Publications by authors named "Samuel D Robinson"

40 Publications

Venom chemistry underlying the painful stings of velvet ants (Hymenoptera: Mutillidae).

Cell Mol Life Sci 2021 May 10. Epub 2021 May 10.

Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD, 4072, Australia.

Velvet ants (Hymenoptera: Mutillidae) are a family of solitary parasitoid wasps that are renowned for their painful stings. We explored the chemistry underlying the stings of mutillid wasps of the genus Dasymutilla Ashmead. Detailed analyses of the venom composition of five species revealed that they are composed primarily of peptides. We found that two kinds of mutillid venom peptide appear to be primarily responsible for the painful effects of envenomation. These same peptides also have defensive utility against invertebrates, since they were able to incapacitate and kill honeybees. Both act directly on cell membranes where they directly increase ion conductivity. The defensive venom peptides of Dasymutilla bear a striking similarity, in structure and mode of action, to those of the ant Myrmecia gulosa (Fabricius), suggesting either retention of ancestral toxins, or convergence driven by similar life histories and defensive selection pressures. Finally, we propose that other highly expressed Dasymutilla venom peptides may play a role in parasitisation, possible in delay or arrest of host development. This study represents the first detailed account of the composition and function of the venoms of the Mutillidae.
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http://dx.doi.org/10.1007/s00018-021-03847-1DOI Listing
May 2021

Production, composition, and mode of action of the painful defensive venom produced by a limacodid caterpillar, .

Proc Natl Acad Sci U S A 2021 May;118(18)

Institute for Molecular Bioscience, The University of Queensland, St Lucia, QLD, 4072, Australia;

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, Contrary to dogma that defensive venoms are simple in composition, 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 Our data reveal convergent molecular evolution between limacodids, hymenopterans, and arachnids and demonstrate that lepidopteran venoms are an untapped source of novel bioactive peptides.
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http://dx.doi.org/10.1073/pnas.2023815118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8106304PMC
May 2021

Alkyne-Bridged α-Conotoxin Vc1.1 Potently Reverses Mechanical Allodynia in Neuropathic Pain Models.

J Med Chem 2021 03 16;64(6):3222-3233. Epub 2021 Mar 16.

School of Chemistry, Monash University, Clayton, Victoria 3800, Australia.

Several -derived venom peptides are promising lead compounds for the management of neuropathic pain, with α-conotoxins being of particular interest. Modification of the interlocked disulfide framework of α-conotoxin Vc1.1 has been achieved using on-resin alkyne metathesis. Although introduction of a metabolically stable alkyne motif significantly disrupts backbone topography, the structural modification generates a potent and selective GABA receptor agonist that inhibits Ca2.2 channels and exhibits dose-dependent reversal of mechanical allodynia in a behavioral rat model of neuropathic pain. The findings herein support the hypothesis that analgesia can be achieved via activation of GABARs expressed in dorsal root ganglion (DRG) sensory neurons.
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http://dx.doi.org/10.1021/acs.jmedchem.0c02151DOI Listing
March 2021

The zebrafish mutant uncovers an evolutionarily conserved role for Tmem161b in the control of cardiac rhythm.

Proc Natl Acad Sci U S A 2021 Mar;118(9)

Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD 4072, Australia;

The establishment of cardiac function in the developing embryo is essential to ensure blood flow and, therefore, growth and survival of the animal. The molecular mechanisms controlling normal cardiac rhythm remain to be fully elucidated. From a forward genetic screen, we identified a unique mutant, that displayed a specific cardiac arrhythmia phenotype. We show that loss-of-function mutations in are responsible for the phenotype, identifying Tmem161b as a regulator of cardiac rhythm in zebrafish. To examine the evolutionary conservation of this function, we generated knockout mice for Tmem161b. Tmem161b knockout mice are neonatal lethal and cardiomyocytes exhibit arrhythmic calcium oscillations. Mechanistically, we find that Tmem161b is expressed at the cell membrane of excitable cells and live imaging shows it is required for action potential repolarization in the developing heart. Electrophysiology on isolated cardiomyocytes demonstrates that Tmem161b is essential to inhibit Ca and K currents in cardiomyocytes. Importantly, Tmem161b haploinsufficiency leads to cardiac rhythm phenotypes, implicating it as a candidate gene in heritable cardiac arrhythmia. Overall, these data describe Tmem161b as a highly conserved regulator of cardiac rhythm that functions to modulate ion channel activity in zebrafish and mice.
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http://dx.doi.org/10.1073/pnas.2018220118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7936323PMC
March 2021

Neurotoxic peptides from the venom of the giant Australian stinging tree.

Sci Adv 2020 Sep 16;6(38). Epub 2020 Sep 16.

Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD 4072, Australia.

