Using Constellation Pharmacology to Characterize a Novel α-Conotoxin from Conus ateralbus.

The venom of cone snails has been proven to be a rich source of bioactive peptides that target a variety of ion channels and receptors. α-Conotoxins (αCtx) interact with nicotinic acetylcholine receptors (nAChRs) and are powerful tools for investigating the structure and function of the various nAChR subtypes. By studying how conotoxins interact with nAChRs, we can improve our understanding of these receptors, leading to new insights into neurological diseases associated with nAChRs. Here, we describe the discovery and characterization of a novel conotoxin from Conus ateralbus, αCtx-AtIA, which has an amino acid sequence homologous to the well-described αCtx-PeIA, but with a different selectivity profile towards nAChRs. We tested the synthetic αCtx-AtIA using the calcium imaging-based Constellation Pharmacology assay on mouse DRG neurons and found that αCtx-AtIA significantly inhibited ACh-induced calcium influx in the presence of an α7 positive allosteric modulator, PNU-120596 (PNU). However, αCtx-AtIA did not display any activity in the absence of PNU. These findings were further validated using two-electrode voltage clamp electrophysiology performed on oocytes overexpressing mouse α3ß4, α6/α3ß4 and α7 nAChRs subtypes. We observed that αCtx-AtIA displayed no or low potency in blocking α3ß4 and α6/α3ß4 receptors, respectively, but improved potency and selectivity to block α7 nAChRs when compared with αCtx-PeIA. Through the synthesis of two additional analogs of αCtx-AtIA and subsequent characterization using Constellation Pharmacology, we were able to identify residue Trp18 as a major contributor to the activity of the peptide.

Analogs of α-conotoxin PnIC selectively inhibit α7ß2- over α7-only subtype nicotinic acetylcholine receptors via a novel allosteric mechanism.

This study was undertaken to identify and characterize the first ligands capable of selectively identifying nicotinic acetylcholine receptors containing α7 and ß2 subunits (α7ß2-nAChR subtype). Basal forebrain cholinergic neurons express α7ß2-nAChR. Here, they appear to mediate neuronal dysfunction induced by the elevated levels of oligomeric amyloid-ß associated with early Alzheimer's disease. Additional work indicates that α7ß2-nAChR are expressed across several further critically important cholinergic and GABAergic neuronal circuits within the central nervous system. Further studies, however, are significantly hindered by the inability of currently available ligands to distinguish heteromeric α7ß2-nAChR from the closely related and more widespread homomeric α7-only-nAChR subtype. Functional screening using two-electrode voltage-clamp electrophysiology identified a family of α7ß2-nAChR-selective analogs of α-conotoxin PnIC (α-CtxPnIC). A combined electrophysiology, functional kinetics, site-directed mutagenesis, and molecular dynamics approach was used to further characterize the α7ß2-nAChR selectivity and site of action of these α-CtxPnIC analogs. We determined that α7ß2-nAChR selectivity of α-CtxPnIC analogs arises from interactions at a site distinct from the orthosteric agonist-binding site shared between α7ß2- and α7-only-nAChR. As numerous previously identified α-Ctx ligands are competitive antagonists of orthosteric agonist-binding sites, this study profoundly expands the scope of use of α-Ctx ligands (which have already provided important nAChR research and translational breakthroughs). More immediately, analogs of α-CtxPnIC promise to enable, for the first time, both comprehensive mapping of the distribution of α7ß2-nAChR and detailed investigations of their physiological roles.

Somatostatin venom analogs evolved by fish-hunting cone snails: From prey capture behavior to identifying drug leads.

Somatostatin (SS) is a peptide hormone with diverse physiological roles. By investigating a deep-water clade of fish-hunting cone snails, we show that predator-prey evolution has generated a diverse set of SS analogs, each optimized to elicit specific systemic physiological effects in prey. The increased metabolic stability, distinct SS receptor activation profiles, and chemical diversity of the venom analogs make them suitable leads for therapeutic application, including pain, cancer, and endocrine disorders. Our findings not only establish the existence of SS-like peptides in animal venoms but also serve as a model for the synergy gained from combining molecular phylogenetics and behavioral observations to optimize the discovery of natural products with biomedical potential.

