolic state of the individual. The excitability of the major output neurons of the olfactory bulb (OB) can be modulated through targeting a voltage-dependent potassium channel, Kv1.3, which responds to changes in metabolic factors such as insulin, glucose, and glucagon-like peptide-1. Because gene-targeted deletion or inhibition of Kv1.3 in the periphery has been found to increase energy metabolism and decrease body weight, we hypothesized that inhibition of Kv1.3 selectively in the OB could enhance excitability of the output neurons to evoke changes in energy homeostasis. We thereby employed metal-histidine coordination to self-assemble the Kv1.3 inhibitor margatoxin (MgTx) to fluorescent quantum dots (QDMgTx) as a means to label cells in vivo and test changes in neuronal excitability and metabolism when delivered to the OB. Using patch-clamp electrophysiology to measure Kv1.3 properties in heterologously expressed cells and native mitral cells in OB slices, we found that QDMgTx had a fast rate of inhibition, but with a reduced IC 50, and increased action potential firing frequency. QDMgTx was capable of labeling cloned Kv1.3 channels but was not visible when delivered to native Kv1.3 in the OB. Diet-induced obese mice were observed to reduce body weight and clear glucose more quickly following osmotic mini-pump delivery of QDMgTx/MgTx to the OB, and following MgTx delivery, they increased the use of fats as fuels (reduced respiratory exchange ratio). These results suggest that enhanced excitability of bulbar output neurons can drive metabolic responses., (© 2020 International Society for Neurochemistry.)

Venoms have evolved independently several times in Lepidoptera. Limacodidae is a family with worldwide distribution, many of which are venomous in the larval stage, but the composition and mode of action of their venom is unknown. Here, we use imaging technologies, transcriptomics, proteomics, and functional assays to provide a holistic picture of the venom system of a limacodid caterpillar, Doratifera vulnerans Contrary to dogma that defensive venoms are simple in composition, D. vulnerans produces a complex venom containing 151 proteinaceous toxins spanning 59 families, most of which are peptides <10 kDa. Three of the most abundant families of venom peptides (vulnericins) are 1) analogs of the adipokinetic hormone/corazonin-related neuropeptide, some of which are picomolar agonists of the endogenous insect receptor; 2) linear cationic peptides derived from cecropin, an insect innate immune peptide that kills bacteria and parasites by disrupting cell membranes; and 3) disulfide-rich knottins similar to those that dominate spider venoms. Using venom fractionation and a suite of synthetic venom peptides, we demonstrate that the cecropin-like peptides are responsible for the dominant pain effect observed in mammalian in vitro and in vivo nociception assays and therefore are likely to cause pain after natural envenomations by D. vulnerans Our data reveal convergent molecular evolution between limacodids, hymenopterans, and arachnids and demonstrate that lepidopteran venoms are an untapped source of novel bioactive peptides., Competing Interests: The authors declare no competing interest.