Stinging trees from Australasia produce remarkably persistent and painful stings upon contact of their stiff epidermal hairs, called trichomes, with mammalian skin. -induced acute pain typically lasts for several hours, and intermittent painful flares can persist for days and weeks. Pharmacological activity has been attributed to small-molecule neurotransmitters and inflammatory mediators, but these compounds alone cannot explain the observed sensory effects. We show here that the venoms of Australian species contain heretofore unknown pain-inducing peptides that potently activate mouse sensory neurons and delay inactivation of voltage-gated sodium channels. These neurotoxins localize specifically to the stinging hairs and are miniproteins of 4 kDa, whose 3D structure is stabilized in an inhibitory cystine knot motif, a characteristic shared with neurotoxins found in spider and cone snail venoms. Our results provide an intriguing example of inter-kingdom convergent evolution of animal and plant venoms with shared modes of delivery, molecular structure, and pharmacology.
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http://dx.doi.org/10.1126/sciadv.abb8828DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7494335PMC
September 2020

Pharmacology and therapeutic potential of venom peptides.

Biochem Pharmacol 2020 11 26;181:114207. Epub 2020 Aug 26.

Institute for Molecular Bioscience, The University of Queensland, St. Lucia, QLD, Australia; School of Pharmacy, Pharmacy Australia Centre of Excellence, The University of Queensland, Woolloongabba, QLD, Australia. Electronic address:

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http://dx.doi.org/10.1016/j.bcp.2020.114207DOI Listing
November 2020

Deadly Proteomes: A Practical Guide to Proteotranscriptomics of Animal Venoms.

Proteomics 2020 09;20(17-18):e1900324

Institute for Molecular Bioscience, The University of Queensland, St. Lucia, Queensland, 4072, Australia.

Animal venoms are renowned for their toxicity, biochemical complexity, and as a source of compounds with potential applications in medicine, agriculture, and industry. Polypeptides underlie much of the pharmacology of animal venoms, and elucidating these arsenals of polypeptide toxins-known as the venom proteome or venome-is an important step in venom research. Proteomics is used for the identification of venom toxins, determination of their primary structure including post-translational modifications, as well as investigations into the physiology underlying their production and delivery. Advances in proteomics and adjacent technologies has led to a recent upsurge in publications reporting venom proteomes. Improved mass spectrometers, better proteomic workflows, and the integration of next-generation sequencing of venom-gland transcriptomes and venomous animal genomes allow quicker and more accurate profiling of venom proteomes with greatly reduced starting material. Technologies such as imaging mass spectrometry are revealing additional insights into the mechanism, location, and kinetics of venom toxin production. However, these numerous new developments may be overwhelming for researchers designing venom proteome studies. Here, the field of venom proteomics is reviewed and some practical solutions for simplifying mass spectrometry workflows to study animal venoms are offered.
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http://dx.doi.org/10.1002/pmic.201900324DOI Listing
September 2020

It Takes Two: Dimerization Is Essential for the Broad-Spectrum Predatory and Defensive Activities of the Venom Peptide Mp1a from the Jack Jumper Ant .

Biomedicines 2020 Jun 30;8(7). Epub 2020 Jun 30.

Institute for Molecular Bioscience, The University of Queensland, St Lucia, QLD 4072, Australia.

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, , 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 , the fruit fly , 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.
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http://dx.doi.org/10.3390/biomedicines8070185DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7400207PMC
June 2020

Characterization of Synthetic Tf2 as a Na1.3 Selective Pharmacological Probe.

Biomedicines 2020 Jun 11;8(6). Epub 2020 Jun 11.

Institute for Molecular Bioscience, The University of Queensland, St. Lucia, QLD 4072, Australia.

Na1.3 is a subtype of the voltage-gated sodium channel family. It has been implicated in the pathogenesis of neuropathic pain, although the contribution of this channel to neuronal excitability is not well understood. Tf2, a β-scorpion toxin previously identified from the venom of , has been reported to selectively activate Na1.3. Here, we describe the activity of synthetic Tf2 and assess its suitability as a pharmacological probe for Na1.3. As described for the native toxin, synthetic Tf2 (1 µM) caused early channel opening, decreased the peak current, and shifted the voltage dependence of Na1.3 activation in the hyperpolarizing direction by -11.3 mV, with no activity at Na1.1, Na1.2, and Na1.4-Na1.8. Additional activity was found at Na1.9, tested using the hNav1.9_C4 chimera, where Tf2 (1 µM) shifted the voltage dependence of activation by -6.3 mV. In an attempt to convert Tf2 into an Na1.3 inhibitor, we synthetized the analogue Tf2[S14R], a mutation previously described to remove the excitatory activity of related β-scorpion toxins. Indeed, Tf2[S14R](10 µM) had reduced excitatory activity at Na1.3, although it still caused a small -5.8 mV shift in the voltage dependence of activation. Intraplantar injection of Tf2 (1 µM) in mice caused spontaneous flinching and swelling, which was not reduced by the Na1.1/1.3 inhibitor ICA-121431 nor in Na1.9 mice, suggesting off-target activity. In addition, despite a loss of excitatory activity, intraplantar injection of Tf2[S14R](10 µM) still caused swelling, providing strong evidence that Tf2 has additional off-target activity at one or more non-neuronal targets. Therefore, due to activity at Na1.9 and other yet to be identified target(s), the use of Tf2 as a selective pharmacological probe may be limited.
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http://dx.doi.org/10.3390/biomedicines8060155DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7345637PMC
June 2020

An Integrated Proteomic and Transcriptomic Analysis Reveals the Venom Complexity of the Bullet Ant .

