Samuel D. Robinson

Diversity of Conotoxin Gene Superfamilies in the Venomous Snail, Conus victoriae

Animal venoms represent a vast library of bioactive peptides and proteins with proven potential, not only as research tools but also as drug leads and therapeutics. This is illustrated clearly by marine cone snails (genus Conus), whose venoms consist of mixtures of hundreds of peptides (conotoxins) with a diverse array of molecular targets, including voltage- and ligand-gated ion channels, G-protein coupled receptors and neurotransmitter transporters. Several conotoxins have found applications as research tools, with some being used or developed as therapeutics. The primary objective of this study was the large-scale discovery of conotoxin sequences from the venom gland of an Australian cone snail species, Conus victoriae. Using cDNA library normalization, high-throughput 454 sequencing, de novo transcriptome assembly and annotation with BLASTX and profile hidden Markov models, we discovered over 100 unique conotoxin sequences from 20 gene superfamilies, the highest diversity of conotoxins so far reported in a single study. Many of the sequences identified are new members of known conotoxin superfamilies, some help to redefine these superfamilies and others represent altogether new classes of conotoxins. In addition, we have demonstrated an efficient combination of methods to mine an animal venom gland and generate a library of sequences encoding bioactive peptides.

Specialized insulin is used for chemical warfare by fish-hunting cone snails

Significance The discovery and characterization of insulin, a key hormone of energy metabolism, provided a life-saving drug for diabetics. We show that insulin can be subverted for nefarious biological purposes: Venomous cone snails use specialized insulins to elicit hypoglycemic shock, facilitating capture of their fish prey. This finding extends our understanding of the chemical and functional diversity of venom components, such that the snail’s arsenal includes a diverse set of neurotoxins that alters neuronal circuitry, as well as components that override glucose homeostasis. The highly expressed venom insulins are distinct from molluscan insulins and exhibit remarkable similarity to fish insulins. They are the smallest of all insulins characterized from any source, potentially providing new insights into structure-function elements of insulin action. More than 100 species of venomous cone snails (genus Conus) are highly effective predators of fish. The vast majority of venom components identified and functionally characterized to date are neurotoxins specifically targeted to receptors, ion channels, and transporters in the nervous system of prey, predators, or competitors. Here we describe a venom component targeting energy metabolism, a radically different mechanism. Two fish-hunting cone snails, Conus geographus and Conus tulipa, have evolved specialized insulins that are expressed as major components of their venoms. These insulins are distinctive in having much greater similarity to fish insulins than to the molluscan hormone and are unique in that posttranslational modifications characteristic of conotoxins (hydroxyproline, γ-carboxyglutamate) are present. When injected into fish, the venom insulin elicits hypoglycemic shock, a condition characterized by dangerously low blood glucose. Our evidence suggests that insulin is specifically used as a weapon for prey capture by a subset of fish-hunting cone snails that use a net strategy to capture prey. Insulin appears to be a component of the nirvana cabal, a toxin combination in these venoms that is released into the water to disorient schools of small fish, making them easier to engulf with the snail’s distended false mouth, which functions as a net. If an entire school of fish simultaneously experiences hypoglycemic shock, this should directly facilitate capture by the predatory snail.

Conotoxin Gene Superfamilies

Conotoxins are the peptidic components of the venoms of marine cone snails (genus Conus). They are remarkably diverse in terms of structure and function. Unique potency and selectivity profiles for a range of neuronal targets have made several conotoxins valuable as research tools, drug leads and even therapeutics, and has resulted in a concerted and increasing drive to identify and characterise new conotoxins. Conotoxins are translated from mRNA as peptide precursors, and cDNA sequencing is now the primary method for identification of new conotoxin sequences. As a result, gene superfamily, a classification based on precursor signal peptide identity, has become the most convenient method of conotoxin classification. Here we review each of the described conotoxin gene superfamilies, with a focus on the structural and functional diversity present in each. This review is intended to serve as a practical guide to conotoxin superfamilies and to facilitate interpretation of the increasing number of conotoxin precursor sequences being identified by targeted-cDNA sequencing and more recently high-throughput transcriptome sequencing.

Dicarba α-conotoxin Vc1.1 analogues with differential selectivity for nicotinic acetylcholine and GABAB receptors.