Vasopressin (VP) and oxytocin (OT) are cyclic neuropeptides that regulate fundamental physiological functions via four G protein-coupled receptors, V 1a R, V 1b R, V 2 R, and OTR. Ligand development remains challenging for these receptors due to complex structure-activity relationships. Here, we investigated dimerization as a strategy for developing ligands with novel pharmacology. We regioselectively synthesised and systematically studied parallel, antiparallel and N- to C-terminal cyclized homo- and heterodimer constructs of VP, OT and dVDAVP (1-deamino-4-valine-8-d-arginine-VP). All disulfide-linked dimers, except for the head-to-tail cyclized constructs, retained nanomolar potency despite the structural implications of dimerization. Our results support a single chain interaction for receptor activation. Dimer orientation had little impact on activity, except for the dVDAVP homodimers, where an antagonist to agonist switch was observed at the V 1a R. This study provides novel insights into the structural requirements of VP/OT receptor activation and spotlights dimerization as a strategy to modulate pharmacology, a concept also frequently observed in nature., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Management of chronic pain presents a major challenge, since many currently available treatments lack efficacy and have problems such as addiction and tolerance. Loss of function mutations in the SCN9A gene lead to a congenital inability to feel pain, with no other sensory deficits aside from anosmia. SCN9A encodes the voltage-gated sodium (Na V ) channel 1.7 (Na V 1.7), which has been identified as a primary pain target. However, in developing Na V 1.7-targeted analgesics, extreme care must to be taken to avoid off-target activity on other Na V subtypes that are critical for survival. Since spider venoms are an excellent source of Na V channel modulators, we screened a panel of spider venoms to identify selective Na V 1.7 inhibitors. This led to identification of two novel Na V modulating venom peptides (β/μ-theraphotoxin-Pe1a and β/μ-theraphotoxin-Pe1b (Pe1b) from the arboreal tarantula Phormingochilus everetti. A third peptide isolated from the tarantula Bumba pulcherrimaklaasi was identical to the well-known ProTx-I (β/ω-theraphotoxin-Tp1a) from the tarantula Thrixopelma pruriens. A tethered toxin (t-toxin)-based alanine scanning strategy was used to determine the Na V 1.7 pharmacophore of ProTx-I. We designed several ProTx-I and Pe1b analogues, and tested them for activity and Na V channel subtype selectivity. Several analogues had improved potency against Na V 1.7, and altered specificity against other Na V channels. These analogues provide a foundation for development of Pe1b as a lead molecule for therapeutic inhibition of Na V 1.7., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Ant venoms have recently attracted increased attention due to their chemical complexity, novel molecular frameworks, and diverse biological activities. The heterodimeric peptide ∆-myrtoxin-Mp1a (Mp1a) from the venom of the Australian jack jumper ant, Myrmecia pilosula , exhibits antimicrobial, membrane-disrupting, and pain-inducing activities. In the present study, we examined the activity of Mp1a and a panel of synthetic analogues against the gastrointestinal parasitic nematode Haemonchus contortus , the fruit fly Drosophila melanogaster , and for their ability to stimulate pain-sensing neurons. Mp1a was found to be both insecticidal and anthelmintic, and it robustly activated mammalian sensory neurons at concentrations similar to those reported to elicit antimicrobial and cytotoxic activity. The native antiparallel Mp1a heterodimer was more potent than heterodimers with alternative disulfide connectivity, as well as monomeric analogues. We conclude that the membrane-disrupting effects of Mp1a confer broad-spectrum biological activities that facilitate both predation and defense for the ant. Our structure-activity data also provide a foundation for the rational engineering of analogues with selectivity for particular cell types., Competing Interests: The authors declare no conflict of interest.

Background and Purpose: Animal toxins have contributed significantly to our understanding of the neurobiology of receptors and ion channels. We studied the venom of the coral snake Micrurus fulvius fulvius and identified and characterized the structure and pharmacology of a new homodimeric neurotoxin, fulditoxin, that exhibited novel pharmacology at nicotinic ACh receptors (nAChRs)., Experimental Approach: Fulditoxin was isolated by chromatography, chemically synthesized, its structure determined by X-ray crystallography, and its pharmacological actions on nAChRs characterized by organ bath assays and two-electrode voltage clamp electrophysiology., Key Results: Fulditoxin's distinct 1.95-Å quaternary structure revealed two short-chain three-finger α-neurotoxins (α-3FNTxs) non-covalently bound by hydrophobic interactions and an ability to bind metal and form tetrameric complexes, not reported previously for three-finger proteins. Although fulditoxin lacked all conserved amino acids canonically important for inhibiting nAChRs, it produced postsynaptic neuromuscular blockade of chick muscle at nanomolar concentrations, comparable to the prototypical α-bungarotoxin. This neuromuscular blockade was completely reversible, which is unusual for snake α-3FNTxs. Fulditoxin, therefore, interacts with nAChRs by utilizing a different pharmacophore. Unlike short-chain α-3FNTxs that bind only to muscle nAChRs, fulditoxin utilizes dimerization to expand its pharmacological targets to include human neuronal α4β2, α7, and α3β2 nAChRs which it blocked with IC 50 values of 1.8, 7, and 12 μM respectively., Conclusions and Implications: Based on its distinct quaternary structure and unusual pharmacology, we named this new class of dimeric Micrurus neurotoxins represented by fulditoxin as Σ-neurotoxins, which offers greater insight into understanding the interactions between nAChRs and peptide antagonists., (© 2019 The British Pharmacological Society.)