Toxins (Basel) 2020 05 14;12(5). Epub 2020 May 14.

School of Life Sciences, University of Technology Sydney, Broadway, NSW 2007, Australia.

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 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.
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http://dx.doi.org/10.3390/toxins12050324DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7290781PMC
May 2020

Transcriptomic Profiling Reveals Extraordinary Diversity of Venom Peptides in Unexplored Predatory Gastropods of the Genus Clavus.

Genome Biol Evol 2020 05;12(5):684-700

A.N. Severtsov Institute of Ecology and Evolution, Russian Academy of Science, Moscow, Russia.

Predatory gastropods of the superfamily Conoidea number over 12,000 living species. The evolutionary success of this lineage can be explained by the ability of conoideans to produce complex venoms for hunting, defense, and competitive interactions. Whereas venoms of cone snails (family Conidae) have become increasingly well studied, the venoms of most other conoidean lineages remain largely uncharacterized. In the present study, we present the venom gland transcriptomes of two species of the genus Clavus that belong to the family Drilliidae. Venom gland transcriptomes of two specimens of Clavus canalicularis and two specimens of Clavus davidgilmouri were analyzed, leading to the identification of a total of 1,176 putative venom peptide toxins (drillipeptides). Based on the combined evidence of secretion signal sequence identity, entire precursor similarity search (BLAST), and the orthology inference, putative Clavus toxins were assigned to 158 different gene families. The majority of identified transcripts comprise signal, pro-, mature peptide, and post-regions, with a typically short (<50 amino acids) and cysteine-rich mature peptide region. Thus, drillipeptides are structurally similar to conotoxins. However, convincing homology with known groups of Conus toxins was only detected for very few toxin families. Among these are Clavus counterparts of Conus venom insulins (drillinsulins), porins (drilliporins), and highly diversified lectins (drillilectins). The short size of most drillipeptides and structural similarity to conotoxins were unexpected, given that most related conoidean gastropod families (Terebridae and Turridae) possess longer mature peptide regions. Our findings indicate that, similar to conotoxins, drillipeptides may represent a valuable resource for future pharmacological exploration.
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http://dx.doi.org/10.1093/gbe/evaa083DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7259678PMC
May 2020

Missiles of Mass Disruption: Composition and Glandular Origin of Venom Used as a Projectile Defensive Weapon by the Assassin Bug .

Toxins (Basel) 2019 11 18;11(11). Epub 2019 Nov 18.

Institute for Molecular Bioscience, the University of Queensland, St. Lucia, Queensland 4072, Australia.

Assassin bugs (Reduviidae) produce venoms that are insecticidal, and which induce pain in predators, but the composition and function of their individual venom components is poorly understood. We report findings on the venom system of the red-spotted assassin bug , a large species of African origin that is unique in propelling venom as a projectile weapon when threatened. We performed RNA sequencing experiments on venom glands (separate transcriptomes of the posterior main gland, PMG, and the anterior main gland, AMG), and proteomic experiments on venom that was either defensively propelled or collected from the proboscis in response to electrostimulation. We resolved a venom proteome comprising 166 polypeptides. Both defensively propelled venom and most venom samples collected in response to electrostimulation show a protein profile similar to the predicted secretory products of the PMG, with a smaller contribution from the AMG. Pooled venom samples induce calcium influx via membrane lysis when applied to mammalian neuronal cells, consistent with their ability to cause pain when propelled into the eyes or mucus membranes of potential predators. The same venom induces rapid paralysis and death when injected into fruit flies. These data suggest that the cytolytic, insecticidal venom used by reduviids to capture prey is also a highly effective defensive weapon when propelled at predators.
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http://dx.doi.org/10.3390/toxins11110673DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6891600PMC
November 2019

High-Throughput Fluorescence Assays for Ion Channels and GPCRs.

Adv Exp Med Biol 2020 ;1131:27-72

Institute for Molecular Bioscience, The University of Queensland, St. Lucia, QLD, Australia.

Ca, Na and K- permeable ion channels as well as GPCRs linked to Ca release are important drug targets. Accordingly, high-throughput fluorescence plate reader assays have contributed substantially to drug discovery efforts and pharmacological characterization of these receptors and ion channels. This chapter describes some of the basic properties of the fluorescent dyes facilitating these assay approaches as well as general methods for establishment and optimisation of fluorescence assays for ion channels and G-coupled GPCRs.
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http://dx.doi.org/10.1007/978-3-030-12457-1_3DOI Listing
October 2019

Characterization of the First Conotoxin from , a Vermivorous Cone Snail from the Cabo Verde Archipelago.

