Singapore blue arboreal tarantula peptide Lv1a preferentially targets NaV1.6 with new world-like pharmacological mechanism.

Pain is a significant public health burden in the United States, and current treatment approaches rely heavily on opioids, which often have limited efficacy and can lead to addiction. In humans, functional loss of the voltage-gated sodium channel Nav1.7 leads to pain insensitivity without deficits in the central nervous system. Accordingly, discovery of a selective Nav1.7 antagonist should provide an analgesic without abuse liability and an improved side-effect profile. Huwentoxin-IV, a component of tarantula venom, potently blocks sodium channels and is an attractive scaffold for engineering a Nav1.7-selective molecule. To define the functional impact of alterations in huwentoxin-IV sequence, we produced a library of 373 point mutants and tested them for Nav1.7 and Nav1.2 activity. We then combined favorable individual changes to produce combinatorial mutants that showed further improvements in Nav1.7 potency (E1N, E4D, Y33W, Q34S–Nav1.7 pIC50 = 8.1 ± 0.08) and increased selectivity over other Nav isoforms (E1N, R26K, Q34S, G36I, Nav1.7 pIC50 = 7.2 ± 0.1, Nav1.2 pIC50 = 6.1 ± 0.18, Nav1.3 pIC50 = 6.4 ± 1.0), Nav1.4 is inactive at 3 μm, and Nav1.5 is inactive at 10 μm. We also substituted noncoded amino acids at select positions in huwentoxin-IV. Based on these results, we identify key determinants of huwentoxin's Nav1.7 inhibition and propose a model for huwentoxin-IV's interaction with Nav1.7. These findings uncover fundamental features of huwentoxin involved in Nav1.7 blockade, provide a foundation for additional optimization of this molecule, and offer a basis for the development of a safe and effective analgesic. Pain is a significant public health burden in the United States, and current treatment approaches rely heavily on opioids, which often have limited efficacy and can lead to addiction. In humans, functional loss of the voltage-gated sodium channel Nav1.7 leads to pain insensitivity without deficits in the central nervous system. Accordingly, discovery of a selective Nav1.7 antagonist should provide an analgesic without abuse liability and an improved side-effect profile. Huwentoxin-IV, a component of tarantula venom, potently blocks sodium channels and is an attractive scaffold for engineering a Nav1.7-selective molecule. To define the functional impact of alterations in huwentoxin-IV sequence, we produced a library of 373 point mutants and tested them for Nav1.7 and Nav1.2 activity. We then combined favorable individual changes to produce combinatorial mutants that showed further improvements in Nav1.7 potency (E1N, E4D, Y33W, Q34S–Nav1.7 pIC50 = 8.1 ± 0.08) and increased selectivity over other Nav isoforms (E1N, R26K, Q34S, G36I, Nav1.7 pIC50 = 7.2 ± 0.1, Nav1.2 pIC50 = 6.1 ± 0.18, Nav1.3 pIC50 = 6.4 ± 1.0), Nav1.4 is inactive at 3 μm, and Nav1.5 is inactive at 10 μm. We also substituted noncoded amino acids at select positions in huwentoxin-IV. Based on these results, we identify key determinants of huwentoxin's Nav1.7 inhibition and propose a model for huwentoxin-IV's interaction with Nav1.7. These findings uncover fundamental features of huwentoxin involved in Nav1.7 blockade, provide a foundation for additional optimization of this molecule, and offer a basis for the development of a safe and effective analgesic.