Compelling human genetic studies have identified the voltage-gated sodium channel Na V 1.7 as a promising therapeutic target for the treatment of pain. The analgesic spider-venom-derived peptide μ-theraphotoxin-Pn3a is an exceptionally potent and selective inhibitor of Na V 1.7; however, little is known about the structure-activity relationships or channel interactions that define this activity. We rationally designed 17 Pn3a analogues and determined their activity at hNa V 1.7 using patch-clamp electrophysiology. The positively charged amino acids K22 and K24 were identified as crucial for Pn3a activity, with molecular modeling identifying interactions of these residues with the S3-S4 loop of domain II of hNa V 1.7. Removal of hydrophobic residues Y4, Y27, and W30 led to a loss of potency (>250-fold), while replacement of negatively charged D1 and D8 residues with a positively charged lysine led to increased potencies (>13-fold), likely through alterations in membrane lipid interactions. Mutating D8 to an asparagine led to the greatest improvement in Pn3a potency at Na V 1.7 (20-fold), while maintaining >100-fold selectivity over the major off-targets Na V 1.4, Na V 1.5, and Na V 1.6. The Pn3a[D8N] mutant retained analgesic activity in vivo , significantly attenuating mechanical allodynia in a clinically relevant mouse model of postsurgical pain at doses 3-fold lower than those with wild-type Pn3a, without causing motor-adverse effects. Results from this study will facilitate future rational design of potent and selective peptidic Na V 1.7 inhibitors for the development of more efficacious and safer analgesics as well as to further investigate the involvement of Na V 1.7 in pain., Competing Interests: The authors declare no competing financial interest., (Copyright © 2020 American Chemical Society.)

Spider venom is a novel source of disulfide-rich peptides with potent and selective activity at voltage-gated sodium channels (Na V ). Here, we describe the discovery of μ-theraphotoxin-Pme1a and μ/ δ -theraphotoxin-Pme2a, two novel peptides from the venom of the Gooty Ornamental tarantula Poecilotheria metallica that modulate Na V channels. Pme1a is a 35 residue peptide that inhibits Na V 1.7 peak current (IC 50 334 ± 114 nM) and shifts the voltage dependence of activation to more depolarised membrane potentials (V 1/2 activation: Δ = +11.6 mV). Pme2a is a 33 residue peptide that delays fast inactivation and inhibits Na V 1.7 peak current (EC 50 > 10 μM). Synthesis of a [+22K]Pme2a analogue increased potency at Na V 1.7 (IC 50 5.6 ± 1.1 μM) and removed the effect of the native peptide on fast inactivation, indicating that a lysine at position 22 (Pme2a numbering) is important for inhibitory activity. Results from this study may be used to guide the rational design of spider venom-derived peptides with improved potency and selectivity at Na V channels in the future., Competing Interests: The authors declare no conflict of interest

Cone snails use separately evolved venoms for prey capture and defence. While most use a harpoon for prey capture, the Gastridium clade that includes the well-studied Conus geographus and Conus tulipa, have developed a net hunting strategy to catch fish. This unique feeding behaviour requires secretion of "nirvana cabal" peptides to dampen the escape response of targeted fish allowing for their capture directly by mouth. However, the active components of the nirvana cabal remain poorly defined. In this study, we evaluated the behavioural effects of likely nirvana cabal peptides on the teleost model, Danio rerio (zebrafish). Surprisingly, the conantokins (NMDA receptor antagonists) and/or conopressins (vasopressin receptor agonists and antagonists) found in C. geographus and C. tulipa venom failed to produce a nirvana cabal-like effect in zebrafish. In contrast, low concentrations of the non-competitive adrenoceptor antagonist ρ-TIA found in C. tulipa venom (EC 50  = 190 nM) dramatically reduced the escape response of zebrafish larvae when added directly to aquarium water. ρ-TIA inhibited the zebrafish α 1 -adrenoceptor, confirming ρ-TIA has the potential to reverse the known stimulating effects of norepinephrine on fish behaviour. ρ-TIA may act alone and not as part of a cabal, since it did not synergise with conopressins and/or conantokins. This study highlights the importance of using ecologically relevant animal behaviour models to decipher the complex neurobiology underlying the prey capture and defensive strategies of cone snails.