Mar Drugs 2019 Jul 24;17(8). Epub 2019 Jul 24.

Department of Biology, University of Utah, 257 S 1400 E, Salt Lake City, UT 84112, USA.

is a cone snail endemic to the west side of the island of Sal, in the Cabo Verde Archipelago off West Africa. We describe the isolation and characterization of the first bioactive peptide from the venom of this species. This 30AA venom peptide is named conotoxin AtVIA (δ-conotoxin-like). An excitatory activity was manifested by the peptide on a majority of mouse lumbar dorsal root ganglion neurons. An analog of AtVIA with conservative changes on three amino acid residues at the C-terminal region was synthesized and this analog produced an identical effect on the mouse neurons. AtVIA has homology with δ-conotoxins from other worm-hunters, which include conserved sequence elements that are shared with δ-conotoxins from fish-hunting . In contrast, there is no comparable sequence similarity with δ-conotoxins from the venoms of molluscivorous species. A rationale for the potential presence of δ-conotoxins, that are potent in vertebrate systems in two different lineages of worm-hunting cone snails, is discussed.
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http://dx.doi.org/10.3390/md17080432DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6723684PMC
July 2019

Na 1.6 regulates excitability of mechanosensitive sensory neurons.

J Physiol 2019 07 13;597(14):3751-3768. Epub 2019 May 13.

IMB Centre for Pain Research, Institute for Molecular Bioscience, 306 Carmody Rd (Building 80), University of Queensland, Brisbane, Queensland, 4072, Australia.

Key Points: Voltage-gated sodium channels are critical for peripheral sensory neuron transduction and have been implicated in a number of painful and painless disorders. The β-scorpion toxin, Cn2, is selective for Na 1.6 in dorsal root ganglion neurons. Na 1.6 plays an essential role in peripheral sensory neurons, specifically at the distal terminals of mechanosensing fibres innervating the skin and colon. Na 1.6 activation also leads to enhanced response to mechanical stimulus in vivo. This works highlights the use of toxins in elucidating pain pathways moreover the importance of non-peripherally restricted Na isoforms in pain generation.

Abstract: Peripheral sensory neurons express multiple voltage-gated sodium channels (Na ) critical for the initiation and propagation of action potentials and transmission of sensory input. Three pore-forming sodium channel isoforms are primarily expressed in the peripheral nervous system (PNS): Na 1.7, Na 1.8 and Na 1.9. These sodium channels have been implicated in painful and painless channelopathies and there has been intense interest in them as potential therapeutic targets in human pain. Emerging evidence suggests Na 1.6 channels are an important isoform in pain sensing. This study aimed to assess, using pharmacological approaches, the function of Na 1.6 channels in peripheral sensory neurons. The potent and Na 1.6 selective β-scorpion toxin Cn2 was used to assess the effect of Na 1.6 channel activation in the PNS. The multidisciplinary approach included Ca imaging, whole-cell patch-clamp recordings, skin-nerve and gut-nerve preparations and in vivo behavioural assessment of pain. Cn2 facilitates Na 1.6 early channel opening, and increased persistent and resurgent currents in large-diameter dorsal root ganglion (DRG) neurons. This promotes enhanced excitatory drive and tonic action potential firing in these neurons. In addition, Na 1.6 channel activation in the skin and gut leads to increased response to mechanical stimuli. Finally, intra-plantar injection of Cn2 causes mechanical but not thermal allodynia. This study confirms selectivity of Cn2 on Na 1.6 channels in sensory neurons. Activation of Na 1.6 channels, in terminals of the skin and viscera, leads to profound changes in neuronal responses to mechanical stimuli. In conclusion, sensory neurons expressing Na 1.6 are important for the transduction of mechanical information in sensory afferents innervating the skin and viscera.
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http://dx.doi.org/10.1113/JP278148DOI Listing
July 2019

The three-dimensional structure of an H-superfamily conotoxin reveals a granulin fold arising from a common ICK cysteine framework.

J Biol Chem 2019 05 11;294(22):8745-8759. Epub 2019 Apr 11.