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

Understanding the relationship between molecule structure and function underpins both biochemistry and chemical biology, and has enabled the discovery of numerous agricultural, diagnostic and therapeutic agents. A subset of this chemical diversity is found in cysteine-rich proteins and peptides. By forming intra-molecular covalent disulfide bridges between their cysteine residues, monomeric peptides (up to about 50 amino acids in length) are able to adopt precise and stable globular conformations allowing interactions of high affinity with various molecular targets involved in the premise of specific biochemical pathways and physiological responses. The first part of this thesis work investigates the structure and function of secreted human cysteine-rich mini-proteins. The design of a high-throughput algorithm allowed the isolation of 53 cysteine frameworks spread over 378 mature forms of secreted mini-proteins, from which all the metadata relative to these active regions were used for classifying them into 21 pharmacological families. A deeper analysis of the molecular targets of these cysteine-rich mini-proteins (up to 200 amino acid in length, containing an even number of cysteine <20 all engaged in intra-chain disulfide bridges) shows that they are frequently ligands for G protein- and enzyme-coupled receptors, transporters, extracellular enzyme inhibitors, and antimicrobial peptides. The second and third chapters rely on a large-scale analysis of the structure-activity relationships of cysteine-rich venom peptides, with an emphasis on molecules specifically directed toward targets of the nociceptive pathways. These analyses demonstrate the preference for recruitment into the venom of highly stable peptides with particular cysteine scaffolds often cross-braced by one or more disulfide bridges that shape well-defined tertiary structures such as inhibitory cysteine knots, Kunitz inhibitor, Kazal, or WAP domains and dictate their specificity of action. This diversity of disulfide-rich architectures that confers venom peptides an important stability against enzymatic degradation or extreme pH and temperatures, has already served as templates for designing molecules of diagnostic and therapeutic interests such as anti-nociceptive or anti-cancer drugs directed toward GABA or natriuretic receptors, as well as sodium channels. The fourth chapter describes the conception of a high-throughput bioinformatic program, called ConoSorter, for fast and precise de novo identification and classification of toxins produced by venomous marine cone snails sequenced with next-generation transcriptomic and proteomic platforms. This published work notably shows the efficiency and specificity of two complementary searching strategies based on regular expressions and profile Hidden Markov Models that allow the recognition of 100% of known superfamilies and classes with a minimum species specificity of 99%. A re-analysis of the Conus marmoreus venom duct transcriptome also allowed the discovery of 158 new toxin sequences (106 confirmed by mass spectrometry), as well as 13 novel gene superfamilies. Finally, the last chapter of this thesis describes a high-resolution interrogation of the Conus episcopatus venom duct, salivary gland, and radular sac transcriptomes and proteomes, supported by a meticulous and efficient bioinformatics methodology. This work has by far unveiled the highest number of conopeptides discovered in a single Conus species to date, by revealing 3,305 novel precursor conopeptide sequences identified from transcriptomic data, 144 of which validated by protein mass spectrometry. In addition, we describe for the first time a large population of venom peptides containing the pharmacologically active C-C-CC-C-C inhibitory cysteine knot (168 molecules) or CC-C-C (45) cysteine frameworks. We also describe six cysteine frameworks novel to cone snails - four of which are ubiquitous in nature, one which is highly abundant in snake C-type lectins, and one containing 10 cysteines which is previously undescribed. These data indicate that sequence hypervariablity of conotoxins originates from codon usage bias at the gene level, and support the creation of 16 novel cone snail gene superfamilies that could be directed toward new classes of targets. The novel conopeptides described here are strong candidates to act as molecular templates for the development of diagnostic and therapeutic tools. Taken together this thesis work depicts the journey of the biodiscovery of numerous novel toxin sequences, some of them characterized by unique cysteine scaffolds present in pharmacologically active molecules, and also demonstrates the power of the transcriptome/proteome sequencing and matching approach for detecting low expressed proteins and potential new drug leads.

Nodes of Ranvier Act as Barriers to Restrict Invasion of Flanking Paranodal Domains in Myelinated Axons

Voltage-gated sodium channels (VGSCs; NaV1.1–NaV1.9) have been proven to be critical in controlling the function of excitable cells, and human genetic evidence shows that aberrant function of these channels causes channelopathies, including epilepsy, arrhythmia, paralytic myotonia, and pain. The effects of peptide toxins, especially those isolated from spider venom, have shed light on the structure–function relationship of these channels. However, most of these toxins have not been analyzed in detail. In particular, the bioactive faces of these toxins have not been determined. Jingzhaotoxin (JZTX)-V (also known as β-theraphotoxin-Cj2a) is a 29-amino acid peptide toxin isolated from the venom of the spider Chilobrachys jingzhao. JZTX-V adopts an inhibitory cysteine knot (ICK) motif and has an inhibitory effect on voltage-gated sodium and potassium channels. Previous experiments have shown that JZTX-V has an inhibitory effect on TTX-S and TTX-R sodium currents on rat DRG cells with IC50 values of 27.6 and 30.2 nM, respectively, and is able to shift the activation and inactivation curves to the depolarizing and the hyperpolarizing direction, respectively. Here, we show that JZTX-V has a much stronger inhibitory effect on NaV1.4, the isoform of voltage-gated sodium channels predominantly expressed in skeletal muscle cells, with an IC50 value of 5.12 nM, compared with IC50 values of 61.7–2700 nM for other heterologously expressed NaV1 subtypes. Furthermore, we investigated the bioactive surface of JZTX-V by alanine-scanning the effect of toxin on NaV1.4 and demonstrate that the bioactive face of JZTX-V is composed of three hydrophobic (W5, M6, and W7) and two cationic (R20 and K22) residues. Our results establish that, consistent with previous assumptions, JZTX-V is a Janus-faced toxin which may be a useful tool for the further investigation of the structure and function of sodium channels.