An Australian estuarine isolate of Penicillium sp. MST-MF667 yielded 3 tetrapeptides named the bilaids with an unusual alternating LDLD chirality. Given their resemblance to known short peptide opioid agonists, we elucidated that they were weak ( K i low micromolar) μ-opioid agonists, which led to the design of bilorphin, a potent and selective μ-opioid receptor (MOPr) agonist ( K i 1.1 nM). In sharp contrast to all-natural product opioid peptides that efficaciously recruit β-arrestin, bilorphin is G protein biased, weakly phosphorylating the MOPr and marginally recruiting β-arrestin, with no receptor internalization. Importantly, bilorphin exhibits a similar G protein bias to oliceridine, a small nonpeptide with improved overdose safety. Molecular dynamics simulations of bilorphin and the strongly arrestin-biased endomorphin-2 with the MOPr indicate distinct receptor interactions and receptor conformations that could underlie their large differences in bias. Whereas bilorphin is systemically inactive, a glycosylated analog, bilactorphin, is orally active with similar in vivo potency to morphine. Bilorphin is both a unique molecular tool that enhances understanding of MOPr biased signaling and a promising lead in the development of next generation analgesics., Competing Interests: Competing interest statement: Patent Application(s) corresponding to Australian Patent Application 2018901944 The University of Sydney and The University of Queensland has been filed concerning this peptide. Title: Analgesics and Methods of Use Thereof., (Copyright © 2019 the Author(s). Published by PNAS.)

Pain is the leading cause of disability in the developed world but remains a poorly treated condition. Specifically, postsurgical pain continues to be a frequent and undermanaged condition. Here, we investigate the analgesic potential of pharmacological NaV1.7 inhibition in a mouse model of acute postsurgical pain, based on incision of the plantar skin and underlying muscle of the hind paw. We demonstrate that local and systemic treatment with the selective NaV1.7 inhibitor μ-theraphotoxin-Pn3a is effectively antiallodynic in this model and completely reverses mechanical hypersensitivity in the absence of motor adverse effects. In addition, the selective NaV1.7 inhibitors ProTx-II and PF-04856264 as well as the clinical candidate CNV1014802 also reduced mechanical allodynia. Interestingly, co-administration of the opioid receptor antagonist naloxone completely reversed analgesic effects of Pn3a, indicating an involvement of endogenous opioids in the analgesic activity of Pn3a. In addition, we found superadditive antinociceptive effects of subtherapeutic Pn3a doses not only with the opioid oxycodone but also with the GABAB receptor agonist baclofen. Transcriptomic analysis of gene expression changes in dorsal root ganglia of mice after surgery did not reveal any changes in mRNA expression of endogenous opioids or opioid receptors; however, several genes involved in pain, including Runx1 (Runt related transcription factor 1), Cacna1a (CaV2.1), and Cacna1b (CaV2.2), were downregulated. In summary, these findings suggest that pain after surgery can be successfully treated with NaV1.7 inhibitors alone or in combination with baclofen or opioids, which may present a novel and safe treatment strategy for this frequent and poorly managed condition.

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.

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.)

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.)

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.

Voltage-gated sodium (Nav) channels initiate action potentials in most neurons, including primary afferent nerve fibres of the pain pathway. Local anaesthetics block pain through non-specific actions at all Nav channels, but the discovery of selective modulators would facilitate the analysis of individual subtypes of these channels and their contributions to chemical, mechanical, or thermal pain. Here we identify and characterize spider (Heteroscodra maculata) toxins that selectively activate the Nav1.1 subtype, the role of which in nociception and pain has not been elucidated. We use these probes to show that Nav1.1-expressing fibres are modality-specific nociceptors: their activation elicits robust pain behaviours without neurogenic inflammation and produces profound hypersensitivity to mechanical, but not thermal, stimuli. In the gut, high-threshold mechanosensitive fibres also express Nav1.1 and show enhanced toxin sensitivity in a mouse model of irritable bowel syndrome. Together, these findings establish an unexpected role for Nav1.1 channels in regulating the excitability of sensory nerve fibres that mediate mechanical pain.

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.)

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.