Cy3-RgIA-5727 Labels and Inhibits α9-Containing nAChRs of Cochlear Hair Cells.

Efferent cholinergic neurons inhibit sensory hair cells of the vertebrate inner ear through the combined action of calcium-permeable α9α10-containing nicotinic acetylcholine receptors (nAChRs) and associated calcium-dependent potassium channels. The venom of cone snails is a rich repository of bioactive peptides, many with channel blocking activities. The conopeptide analog, RgIA-5474, is a specific and potent antagonist of α9α10-containing nAChRs. We added an alkyl functional group to the N-terminus of the RgIA-5474, to enable click chemistry addition of the fluorescent cyanine dye, Cy3. The resulting peptide, Cy3-RgIA-5727, potently blocked mouse α9α10 nAChRs expressed in Xenopus oocytes (IC50 23 pM), with 290-fold less activity on α7 nAChRs and 40,000-fold less activity on all other tested nAChR subtypes. The tight binding of Cy3-RgIA-5727 provided robust visualization of hair cell nAChRs juxtaposed to cholinergic efferent terminals in excised, unfixed cochlear tissue from mice. Presumptive postsynaptic sites on outer hair cells (OHCs) were labeled, but absent from inner hair cells (IHCs) and from OHCs in cochlear tissue from α9-null mice and in cochlear tissue pre-incubated with non-Cy3-conjugated RgIA-5474. In cochlear tissue from younger (postnatal day 10) mice, Cy3-RgIA-5727 also labeled IHCs, corresponding to transient efferent innervation at that age. Cy3 puncta in Kölliker's organ remained in the α9-null tissue. Pre-exposure with non-Cy3-conjugated RgIA-5474 or bovine serum albumin reduced this non-specific labeling to variable extents in different preparations. Cy3-RgIA-5727 and RgIA-5474 blocked the native hair cell nAChRs, within the constraints of application to the excised cochlear tissue. Cy3-RgIA-5727 or RgIA-5474 block of efferent synaptic currents in young IHCs was not relieved after 50 min washing, so effectively irreversible.

Selective Penicillamine Substitution Enables Development of a Potent Analgesic Peptide that Acts through a Non-Opioid-Based Mechanism.

Venom-derived compounds are of broad interest in neuropharmacology and drug development. α-Conotoxins are small disulfide-containing peptides from Conus snails that target nicotinic acetylcholine receptors (nAChRs) and are in clinical development for non-opioid-based treatment of intractable pain. Although refined by evolution for interaction with target prey receptors, enhancements of pharmacological properties are needed for use in mammalian systems. Therefore, we synthesized analogues of α-conotoxin RgIA using a combination of selective penicillamine substitutions together with natural and non-natural amino acid replacements. This approach resulted in a peptide with 9000-fold increased potency on the human α9α10 nAChR and improved resistance to disulfide shuffling compared to the native peptide. The lead analogue, RgIA-5474, potently blocked α9α10 nAChRs, but not opioid- or other pain-related targets. In addition, RgIA-5474 effectively reversed chemotherapy-induced neuropathic pain.

Discovery of a Potent Conorfamide from Conus episcopatus Using a Novel Zebrafish Larvae Assay.

Natural products such as conotoxins have tremendous potential as tools for biomedical research and for the treatment of different human diseases. Conotoxins are peptides present in the venoms of predatory cone snails that have a rich diversity of pharmacological functions. One of the major bottlenecks in natural products research is the rapid identification and evaluation of bioactive molecules. To overcome this limitation, we designed a set of light-induced behavioral assays in zebrafish larvae to screen for bioactive conotoxins. We used this screening approach to test several unique conotoxins derived from different cone snail clades and discovered that a conorfamide from Conus episcopatus, CNF-Ep1, had the most dramatic alterations in the locomotor behavior of zebrafish larvae. Interestingly, CNF-Ep1 is also bioactive in several mouse assay systems when tested in vitro and in vivo. Our novel screening platform can thus accelerate the identification of bioactive marine natural products, and the first compound discovered using this assay has intriguing properties that may uncover novel neuronal circuitry.