Conotoxins have emerged as useful leads for the development of novel therapeutic analgesics. These peptides, isolated from marine molluscs of the genus Conus, have evolved exquisite selectivity for receptors and ion channels of excitable tissue. One such peptide, α-conotoxin Vc1.1, is a 16-mer possessing an interlocked disulfide framework. Despite its emergence as a potent analgesic lead, the molecular target and mechanism of action of Vc1.1 have not been elucidated to date. In this paper we describe the regioselective synthesis of dicarba analogues of Vc1.1 using olefin metathesis. The ability of these peptides to inhibit acetylcholine-evoked current at rat α9α10 and α3β4 nicotinic acetylcholine receptors (nAChR) expressed in Xenopus oocytes has been assessed in addition to their ability to inhibit high voltage-activated (HVA) calcium channel current in isolated rat DRG neurons. Their solution structures were determined by NMR spectroscopy. Significantly, we have found that regioselective replacement of the native cystine framework with a dicarba bridge can be used to selectively tune the cyclic peptides innate biological activity for one receptor over another. The 2,8-dicarba Vc1.1 isomer retains activity at γ-aminobutyric acid (GABAB) G protein-coupled receptors, whereas the isomeric 3,16-dicarba Vc1.1 peptide retains activity at the α9α10 nAChR subtype. These singularly acting analogues will enable the elucidation of the biological target responsible for the peptides potent analgesic activity.

Dicarba Analogues of α-Conotoxin RgIA. Structure, Stability, and Activity at Potential Pain Targets

α-Conotoxin RgIA is both an antagonist of the α9α10 nicotinic acetylcholine receptor (nAChR) subtype and an inhibitor of high-voltage-activated N-type calcium channel currents. RgIA has therapeutic potential for the treatment of pain, but reduction of the disulfide bond framework under physiological conditions represents a potential liability for clinical applications. We synthesized four RgIA analogues that replaced native disulfide pairs with nonreducible dicarba bridges. Solution structures were determined by NMR, activity assessed against biological targets, and stability evaluated in human serum. [3,12]-Dicarba analogues retained inhibition of ACh-evoked currents at α9α10 nAChRs but not N-type calcium channel currents, whereas [2,8]-dicarba analogues displayed the opposite pattern of selectivity. The [2,8]-dicarba RgIA analogues were effective in HEK293 cells stably expressing human Cav2.2 channels and transfected with human GABAB receptors. The analogues also exhibited improved serum stability over the native peptide. These selectively acting dicarba analogues may represent mechanistic probes to explore analgesia-related biological receptors.

Novel Peptide Antagonists of Adrenomedullin and Calcitonin Gene-Related Peptide Receptors: Identification, Pharmacological Characterization, and Interactions with Position 74 in Receptor Activity-Modifying Protein 1/3

Human adrenomedullin (AM) is a 52-amino acid peptide belonging to the calcitonin peptide family, which also includes calcitonin gene-related peptide (CGRP) and AM2. The two AM receptors, AM1 and AM2, are calcitonin receptor-like receptor (CL)/receptor activity-modifying protein (RAMP) (RAMP2 and RAMP3, respectively) heterodimers. CGRP receptors comprise CL/RAMP1. The only human AM receptor antagonist (AM22–52) is a truncated form of AM; it has low affinity and is only weakly selective for AM1 over AM2 receptors. To develop novel AM receptor antagonists, we explored the importance of different regions of AM in interactions with AM1, AM2, and CGRP receptors. AM22–52 was the framework for generating further AM fragments (AM26–52 and AM30–52), novel AM/αCGRP chimeras (C1–C5 and C9), and AM/AM2 chimeras (C6–C8). cAMP assays were used to screen the antagonists at all receptors to determine their affinity and selectivity. Circular dichroism spectroscopy was used to investigate the secondary structures of AM and its related peptides. The data indicate that the structures of AM, AM2, and αCGRP differ from one another. Our chimeric approach enabled the identification of two nonselective high-affinity antagonists of AM1, AM2, and CGRP receptors (C2 and C6), one high-affinity antagonist of AM2 receptors (C7), and a weak antagonist selective for the CGRP receptor (C5). By use of receptor mutagenesis, we also determined that the C-terminal nine amino acids of AM seem to be responsible for its interaction with Glu74 of RAMP3. We provide new information on the structure-activity relationship of AM, αCGRP, and AM2 and how AM interacts with CGRP and AM2 receptors.