From the Department of Biology, Linderstrøm-Lang Centre for Protein Science, University of Copenhagen, 2200 Copenhagen N., Denmark,

Venomous marine cone snails produce peptide toxins (conotoxins) that bind ion channels and receptors with high specificity and therefore are important pharmacological tools. Conotoxins contain conserved cysteine residues that form disulfide bonds that stabilize their structures. To gain structural insight into the large, yet poorly characterized conotoxin H-superfamily, we used NMR and CD spectroscopy along with MS-based analyses to investigate H-Vc7.2 from , a peptide with a VI/VII cysteine framework. This framework has Cys-Cys/Cys-Cys/Cys-Cys connectivities, which have invariably been associated with the inhibitor cystine knot (ICK) fold. However, the solution structure of recombinantly expressed and purified H-Vc7.2 revealed that although it displays the expected cysteine connectivities, H-Vc7.2 adopts a different fold consisting of two stacked β-hairpins with opposing β-strands connected by two parallel disulfide bonds, a structure homologous to the N-terminal region of the human granulin protein. Using structural comparisons, we subsequently identified several toxins and nontoxin proteins with this "mini-granulin" fold. These findings raise fundamental questions concerning sequence-structure relationships within peptides and proteins and the key determinants that specify a given fold.
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http://dx.doi.org/10.1074/jbc.RA119.007491DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6552430PMC
May 2019

Fish-hunting cone snail venoms are a rich source of minimized ligands of the vertebrate insulin receptor.

Elife 2019 02 12;8. Epub 2019 Feb 12.

Department of Biology, University of Utah School of Medicine, Salt Lake City, United States.

The fish-hunting marine cone snail uses a specialized venom insulin to induce hypoglycemic shock in its prey. We recently showed that this venom insulin, Con-Ins G1, has unique characteristics relevant to the design of new insulin therapeutics. Here, we show that fish-hunting cone snails provide a rich source of minimized ligands of the vertebrate insulin receptor. Insulins from , and exhibit diverse sequences, yet all bind to and activate the human insulin receptor. Molecular dynamics reveal unique modes of action that are distinct from any other insulins known in nature. When tested in zebrafish and mice, venom insulins significantly lower blood glucose in the streptozotocin-induced model of diabetes. Our findings suggest that cone snails have evolved diverse strategies to activate the vertebrate insulin receptor and provide unique insight into the design of novel drugs for the treatment of diabetes.
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http://dx.doi.org/10.7554/eLife.41574DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6372279PMC
February 2019

Discovery of Novel Conotoxin Candidates Using Machine Learning.

Toxins (Basel) 2018 12 1;10(12). Epub 2018 Dec 1.

Eccles Institute of Human Genetics, University of Utah, Salt Lake City, UT 84112, USA.

Cone snails (genus ) are venomous marine snails that inject prey with a lethal cocktail of conotoxins, small, secreted, and cysteine-rich peptides. Given the diversity and often high affinity for their molecular targets, consisting of ion channels, receptors or transporters, many conotoxins have become invaluable pharmacological probes, drug leads, and therapeutics. Transcriptome sequencing of venom glands followed by de novo assembly and homology-based toxin identification and annotation is currently the state-of-the-art for discovery of new conotoxins. However, homology-based search techniques, by definition, can only detect novel toxins that are homologous to previously reported conotoxins. To overcome these obstacles for discovery, we have created Pipe, a machine learning tool that utilizes prominent chemical characters of conotoxins to predict whether a certain transcript in a transcriptome, which has no otherwise detectable homologs in current reference databases, is a putative conotoxin. By using Pipe on RNASeq data of 10 species, we report 5148 new putative conotoxin transcripts that have no homologues in current reference databases. 896 of these were identified by at least three out of four models used. These data significantly expand current publicly available conotoxin datasets and our approach provides a new computational avenue for the discovery of novel toxin families.
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http://dx.doi.org/10.3390/toxins10120503DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6315676PMC
December 2018

Buzz Kill: Function and Proteomic Composition of Venom from the Giant Assassin Fly (Diptera: Asilidae).

Toxins (Basel) 2018 Nov 5;10(11). Epub 2018 Nov 5.

Venom Evolution Lab, School of Biological Sciences, The University of Queensland, St Lucia, QLD 4072, Australia.

Assassin flies (Diptera: Asilidae) inject paralysing venom into insect prey during hunting, but their venoms are poorly characterised in comparison to those produced by spiders, scorpions, or hymenopteran insects. Here we investigated the composition of the venom of the giant Australian assassin fly using a combination of insect microinjection assays, calcium imaging assays of mammalian sensory neurons, proteomics and transcriptomics. Injection of venom into blowflies () produced rapid contractile paralysis (PD at 1 min = 3.1 μg per fly) followed by death, and also caused immediate activation of mouse dorsal root ganglion neurons (at 50 ng/μL). These results are consistent with venom use for both prey capture and predator deterrence. Paragon searches of tandem mass spectra of venom against a translated thoracic gland RNA-Seq database identified 122 polypeptides present in the venom, including six linear and 21 disulfide-rich peptides. Some of these disulfide-rich peptides display sequence homology to peptide families independently recruited into other animal venoms, including inhibitor cystine knots, cystine-stabilised α/β defensins, Kazal peptides, and von Willebrand factors. Numerous enzymes are present in the venom, including 35 proteases of the S1 family, proteases of the S10, C1A, M12A, M14, and M17 families, and phosphatase, amylase, hydrolase, nuclease, and dehydrogenase-like proteins. These results highlight convergent molecular evolution between the assassin flies and other venomous animals, as well as the unique and rich molecular composition of assassin fly venom.
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http://dx.doi.org/10.3390/toxins10110456DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6266666PMC
November 2018

Entomo-venomics: The evolution, biology and biochemistry of insect venoms.