Computational and Functional Mapping of Human and Rat α6ß4 Nicotinic Acetylcholine Receptors Reveals Species-Specific Ligand-Binding Motifs.

Nicotinic acetylcholine receptors (nAChRs) are pharmacological targets for the treatment of neuropathic pain, and the α6ß4 subtype has been identified as particularly promising. Rat α6ß4 nAChRs are less sensitive to some ligands than the human homologue potentially complicating the use of rodent α6ß4 receptors for screening therapeutic compounds. We used molecular dynamics simulations coupled with functional assays to study the interaction between α-conotoxin PeIA and α6ß4 nAChRs and to identify key ligand-receptor interactions that contribute to species differences in α-conotoxin potency. Our results show that human and rat α6ß4 nAChRs have distinct ligand-binding motifs and show markedly different sensitivities to α-conotoxins. These studies facilitated the creation of PeIA-5667, a peptide that shows 270-fold higher potency for rat α6ß4 nAChRs over native PeIA and similar potency for the human homologue. Our results may inform the design of therapeutic ligands that target α6ß4 nAChRs for the treatment of neuropathic pain.

Purification and Characterization of the Pink-Floyd Drillipeptide, a Bioactive Venom Peptide from Clavus davidgilmouri (Gastropoda: Conoidea: Drilliidae).

The cone snails (family Conidae) are the best known and most intensively studied venomous marine gastropods. However, of the total biodiversity of venomous marine mollusks (superfamily Conoidea, >20,000 species), cone snails comprise a minor fraction. The venoms of the family Drilliidae, a highly diversified family in Conoidea, have not previously been investigated. In this report, we provide the first biochemical characterization of a component in a Drilliidae venom and define a gene superfamily of venom peptides. A bioactive peptide, cdg14a, was purified from the venom of Clavus davidgilmouri Fedosov and Puillandre, 2020. The peptide is small (23 amino acids), disulfide-rich (4 cysteine residues) and belongs to the J-like drillipeptide gene superfamily. Other members of this superfamily share a conserved signal sequence and the same arrangement of cysteine residues in their predicted mature peptide sequences. The cdg14a peptide was chemically synthesized in its bioactive form. It elicited scratching and hyperactivity, followed by a paw-thumping phenotype in mice. Using the Constellation Pharmacology platform, the cdg14a drillipeptide was shown to cause increased excitability in a majority of non-peptidergic nociceptors, but did not affect other subclasses of dorsal root ganglion (DRG) neurons. This suggests that the cdg14a drillipeptide may be blocking a specific molecular isoform of potassium channels. The potency and selectivity of this biochemically characterized drillipeptide suggest that the venoms of the Drilliidae are a rich source of novel and selective ligands for ion channels and other important signaling molecules in the nervous system.

Studies of Conorfamide-Sr3 on Human Voltage-Gated Kv1 Potassium Channel Subtypes.

Recently, Conorfamide-Sr3 (CNF-Sr3) was isolated from the venom of Conus spurius and was demonstrated to have an inhibitory concentration-dependent effect on the Shaker K+ channel. The voltage-gated potassium channels play critical functions on cellular signaling, from the regeneration of action potentials in neurons to the regulation of insulin secretion in pancreatic cells, among others. In mammals, there are at least 40 genes encoding voltage-gated K+ channels and the process of expression of some of them may include alternative splicing. Given the enormous variety of these channels and the proven use of conotoxins as tools to distinguish different ligand- and voltage-gated ion channels, in this work, we explored the possible effect of CNF-Sr3 on four human voltage-gated K+ channel subtypes homologous to the Shaker channel. CNF-Sr3 showed a 10 times higher affinity for the Kv1.6 subtype with respect to Kv1.3 (IC50 = 2.7 and 24 µM, respectively) and no significant effect on Kv1.4 and Kv1.5 at 10 µM. Thus, CNF-Sr3 might become a novel molecular probe to study diverse aspects of human Kv1.3 and Kv1.6 channels.