For the past 30 years, oocytes from Xenopus laevis have been extensively used to express and characterise ion channels in an easily controlled environment. Here we report the first use of oocytes from the closely related species Xenopus borealis as an alternative expression system for neuronal ion channels. Using the two-electrode voltage-clamp technique, we show that a wide variety of voltage- and ligand-gated ion channels have the same channel properties and pharmacological profiles when expressed in either X. laevis or X. borealis oocytes. Potential advantages of the X. borealis oocytes include a smaller endogenous chloride current and the ability to produce more intense fluorescence signals when studied with voltage-clamp fluorometry. Scanning electron microscopy revealed a difference in vitelline membrane structure between the two species, which may be related to the discrepancy in fluorescence signals observed. We demonstrate that X. borealis oocytes are a viable heterologous system for expression of neuronal ion channels with some potential advantages over X. laevis oocytes for certain applications.

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.)

A safety-catch cysteine protecting group, S-4,4'-dimethylsulfinylbenzhydryl (Msbh), was designed and developed to expand the capabilities of synthetic strategies for the regioselective formation of disulfide bonds in cysteine-rich peptides. The directed regioselective synthesis of human hepcidin, which contains four disulfide bonds, was undertaken and led to a high-resolution NMR structure under more physiologically relevant conditions than previously. Conversely, hepcidin synthesized with the formerly assigned vicinal disulfide-bond connectivity displayed significant conformational heterogeneity under similar conditions. The two synthetic forms of human hepcidin induced ferroportin internalization with apparent EC50 values of 2.0 (native fold, 1) and 4.4 nM (non-native fold, 2), with 2 undergoing isomerization to 1 in the presence of ferroportin expressing cells., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: The venoms of predators have been an excellent source of diverse highly specific peptides targeting ion channels. Here we describe the first known peptide antagonist of the nociceptor ion channel transient receptor potential ankyrin 1 (TRPA1)., Results: We constructed a recombinant cDNA library encoding ∼100 diverse GPI-anchored peptide toxins (t-toxins) derived from spider venoms and screened this library by coexpression in Xenopus oocytes with TRPA1. This screen resulted in identification of protoxin-I (ProTx-I), a 35-residue peptide from the venom of the Peruvian green-velvet tarantula, Thrixopelma pruriens, as the first known high-affinity peptide TRPA1 antagonist. ProTx-I was previously identified as an antagonist of voltage-gated sodium (NaV) channels. We constructed a t-toxin library of ProTx-I alanine-scanning mutants and screened this library against NaV1.2 and TRPA1. This revealed distinct partially overlapping surfaces of ProTx-I by which it binds to these two ion channels. Importantly, this mutagenesis yielded two novel ProTx-I variants that are only active against either TRPA1or NaV1.2. By testing its activity against chimeric channels, we identified the extracellular loops of the TRPA1 S1-S4 gating domain as the ProTx-I binding site., Conclusions: These studies establish our approach, which we term "toxineering," as a generally applicable method for isolation of novel ion channel modifiers and design of ion channel modifiers with altered specificity. They also suggest that ProTx-I will be a valuable pharmacological reagent for addressing biophysical mechanisms of TRPA1 gating and the physiology of TRPA1 function in nociceptors, as well as for potential clinical application in the context of pain and inflammation., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Poor oral availability and susceptibility to reduction and protease degradation is a major hurdle in peptide drug development. However, drugable receptors in the gut present an attractive niche for peptide therapeutics. Here we demonstrate, in a mouse model of chronic abdominal pain, that oxytocin receptors are significantly upregulated in nociceptors innervating the colon. Correspondingly, we develop chemical strategies to engineer non-reducible and therefore more stable oxytocin analogues. Chemoselective selenide macrocyclization yields stabilized analogues equipotent to native oxytocin. Ultra-high-field nuclear magnetic resonance structural analysis of native oxytocin and the seleno-oxytocin derivatives reveals that oxytocin has a pre-organized structure in solution, in marked contrast to earlier X-ray crystallography studies. Finally, we show that these seleno-oxytocin analogues potently inhibit colonic nociceptors both in vitro and in vivo in mice with chronic visceral hypersensitivity. Our findings have potentially important implications for clinical use of oxytocin analogues and disulphide-rich peptides in general.