SummaryVoltage-gated sodium (NaV) channels, initially characterized in excitable cells, have been shown to be aberrantly expressed in non-excitable cancer tissues and cells from epithelial origins such as in breast, lung, prostate, colon, and cervix, whereas they are not expressed in cognate non-cancer tissues. Their activity was demonstrated to promote aggressive and invasive potencies of cancer cells, both in vitro and in vivo, whereas their deregulated expression in cancer tissues has been associated with metastatic progression and cancer-related death. This review proposes NaV channels as pharmacological targets for anticancer treatments providing opportunities for repurposing existing NaV-inhibitors or developing new pharmacological and nutritional interventions.

Electrical excitability in neurons depends on the activity of membrane-bound voltage gated sodium channels (Na(v)) that are assembled from an ion conducting α-subunit and often auxiliary β-subunits. The α-subunit isoform Na(v)1.3 occurs in peripheral neurons together with the Na(v) β3-subunit, both of which are coordinately up-regulated in rat dorsal root ganglion neurons after nerve injury. Here we examine the effect of the β3-subunit on the gating behavior of Na(v)1.3 using whole cell patch clamp electrophysiology in HEK-293 cells. We show that β3 depolarizes the voltage sensitivity of Na(v)1.3 activation and inactivation and induces biphasic components of the inactivation curve. We detect both a fast and a novel slower component of inactivation, and we show that the β3-subunit increases the fraction of channels inactivating by the slower component. Using CD and NMR spectroscopy, we report the first structural analysis of the intracellular domain of any Na(v) β-subunit. We infer the presence of a region within the β3-subunit intracellular domain that has a propensity to form a short amphipathic α-helix followed by a structurally disordered sequence, and we demonstrate a role for both of these regions in the selective stabilization of fast inactivation. The complex gating behavior induced by β3 may contribute to the known hyperexcitability of peripheral neurons under those physiological conditions where expression of β3 and Na(v)1.3 are both enhanced.

The three-dimensional (3D) NMR solution structure (MeOH) of the highly hydrophobic delta-conotoxin delta-Am2766 from the molluscivorous snail Conus amadis has been determined. Fifteen converged structures were obtained on the basis of 262 distance constraints, 25 torsion-angle constraints, and ten constraints based on disulfide linkages and H-bonds. The root-mean-square deviations (rmsd) about the averaged coordinates of the backbone (N, C(alpha), C) and (all) heavy atoms were 0.62+/-0.20 and 1.12+/-0.23 A, respectively. The structures determined are of good stereochemical quality, as evidenced by the high percentage (100%) of backbone dihedral angles that occupy favorable and additionally allowed regions of the Ramachandran map. The structure of delta-Am2766 consists of a triple-stranded antiparallel beta-sheet, and of four turns. The three disulfides form the classical 'inhibitory cysteine knot' motif. So far, only one tertiary structure of a delta-conotoxin has been reported; thus, the tertiary structure of delta-Am2766 is the second such example. Another Conus peptide, Am2735 from C. amadis, has also been purified and sequenced. Am2735 shares 96% sequence identity with delta-Am2766. Unlike delta-Am2766, Am2735 does not inhibit the fast inactivation of Na+ currents in rat brain Na(v)1.2 Na+ channels at concentrations up to 200 nM.

Although necessary for human survival, pain may sometimes become pathologic if long-lasting and associated with alterations in its signaling pathway. Opioid painkillers are officially used to treat moderate to severe, and even mild, pain. However, the consequent strong and not so rare complications that occur, including addiction and overdose, combined with pain management costs, remain an important societal and economic concern. In this context, animal venom toxins represent an original source of antinociceptive peptides that mainly target ion channels (such as ASICs as well as TRP, CaV, KV and NaV channels) involved in pain transmission. The present review aims to highlight the NaV1.7 channel subtype as an antinociceptive target for spider toxins in adult dorsal root ganglia neurons. It will detail (i) the characteristics of these primary sensory neurons, the first ones in contact with pain stimulus and conveying the nociceptive message, (ii) the electrophysiological properties of the different NaV channel subtypes expressed in these neurons, with a particular attention on the NaV1.7 subtype, an antinociceptive target of choice that has been validated by human genetic evidence, and (iii) the features of spider venom toxins, shaped of inhibitory cysteine knot motif, that present high affinity for the NaV1.7 subtype associated with evidenced analgesic efficacy in animal models.