Author Correction: A structurally minimized yet fully active insulin based on cone-snail venom insulin principles.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Critical residue properties for potency and selectivity of α-Conotoxin RgIA towards α9α10 nicotinic acetylcholine receptors.

The α9α10 nicotinic acetylcholine receptor (nAChR) has been characterized as an effective anti-pain target that functions through a non-opioid mechanism. However, as a pentameric ion channel comprised of two different subunits, the specific targeting of α9α10 nAChRs has proven challenging. Previously the 13-amino-acid peptide, RgIA, was shown to block α9α10 nAChRs with high potency and specificity. This peptide, characterized from the venom of the carnivorous marine snail, Conus regius, produced analgesia in several rodent models of chronic pain. Despite promising pre-clinical data in behavioral assays, the number of specific α9α10 nAChR antagonists remains small and the physiological mechanisms of analgesia remain cryptic. In this study, we implement amino-acid substitutions to definitively characterize the chemical properties of RgIA that contribute to its activity against α9α10 nAChRs. Using this mutational approach, we determined the vital role of biochemical side-chain properties and amino acids in the second loop that are amenable to substitutions to further engineer next-generation analogs for the blockade of α9α10 nAChRs.

A structurally minimized yet fully active insulin based on cone-snail venom insulin principles.

Human insulin and its current therapeutic analogs all show propensity, albeit varyingly, to self-associate into dimers and hexamers, which delays their onset of action and makes blood glucose management difficult for people with diabetes. Recently, we described a monomeric, insulin-like peptide in cone-snail venom with moderate human insulin-like bioactivity. Here, with insights from structural biology studies, we report the development of mini-Ins-a human des-octapeptide insulin analog-as a structurally minimal, full-potency insulin. Mini-Ins is monomeric and, despite the lack of the canonical B-chain C-terminal octapeptide, has similar receptor binding affinity to human insulin. Four mutations compensate for the lack of contacts normally made by the octapeptide. Mini-Ins also has similar in vitro insulin signaling and in vivo bioactivities to human insulin. The full bioactivity of mini-Ins demonstrates the dispensability of the PheB24-PheB25-TyrB26 aromatic triplet and opens a new direction for therapeutic insulin development.

Expression of α3ß2ß4 nicotinic acetylcholine receptors by rat adrenal chromaffin cells determined using novel conopeptide antagonists.

Adrenal chromaffin cells release neurotransmitters in response to stress and may be involved in conditions such as post-traumatic stress and anxiety disorders. Neurotransmitter release is triggered, in part, by activation of nicotinic acetylcholine receptors (nAChRs). However, despite decades of use as a model system for studying exocytosis, the nAChR subtypes involved have not been pharmacologically identified. Quantitative real-time PCR of rat adrenal medulla revealed an abundance of mRNAs for α3, α7, ß2, and ß4 subunits. Whole-cell patch-clamp electrophysiology of chromaffin cells and subtype-selective ligands were used to probe for nAChRs derived from the mRNAs found in adrenal medulla. A novel conopeptide antagonist, PeIA-5469, was created that is highly selective for α3ß2 over other nAChR subtypes heterologously expressed in Xenopus laevis oocytes. Experiments using PeIA-5469 and the α3ß4-selective α-conotoxin TxID revealed that rat adrenal medulla contain two populations of chromaffin cells that express either α3ß4 nAChRs alone or α3ß4 together with the α3ß2ß4 subtype. Conclusions were derived from observations that acetylcholine-gated currents in some cells were sensitive to inhibition by PeIA-5469 and TxID, while in other cells, currents were sensitive only to TxID. Expression of functional α7 nAChRs was determined using three α7-selective ligands: the agonist PNU282987, the positive allosteric modulator PNU120596, and the antagonist α-conotoxin [V11L,V16D]ArIB. The results of these studies identify for the first time the expression of α3ß2ß4 nAChRs as well as functional α7 nAChRs by rat adrenal chromaffin cells.