The μO-conotoxins are notable for their unique selectivity for Na(v)1.8 over other sodium channel isoforms, making them attractive drug leads for the treatment of neuropathic pain. We describe the discovery of a novel μO-conotoxin, MfVIA, from the venom of Conus magnificus using high-throughput screening approaches. MfVIA was found to be a hydrophobic 32-residue peptide (amino acid sequence RDCQEKWEYCIVPILGFVYCCPGLICGPFVCV) with highest sequence homology to μO-conotoxin MrVIB. To overcome the synthetic challenges posed by μO-conotoxins due to their hydrophobic nature and difficult folding, we developed a novel regioselective approach for the synthesis of μO-conotoxins. Performing selective oxidative deprotections of the cysteine side-chain protecting groups of the fully protected peptide allowed manipulations in organic solvents with no chromatography required between steps. Using this approach, we obtained correctly folded MfVIA with increased synthetic yields. Biological activity of MfVIA was assessed using membrane potential-sensitive dyes and electrophysiological recording techniques. MfVIA preferentially inhibits Na(v)1.8 (IC₅₀ 95.9±74.3 nM) and Na(v)1.4 (IC₅₀ 81±16 nM), with significantly lower affinity for other Na(v) subtypes (IC₅₀ 431-6203 nM; Na(v)1.5>1.6∼1.7∼1.3∼1.1∼1.2). This improved approach to μO-conotoxin synthesis will facilitate the optimization of μO-conotoxins as novel analgesic molecules to improve pain management., (Crown Copyright © 2012. Published by Elsevier Inc. All rights reserved.)

A dual-pharmacophoric peptide was engineered by grafting the integrin binding RGD motif between the C- and N-termini of a disulfide-rich noradrenaline transporter inhibiting χ-conotoxin resulting in a stable backbone cyclized peptide. The construct maintained two independent biological activities and showed increased plasma stability with no adverse effects observed following administration to rats, highlighting the potential value of pharmacophore grafting into constrained peptide scaffolds.

The two disulfide bonds of α-conotoxin ImI, a peptide antagonist of the α7 nicotinic acetylcholine receptor (nAChR), were systematically replaced with isosteric redox-stable cystathionine thioethers. Regioselective thioether formation was accomplished on solid support through substitution of a γ-chlorohomoalanine by an intramolecular cysteine thiol to produce hybrid thioether/disulfide analogues (2 and 3) as well as a dual cystathionine analogue (4) that were found to be structurally homologous to α-conotoxin ImI by (1)H NMR. The antagonistic activity at the α7 nAChR of cystathionine analogue 3 (pIC(50) = 6.41 ± 0.09) was identical to that of α-conotoxin ImI (1, pIC(50) = 6.41 ± 0.09), whereas those of 2 (pIC(50) = 5.96 ± 0.09) and 4 (pIC(50) = 5.89 ± 0.09) showed a modest decrease. The effect of oxidation of the thioethers to sulfoxides was also investigated, with significant changes in the biological activities observed ranging from a >30-fold reduction (2S═O) to a 3-fold increase (3S═O(B)) in potencies.

Disulfide bond engineering is an important approach to improve the metabolic half-life of cysteine-containing peptides. Eleven analogues of oxytocin were synthesized including disulfide bond replacements by thioether, selenylsulfide, diselenide, and ditelluride bridges, and their stabilities in human plasma and activity at the human oxytocin receptor were assessed. The cystathionine (K(i) = 1.5 nM, and EC₅₀ = 32 nM), selenylsulfide (K(i) = 0.29/0.72 nM, and EC₅₀ = 2.6/154 nM), diselenide (K(i) = 11.8 nM, and EC₅₀ = 18 nM), and ditelluride analogues (K(i) = 7.6 nM, and EC₅₀ = 27.3 nM) retained considerable affinity and functional potency as compared to oxytocin (K(i) = 0.79 nM, and EC₅₀ = 15 nM), while shortening the disulfide bridge abolished binding and functional activity. The mimetics showed a 1.5-3-fold enhancement of plasma stability as compared to oxytocin (t(½) = 12 h). By contrast, the all-D-oxytocin and head to tail cyclic oxytocin analogues, while significantly more stable with half-lives greater than 48 h, had little or no detectable binding or functional activity.