Toxicon 2018 Nov 26;154:15-27. Epub 2018 Sep 26.

Institute for Molecular Bioscience, The University of Queensland, St. Lucia, QLD 4072, Australia. Electronic address:

The insects are a hyperdiverse class containing more species than all other animal groups combined-and many employ venom to capture prey, deter predators and micro-organisms, or facilitate parasitism or extra-oral digestion. However, with the exception of those made by Hymenoptera (wasps, ants and bees), little is known about insect venoms. Here, we review the current literature on insects that use venom for prey capture and predator deterrence, finding evidence for fourteen independent origins of venom usage among insects, mostly among the hyperdiverse holometabolan orders. Many lineages, including the True Bugs (Heteroptera), robber flies (Asilidae), and larvae of many Neuroptera, Coleoptera and Diptera, use mouthpart-associated venoms to paralyse and pre-digest prey during hunting. In contrast, some Hymenoptera and larval Lepidoptera, and one species of beetle, use non-mouthpart structures to inject venom in order to cause pain to deter potential predators. Several recently published insect venom proteomes indicate molecular convergence between insects and other venomous animal groups, with all insect venoms studied so far being potently bioactive cocktails containing both peptides and larger proteins, including novel peptide and protein families. This review summarises the current state of the field of entomo-venomics.
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http://dx.doi.org/10.1016/j.toxicon.2018.09.004DOI Listing
November 2018

Conopeptides promote itch through human itch receptor hMgprX1.

Toxicon 2018 Nov 21;154:28-34. Epub 2018 Sep 21.

Department of Anesthesiology and Center for the Study of Itch, Washington University School of Medicine, St. Louis MO 63110, USA. Electronic address:

Members of Mas related G-protein coupled receptors (Mrgpr) are known to mediate itch. To date, several compounds have been shown to activate these receptors, including chloroquine, a common antimalarial drug, and peptides of the RF-amide family. However, specific ligands for these receptors are still lacking and there is a need for novel compounds that can be used to modulate the receptors in order to understand the cellular and molecular mechanism in which they mediate itch. Some cone snail venoms were previously shown to induce itch in mice. Here, we show that the venom of Conus textile induces itch through activation of itch-sensing sensory neurons, marked by their sensitivity to chloroquine. Two RF-amide peptides, CNF-Tx1 and CNF-Tx2, were identified in a C. textile venom gland transcriptome. These belong to the conorfamide family of peptides which includes previously described peptides from the venoms of Conus victoriae (CNF-Vc1) and Conus spurius (CNF-Sr1 and CNF-Sr2). We show that CNF-Vc1 and CNF-Sr1 activate MrgprC11 whereas CNF-Vc1 and CNF-Tx2 activate the human MrgprX1 (hMrgprX1). The peptides CNF-Tx1 and CNF-Sr2 do not activate MrgprC11 or hMrgprX1. Intradermal injection of CNF-Vc1 and CNF-Tx2 into the cheek of a transgenic mouse expressing hMrgprX1 instead of endogenous mouse Mrgprs resulted in itch-related scratching thus demonstrating the in vivo activity of these peptides. Using truncated analogues of CNF-Vc1, we identified amino acids at positions 7-14 as important for activity against hMrgprX1. The conopeptides reported here are tools that can be used to advance our understanding of the cellular and molecular mechanism of itch mediated by Mrgprs.
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http://dx.doi.org/10.1016/j.toxicon.2018.09.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6299835PMC
November 2018

A comprehensive portrait of the venom of the giant red bull ant, , reveals a hyperdiverse hymenopteran toxin gene family.

Sci Adv 2018 09 12;4(9):eaau4640. Epub 2018 Sep 12.

Centre for Advance Imaging, The University of Queensland, St Lucia, Queensland 4072, Australia.

Ants (Hymenoptera: Formicidae) are diverse and ubiquitous, and their ability to sting is familiar to many of us. However, their venoms remain largely unstudied. We provide the first comprehensive characterization of a polypeptidic ant venom, that of the giant red bull ant, . We reveal a suite of novel peptides with a range of posttranslational modifications, including disulfide bond formation, dimerization, and glycosylation. One venom peptide has sequence features consistent with an epidermal growth factor fold, while the remaining peptides have features suggestive of a capacity to form amphipathic helices. We show that these peptides are derived from what appears to be a single, pharmacologically diverse, gene superfamily (aculeatoxins) that includes most venom peptides previously reported from the aculeate Hymenoptera. Two aculeatoxins purified from the venom were found to be capable of activating mammalian sensory neurons, consistent with the capacity to produce pain but via distinct mechanisms of action. Further investigation of the major venom peptide MIITX-Mg1a revealed that it can also incapacitate arthropods, indicative of dual utility in both defense and predation. MIITX-Mg1a accomplishes these functions by generating a leak in membrane ion conductance, which alters membrane potential and triggers neuronal depolarization. Our results provide the first insights into the evolution of the major toxin gene superfamily of the aculeate Hymenoptera and provide a new paradigm in the functional evolution of toxins from animal venoms.
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http://dx.doi.org/10.1126/sciadv.aau4640DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6135544PMC
September 2018

Venom peptides as therapeutics: advances, challenges and the future of venom-peptide discovery.