Voltage-gated sodium channels (NaV) are integral membrane proteins that are responsible for the increase in sodium permeability that initiates and propagates the rising phase of action potentials, carrying electrical signals along nerve fibers and through excitable cells. NaV channels play a diverse role in neurophysiology and neurotransmission, as well as serving as molecular targets for several groups of neurotoxins that bind to different receptor sites and alter voltage-dependent activation, inactivation and conductance. There are nine NaV channel isoforms so far discovered, each of which display distinct functional profiles and tissue-specific expression patterns. The modulation of specific isoforms for therapeutic purposes has become an important research objective for the treatment of conductance diseases exhibiting phenotypes of chronic pain, epilepsy, myotonia, seizure, and cardiac arrhythmia. However, because of the high sequence similarity and structural homology between NaV channel isoforms, many current therapeutics that target NaV channels – the vast majority of which are small molecules – lack specificity between isoforms, or even other voltage-gated ion channels. The current push for greater selectivity while maintaining a relevant degree of potency has led the focus away from small molecules and towards the discovery and development of peptidic ligands for therapeutic use. Venom derived peptides have proven to be naturally potent and selective bioactive molecules, exhibiting inherent secondary structures that add stability through the formation of disulfide bonds. Organisms such as cone snails and spiders have evolved venom for the purpose of prey capture and defense, therefore many of the components exhibit paralytic qualities and specifically target NaV channels. Much of the discovery process has focused on screening crude venoms for a particular function and then isolating the molecule responsible for that function using assay guided purification. The first section of this thesis describes the development of a cell-based, high-throughput functional assay for the detection of NaV modulating venom peptides in crude venom. This assay was successfully implemented and resulted in the isolation and sequencing of MVIA from Conus magus. Initial results and sequence similarity placed MVIA into the δ-conotoxin family, a poorly described class of peptides that inhibits fast inactivation. However, multiple solid-phase synthesis and bacterial recombinant expression methods were unsuccessful in producing enough of the extremely hydrophobic δ-MVIA for further characterization. As no more C. magus crude venom was available, this project was left until optimized methods of production for highly hydrophobic peptides could be developed. An optimized method of bacterial recombinant expression was successful in producing large yields of another venom peptide, β/δ-TRTX-Pre1a, isolated from the spider Psalmopoeus reduncus. The same recombinant expression method was also used to produce uniformly labeled 13C/15N-peptide for determination of a heteronuclear NMR solution structure. Pre1a was revealed to be a sub-micromolar inhibitor of both NaV1.2 and NaV1.7 peak currents, yet demonstrated potent inhibition of fast inactivation of NaV1.3. This dual mode of action on different NaV isoforms had not yet been reported for any known venom peptide. Further, Pre1a exhibited structural heterogeneity as determined by analysis of rpHPLC and NMR 15N-HSQC, which was traced to the contribution of residues making up the all aromatic, hydrophobic face of the peptide. The high sequence similarity of Pre1a to previously discovered spider venom peptides allowed comparative functional analysis and the identification of key residues contributing to NaV isoform selectivity. Mutagenesis focusing on both structural and functional aspects of Pre1a was performed incorporating both alanine and non-alanine substitutions. Through a single mutation a residue critical for the observed conformational heterogeneity was identified. This mutation served as a model for the obtainment of a high-resolution solution structure for comparison with the active Pre1a. Lastly, we identified a single residue on the C-terminus critical for NaV channel isoform selectivity. Substitution with amino acids exhibiting different properties of charge, polarity, and size could modify selectivity and in the process create a highly selective inhibitor of NaV1.2. This study not only revealed a unique mode of action for a venom peptide, but also demonstrated novelty as a proof-of-concept for the rational design and engineering of venom peptides using available information and non-standard methods of selective mutagenesis.

Nuclear Factor-kappaB Gates Nav1.7 Channels in DRG Neurons via Protein-Protein Interaction.