Discovery by proteogenomics and characterization of an RF-amide neuropeptide from cone snail venom

UNLABELLED In this study, a proteogenomic annotation strategy was used to identify a novel bioactive peptide from the venom of the predatory marine snail Conus victoriae. The peptide, conorfamide-Vc1 (CNF-Vc1), defines a new gene family. The encoded mature peptide was unusual for conotoxins in that it was cysteine-free and, despite low overall sequence similarity, contained two short motifs common to known neuropeptides/hormones. One of these was the C-terminal RF-amide motif, commonly observed in neuropeptides from a range of organisms, including humans. The mature venom peptide was synthesized and characterized structurally and functionally. The peptide was bioactive upon injection into mice, and calcium imaging of mouse dorsal root ganglion (DRG) cells revealed that the peptide elicits an increase in intracellular calcium levels in a subset of DRG neurons. Unusually for most Conus venom peptides, it also elicited an increase in intracellular calcium levels in a subset of non-neuronal cells. BIOLOGICAL SIGNIFICANCE Our findings illustrate the utility of proteogenomics for the discovery of novel, functionally relevant genes and their products. CNF-Vc1 should be useful for understanding the physiological role of RF-amide peptides in the molluscan and mammalian nervous systems.

Hormone-like peptides in the venoms of marine cone snails

The venoms of cone snails (genus Conus) are remarkably complex, consisting of hundreds of typically short, disulfide-rich peptides termed conotoxins. These peptides have diverse pharmacological targets, with injection of venom eliciting a range of physiological responses, including sedation, paralysis and sensory overload. Most conotoxins target the preys nervous system but evidence of venom peptides targeting neuroendocrine processes is emerging. Examples include vasopressin, RFamide neuropeptides and recently also insulin. To investigate the diversity of hormone/neuropeptide-like molecules in the venoms of cone snails we systematically mined the venom gland transcriptomes of several cone snail species and examined secreted venom peptides in dissected and injected venom of the Australian cone snail Conus victoriae. Using this approach we identified several novel hormone/neuropeptide-like toxins, including peptides similar to the bee brain hormone prohormone-4, the mollusc ganglia neuropeptide elevenin, and thyrostimulin, a member of the glycoprotein hormone family, and confirmed the presence of insulin. We confirmed that at least two of these peptides are not only expressed in the venom gland but also form part of the injected venom cocktail, unambiguously demonstrating their role in envenomation. Our findings suggest that hormone/neuropeptide-like toxins are a diverse and integral part of the complex envenomation strategy of Conus. Exploration of this group of venom components offers an exciting new avenue for the discovery of novel pharmacological tools and drug candidates, complementary to conotoxins.

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

ABSTRACT 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.

Interactions of disulfide‐deficient selenocysteine analogs of μ‐conotoxin BuIIIB with the α‐subunit of the voltage‐gated sodium channel subtype 1.3

Inhibitors of the α‐subunit of the voltage‐gated sodium channel subtype 1.3 (NaV1.3) are of interest as pharmacological tools for the study of neuropathic pain associated with spinal cord injury and have potential therapeutic applications. The recently described μ‐conotoxin BuIIIB (μ‐BuIIIB) from Conus bullatus was shown to block NaV1.3 with submicromolar potency (Kd = 0.2 μm), making it one of the most potent peptidic inhibitors of this subtype described to date. However, oxidative folding of μ‐BuIIIB results in numerous folding isoforms, making it difficult to obtain sufficient quantities of the active form of the peptide for detailed structure–activity studies. In the present study, we report the synthesis and characterization of μ‐BuIIIB analogs incorporating a disulfide‐deficient, diselenide‐containing scaffold designed to simplify synthesis and facilitate structure–activity studies directed at identifying amino acid residues involved in NaV1.3 blockade. Our results indicate that, similar to other μ‐conotoxins, the C‐terminal residues (Trp16, Arg18 and His20) are most crucial for NaV1 blockade. At the N‐terminus, replacement of Glu3 by Ala resulted in an analog with an increased potency for NaV1.3 (Kd = 0.07 μm), implicating this position as a potential site for modification for increased potency and/or selectivity. Further examination of this position showed that increased negative charge, through γ‐carboxyglutamate replacement, decreased potency (Kd = 0.33 μm), whereas replacement with positively‐charged 2,4‐diamonobutyric acid increased potency (Kd = 0.036 μm). These results provide a foundation for the design and synthesis of μ‐BuIIIB‐based analogs with increased potency against NaV1.3.