Expert Rev Proteomics 2017 10 13;14(10):931-939. Epub 2017 Sep 13.

a Institute for Molecular Bioscience , University of Queensland , St Lucia , Australia.

Introduction: Animal venoms are complex chemical arsenals. Most venoms are rich in bioactive peptides with proven potential as research tools, drug leads and drugs. Areas covered: We review recent advances in venom-peptide discovery, particularly the adoption of combined transcriptomic/proteomic approaches for the exploration of venom composition. Expert commentary: Advances in transcriptomics and proteomics have dramatically altered the manner and rate of venom-peptide discovery. The increasing trend towards a toxin-driven approach, as opposed to traditional target-based screening of venoms, is likely to expedite the discovery of venom-peptides with novel structures and new and unanticipated mechanisms of action. At the same time, these advances will drive the development of higher-throughput approaches for target identification. Taken together, these approaches should enhance our understanding of the natural ecological function of venom peptides and increase the rate of identification of novel venom-derived pharmacological tools, drug leads and drugs.
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http://dx.doi.org/10.1080/14789450.2017.1377613DOI Listing
October 2017

Venom peptides as pharmacological tools and therapeutics for diabetes.

Neuropharmacology 2017 Dec 5;127:79-86. Epub 2017 Jul 5.

Department of Biology, University of Utah, Salt Lake City, UT 84112, USA. Electronic address:

Diabetes mellitus is a chronic disease caused by a deficiency in production of insulin by the beta cells of the pancreas (type 1 diabetes, T1D), or by partial deficiency of insulin production and the ineffectiveness of the insulin produced (type 2 diabetes, T2D). Animal venoms are a unique source of compounds targeting ion channels and receptors in the nervous and cardiovascular systems. In recent years, several venom peptides have also emerged as pharmacological tools and therapeutics for T1D and T2D. Some of these peptides act directly as mimics of endogenous metabolic hormones while others act on ion channels expressed in pancreatic beta cells. Here, we provide an overview of the discovery of these venom peptides, their mechanisms of action in the context of diabetes, and their therapeutic potential for the treatment of this disease. This article is part of the Special Issue entitled 'Venom-derived Peptides as Pharmacological Tools.'
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http://dx.doi.org/10.1016/j.neuropharm.2017.07.001DOI Listing
December 2017

The Venom Repertoire of Conus gloriamaris (Chemnitz, 1777), the Glory of the Sea.

Mar Drugs 2017 May 20;15(5). Epub 2017 May 20.

Department of Biology, University of Utah, Salt Lake City 84112, UT, USA.

The marine cone snail Conus gloriamaris is an iconic species. For over two centuries, its shell was one of the most prized and valuable natural history objects in the world. Today, cone snails have attracted attention for their remarkable venom components. Many conotoxins are proving valuable as research tools, drug leads, and drugs. In this article, we present the venom gland transcriptome of C. gloriamaris, revealing this species' conotoxin repertoire. More than 100 conotoxin sequences were identified, representing a valuable resource for future drug discovery efforts.
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http://dx.doi.org/10.3390/md15050145DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5450551PMC
May 2017

The Single Disulfide-Directed β-Hairpin Fold. Dynamics, Stability, and Engineering.

Biochemistry 2017 05 2;56(19):2455-2466. Epub 2017 May 2.

Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University , Parkville, Victoria 3052, Australia.

Grafting bioactive peptide sequences onto small cysteine-rich scaffolds is a promising strategy for enhancing their stability and value as novel peptide-based therapeutics. However, correctly folded disulfide-rich peptides can be challenging to produce by either recombinant or synthetic means. The single disulfide-directed β-hairpin (SDH) fold, first observed in contryphan-Vc1, provides a potential alternative to complex disulfide-rich scaffolds. We have undertaken recombinant production of full-length contryphan-Vc1 (rCon-Vc1[Z1Q]) and a truncated analogue (rCon-Vc1[Z1Q]), analyzed the backbone dynamics of rCon-Vc1[Z1Q], and probed the conformational and proteolytic stability of these peptides to evaluate the potential of contryphan-Vc1 as a molecular scaffold. Backbone N relaxation measurements for rCon-Vc1[Z1Q] indicate that the N-terminal domain of the peptide is ordered up to Thr19, whereas the remainder of the C-terminal region is highly flexible. The solution structure of truncated rCon-Vc1[Z1Q] was similar to that of the full-length peptide, indicating that the flexible C-terminus does not have any effect on the structured domain of the peptide. Contryphan-Vc1 exhibited excellent proteolytic stability against trypsin and chymotrypsin but was susceptible to pepsin digestion. We have investigated whether contryphan-Vc1 can accept a bioactive epitope while maintaining the structure of the peptide by introducing peptide sequences based on the DINNN motif of inducible nitric oxide synthase. We show that sCon-Vc1[NNN] binds to the iNOS-binding protein SPSB2 with an affinity of 1.3 μM while maintaining the SDH fold. This study serves as a starting point in utilizing the SDH fold as a peptide scaffold.
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http://dx.doi.org/10.1021/acs.biochem.7b00120DOI Listing
May 2017