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

Conus ateralbus 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 Conus. In contrast, there is no comparable sequence similarity with δ-conotoxins from the venoms of molluscivorous Conus 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.

α-Conotoxin VnIB from Conus ventricosus is a potent and selective antagonist of α6ß4* nicotinic acetylcholine receptors.

α6-containing (α6*) nicotinic acetylcholine receptors (nAChRs) are expressed throughout the periphery and the central nervous system and constitute putative therapeutic targets in pain, addiction and movement disorders. The α6ß2* nAChRs are relatively well studied, in part due to the availability of target specific α-conotoxins (α-Ctxs). In contrast, all native α-Ctxs identified that potently block α6ß4 nAChRs exhibit higher potencies for the closely related α6ß2ß3 and/or α3ß4 subtypes. In this study, we have identified a novel peptide from Conus ventricosus with pronounced selectivity for the α6ß4 nAChR. The peptide-encoding gene was cloned from genomic DNA and the predicted mature peptide, α-Ctx VnIB, was synthesized. The functional properties of VnIB were characterized at rat and human nAChRs expressed in Xenopus oocytes by two-electrode voltage clamp electrophysiology. VnIB potently inhibited ACh-evoked currents at rα6ß4 and rα6/α3ß4 nAChRs, displayed ∼20-fold and ∼250-fold lower potencies at rα3ß4 and rα6/α3ß2ß3 receptors, respectively, and exhibited negligible effects at eight other nAChR subtypes. Interestingly, even higher degrees of selectivity were observed for hα6/α3ß4 over hα6/α3ß2ß3 and hα3ß4 receptors. Finally, VnIB displayed fast binding kinetics at rα6/α3ß4 (on-rate t½â€¯= 0.87 min-1, off-rate t½â€¯= 2.7 min-1). The overall preference of VnIB for ß4* over ß2* nAChRs is similar to the selectivity profiles of other 4/6 α-Ctxs. However, in contrast to previously identified native α-Ctxs targeting α6* nAChRs, VnIB displays pronounced selectivity for α6ß4 nAChRs over both α3ß4 and α6ß2ß3 receptors. VnIB thus represents a novel molecular probe for elucidating the physiological role and therapeutic properties of α6ß4* nAChRs.

PeIA-5466: A Novel Peptide Antagonist Containing Non-natural Amino Acids That Selectively Targets α3ß2 Nicotinic Acetylcholine Receptors.

Pharmacologically distinguishing α3ß2 nicotinic acetylcholine receptors (nAChRs) from closely related subtypes, particularly α6ß2, has been challenging due to the lack of subtype-selective ligands. We created analogs of α-conotoxin (α-Ctx) PeIA to identify ligand-receptor interactions that could be exploited to selectively increase potency and selectivity for α3ß2 nAChRs. A series of PeIA analogs were synthesized by replacing amino acid residues in the second disulfide loop with standard or nonstandard residues and assessing their activity on α3ß2 and α6/α3ß2ß3 nAChRs heterologously expressed in Xenopus laevis oocytes. Asparagine11 was found to occupy a pivotal position, and when replaced with negatively charged amino acids, selectivity for α3ß2 over α6/α3ß2ß3 nAChRs was substantially increased. Second generation peptides were then designed to further improve both potency and selectivity. One peptide, PeIA-5466, was ∼300-fold more potent on α3ß2 than α6/α3ß2ß3 and is the most α3ß2-selective antagonist heretofore reported.

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

The fish-hunting marine cone snail Conus geographus 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 C. geographus, Conus tulipa and Conus kinoshitai 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.