A critical hurdle in ant venom proteomic investigations is the lack of databases to comprehensively and specifically identify the sequence and function of venom proteins and peptides. To resolve this, we used venom gland transcriptomics to generate a sequence database that was used to assign the tandem mass spectrometry (MS) fragmentation spectra of venom peptides and proteins to specific transcripts. This was performed alongside a shotgun liquid chromatography–mass spectrometry (LC-MS/MS) analysis of the venom to confirm that these assigned transcripts were expressed as proteins. Through the combined transcriptomic and proteomic investigation of Paraponera clavata venom, we identified four times the number of proteins previously identified using 2D-PAGE alone. In addition to this, by mining the transcriptomic data, we identified several novel peptide sequences for future pharmacological investigations, some of which conform with inhibitor cysteine knot motifs. These types of peptides have the potential to be developed into pharmaceutical or bioinsecticide peptides.

Pholcid spiders (Araneae: Pholcidae), officially “cellar spiders” but popularly known as “daddy long-legs,” are renown for the potential of deadly toxic venom, even though venom composition and potency has never formally been studied. Here we detail the venom composition of male Physocyclus mexicanus using proteomic analyses and venom-gland transcriptomes (“venomics”). We also analyze the venom’s potency on insects, and assemble available evidence regarding mammalian toxicity. The majority of the venom (51% of tryptic polypeptides and 62% of unique tryptic peptides) consists of proteins homologous to known venom toxins including enzymes (astacin metalloproteases, serine proteases and metalloendopeptidases, particularly neprilysins) and venom peptide neurotoxins. We identify 17 new groups of peptides (U1–17-PHTX) most of which are homologs of known venom peptides and are predicted to have an inhibitor cysteine knot fold; of these, 13 are confirmed in the proteome. Neprilysins (M13 peptidases), and astacins (M12 peptidases) are the most abundant venom proteins, respectively representing 15 and 11% of the individual proteins and 32 and 20% of the tryptic peptides detected in crude venom. Comparative evidence suggests that the neprilysin gene family is expressed in venoms across a range of spider taxa, but has undergone an expansion in the venoms of pholcids and may play a central functional role in these spiders. Bioassays of crude venoms on crickets resulted in an effective paralytic dose of 3.9 µg/g, which is comparable to that of crude venoms of Plectreurys tristis and other Synspermiata taxa. However, crickets exhibit flaccid paralysis and regions of darkening that are not observed after P. tristis envenomation. Documented bites on humans make clear that while these spiders can bite, the typical result is a mild sting with no long-lasting effects. Together, the evidence we present indicates pholcid venoms are a source of interesting new peptides and proteins, and effects of bites on humans and other mammals are inconsequential.

Venom based research is exploited to find novel candidates for the development of innovative pharmacological tools, drug candidates and new ingredients for cosmetic and agrochemical industries. Moreover, venomics, as a well-established approach in systems biology, helps to elucidate the genetic mechanisms of the production of such a great molecular biodiversity. Today the advances made in the proteomics, transcriptomics and bioinformatics fields, favor venomics, allowing the in depth study of complex matrices and the elucidation even of minor compounds present in minute biological samples. The present study illustrates a rapid and efficient method developed for the elucidation of venom composition based on NextGen mRNA sequencing of venom glands and LC-MS/MS venom proteome profiling. The analysis of the comprehensive data obtained was focused on cysteine rich peptide toxins from four spider species originating from phylogenetically distant families for comparison purposes. The studied species were Heteropoda davidbowie (Sparassidae), Poecilotheria formosa (Theraphosidae), Viridasius fasciatus (Viridasiidae) and Latrodectus mactans (Theridiidae). This led to a high resolution profiling of 284 characterized cysteine rich peptides, 111 of which belong to the Inhibitor Cysteine Knot (ICK) structural motif. The analysis of H. davidbowie venom revealed a high richness in term of venom diversity: 95 peptide sequences were identified; out of these, 32 peptides presented the ICK structural motif and could be classified in six distinct families. The profiling of P. formosa venom highlighted the presence of 126 peptide sequences, with 52 ICK toxins belonging to three structural distinct families. V. fasciatus venom was shown to contain 49 peptide sequences, out of which 22 presented the ICK structural motif and were attributed to five families. The venom of L. mactans, until now studied for its large neurotoxins (Latrotoxins), revealed the presence of 14 cysteine rich peptides, out of which five were ICK toxins belonging to the CSTX superfamily. This in depth profiling of distinct ICK peptide families identified across the four spider species highlighted the high conservation of these neurotoxins among spider families.

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