Structure and activity of contryphan-Vc2: Importance of the d-amino acid residue.

Toxicon 2017 Apr 17;129:113-122. Epub 2017 Feb 17.

Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville 3052, Victoria, Australia. Electronic address:

In natural proteins and peptides, amino acids exist almost invariably as l-isomers. There are, however, several examples of naturally-occurring peptides containing d-amino acids. In this study we investigated the role of a naturally-occurring d-amino acid in a small peptide identified in the transcriptome of a marine cone snail. This peptide belongs to a family of peptides known as contryphans, all of which contain a single d-amino acid residue. The solution structure of this peptide was solved by NMR, but further investigations with molecular dynamics simulations suggest that its solution behaviour may be more dynamic than suggested by the NMR ensemble. Functional tests in mice uncovered a novel bioactivity, a depressive phenotype that contrasts with the hyperactive phenotypes typically induced by contryphans. Trp3 is important for bioactivity, but this role is independent of the chirality at this position. The d-chirality of Trp3 in this peptide was found to be protective against enzymatic degradation. Analysis by NMR and molecular dynamics simulations indicated an interaction of Trp3 with lipid membranes, suggesting the possibility of a membrane-mediated mechanism of action for this peptide.
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http://dx.doi.org/10.1016/j.toxicon.2017.02.012DOI Listing
April 2017

Insulin as a weapon.

Toxicon 2016 Dec 21;123:56-61. Epub 2016 Oct 21.

Department of Biology, University of Utah, Salt Lake City, UT 84112, USA; Department of Biology, University of Copenhagen, DK-2200, Copenhagen, Denmark. Electronic address:

The discovery of insulin and its use for the treatment of diabetes is undoubtedly one of the true successes of modern medicine. Injectable insulin would prove the first effective treatment for a previously incurable and usually fatal disease. Soon after however, the powerful effects of insulin overdose would be reported, and subsequently exploited for dubious medical and sometimes nefarious purposes. In this article we describe the discovery that certain venomous marine snails of the genus Conus also exploit the powerful effects of insulin overdose, employing it as a weapon for prey capture.
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http://dx.doi.org/10.1016/j.toxicon.2016.10.010DOI Listing
December 2016

Stereoselective synthesis and structural elucidation of dicarba peptides.

Chem Commun (Camb) 2016 Mar;52(24):4446-9

School of Chemistry, Monash University, Clayton 3800, Victoria, Australia.

A facile stereoselective synthesis of cis and trans unsaturated dicarba peptides has been established using preformed diaminosuberic acid derivatives as bridging units. In addition, characteristic spectral differences in the (13)C-NMR spectra of the cis- and trans-isomers show that the chemical shift of carbons in the Δ4,5-diaminosuberic acid residue can be used to assign stereochemistry in unsaturated dicarba peptides formed from ring closing metathesis of linear peptide sequences.
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http://dx.doi.org/10.1039/c5cc10540dDOI Listing
March 2016

A Naturally Occurring Peptide with an Elementary Single Disulfide-Directed β-Hairpin Fold.

Structure 2016 Feb 7;24(2):293-9. Epub 2016 Jan 7.

Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, VIC 3052, Australia. Electronic address:

Certain peptide folds, owing to a combination of intrinsic stability and resilience to amino acid substitutions, are particularly effective for the display of diverse functional groups. Such "privileged scaffolds" are valuable as starting points for the engineering of new bioactive molecules. We have identified a precursor peptide expressed in the venom gland of the marine snail Conus victoriae, which appears to belong to a hitherto undescribed class of molluscan neuropeptides. Mass spectrometry matching with the venom confirmed the complete mature peptide sequence as a 31-residue peptide with a single disulfide bond. Solution structure determination revealed a unique peptide fold that we have designated the single disulfide-directed β hairpin (SDH). The SDH fold is highly resistant to thermal denaturation and forms the core of several other multiple disulfide-containing peptide folds, including the inhibitor cystine knot. This elementary fold may offer a valuable starting point for the design and engineering of new bioactive peptides.
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http://dx.doi.org/10.1016/j.str.2015.11.015DOI Listing
February 2016