Insulin is a hormone critical for maintaining healthy blood sugar levels in humans. When the insulin system becomes faulty, blood sugar levels become too high, which can lead to diabetes. At the moment, the only effective treatment for one of the major types of diabetes are daily insulin injections. However, designing fast-acting insulin drugs has remained a challenge. Insulin molecules form clusters (so-called hexamers) that first have to dissolve in the body to activate the insulin receptor, which plays a key role in regulating the blood sugar levels throughout the body. This can take time and can therefore delay the blood-sugar control. In 2015, researchers discovered that the fish-hunting cone snail Conus geographus uses a specific type of insulin to capture its prey ­ fish. The cone snail releases insulin into the surrounding water and then engulfs its victim with its mouth. This induces dangerously low blood sugar levels in the fish and so makes them an easy target. Unlike the human version, the snail insulin does not cluster, and despite structural differences, can bind to the human insulin receptor. Now, Ahorukomeye, Disotuar et al. ­ including some of the authors involved in the previous study ­ wanted to find out whether other fish-hunting cone snails also make insulins and if they differed from the one previously discovered in C. geographus. The insulin molecules were extracted and analyzed, and the results showed that the three cone snail species had different versions of insulin ­ but none of them formed clusters. Ahorukomeye, Disotuar et al. further revealed that the snail insulins could bind to the human insulin receptors and could also reverse high blood sugar levels in fish and mouse models of the disease. This research may help guide future studies looking into developing fast-acting insulin drugs for diabetic patients. A next step will be to fully understand how snail insulins can be active at the human receptor without forming clusters. Cone snails solved this problem millions of years ago and by understanding how they have done this, researchers are hoping to redesign current diabetic therapeutics. Since the snail insulins do not form clusters and should act faster than currently available insulin drugs, they may lead to better or new diabetes treatments.

Molecular determinants of α-conotoxin potency for inhibition of human and rat α6ß4 nicotinic acetylcholine receptors.

Nicotinic acetylcholine receptors (nAChRs) containing α6 and ß4 subunits are expressed by dorsal root ganglion neurons and have been implicated in neuropathic pain. Rodent models are often used to evaluate the efficacy of analgesic compounds, but species differences may affect the activity of some nAChR ligands. A previous candidate α-conotoxin-based therapeutic yielded promising results in rodent models, but failed in human clinical trials, emphasizing the importance of understanding species differences in ligand activity. Here, we show that human and rat α6/α3ß4 nAChRs expressed in Xenopus laevis oocytes exhibit differential sensitivity to α-conotoxins. Sequence homology comparisons of human and rat α6ß4 nAChR subunits indicated that α6 residues forming the ligand-binding pocket are highly conserved between the two species, but several residues of ß4 differed, including a Leu-Gln difference at position 119. X-ray crystallography of α-conotoxin PeIA complexed with the Aplysia californica acetylcholine-binding protein (AChBP) revealed that binding of PeIA orients Pro13 in close proximity to residue 119 of the AChBP complementary subunit. Site-directed mutagenesis studies revealed that Leu119 of human ß4 contributes to higher sensitivity of human α6/α3ß4 nAChRs to α-conotoxins, and structure-activity studies indicated that PeIA Pro13 is critical for high potency. Human and rat α6/α3ß4 nAChRs displayed differential sensitivities to perturbations of the interaction between PeIA Pro13 and residue 119 of the ß4 subunit. These results highlight the potential significance of species differences in α6ß4 nAChR pharmacology that should be taken into consideration when evaluating the activity of candidate human therapeutics in rodent models.

Conopeptides promote itch through human itch receptor hMgprX1.

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.

Structure and Biological Activity of a Turripeptide from Unedogemmula bisaya Venom.

The turripeptide ubi3a was isolated from the venom of the marine gastropod Unedogemmula bisaya, family Turridae, by bioassay-guided purification; both native and synthetic ubi3a elicited prolonged tremors when injected intracranially into mice. The sequence of the peptide, DCCOCOAGAVRCRFACC-NH2 (O = 4-hydroxyproline) follows the framework III pattern for cysteines (CC-C-C-CC) in the M-superfamily of conopeptides. The three-dimensional structure determined by NMR spectroscopy indicated a disulfide connectivity that is not found in conopeptides with the cysteine framework III: C1-C4, C2-C6, C3-C5. The peptide inhibited the activity of the α9α10 nicotinic acetylcholine receptor with relatively low affinity (IC50, 10.2 µM). Initial Constellation Pharmacology data revealed an excitatory activity of ubi3a on a specific subset of mouse dorsal root ganglion neurons.