A Novel α4/7-Conotoxin QuIA Selectively Inhibits α3ß2 and α6/α3ß4 Nicotinic Acetylcholine Receptor Subtypes with High Efficacy.

α6ß4 nAChR is expressed in the peripheral and central nervous systems and is associated with pain, addiction, and movement disorders. Natural α-conotoxins (α-CTxs) can effectively block different nAChR subtypes with higher efficacy and selectivity. However, the research on α6ß4 nAChR is relatively poor, partly because of the lack of available target-specific α-CTxs. In this study, we synthesized a novel α-4/7 conotoxin QuIA that was found from Conus quercinus. We investigated the efficacy of this peptide to different nAChR subtypes using a two-electrode voltage-clamp technique. Remarkably, we found α-QuIA inhibited the neuronal α3ß2 and α6/α3ß4 nAChR subtypes with significantly high affinity (IC50 was 55.7 nM and 90.68 nM, respectively), and did not block other nAChR subtypes even at a high concentration of 10 µM. In contrast, most α-CTxs have been determined so far to effectively block the α6/α3ß4 nAChR subtype while also maintaining a similar higher efficacy against the closely related α6ß2ß3 and/or α3ß4 subtypes, which are different from QuIA. In conclusion, α-QuIA is a novel α4/7-CTx, which has the potential to develop as an effective neuropharmacology tool to detect the function of α6ß4 nAChR.

Isolation and characterization of five novel disulfide-poor conopeptides from Conus marmoreus venom

Background: Conopeptides from cone snail venom have aroused great interest related to the discovery of novel bioactive candidates, due to their excellent prospects for the treatment of various health problems such as pain, addiction, psychosis and epilepsy. In order to explore novel biopeptides, we investigated the structure and function of five novel conopeptides isolated from the venom of Conus marmoreus from South China Sea. Methods: C. marmoreus crude venom was prepared, fractionated and purified by HPLC system. The primary sequences of the five novel disulfide-poor conopeptides Mr-1 to Mr-5 were identified by comprehensive analysis of de novo MALDI-TOF tandem mass spectrometry and Edman degradation data. In order to investigate their function, these five conopeptides were synthesized by Fmoc-SPPS chemistry, and their biological effects at several heterologous rat nicotinic acetylcholine receptor (nAChR) subtypes (α1β1δε, α3β2, α3β4, α4β2) were determined by electrophysiological technique. Results: Five novel disulfide-poor conopeptides were identified and named as follows: Mr-1 (DWEYHAHPKPNSFWT), Mr-2 (YPTRAYPSNKFG), Mr-3 (NVIQAPAQSVAPP NTST), Mr-4 [KENVLNKLKSK(L/I)] and Mr-5 [NAVAAAN(L/I)PG(L/I)V]. None of them contains a disulfide bond. The sequences of conopeptides Mr-2 to Mr-5 do not belong to any category of the known disulfide-poor conopeptides. No significant activity against the above nAChR subtypes were observed for the five conopeptides at 100 µM. Conclusion: We purified and structurally characterized five novel disulfide-poor conopeptides from C. marmoreus crude venom and first investigated their nAChR inhibitory effects. This work expanded our knowledge on the structure and function of disulfide-poor conopeptides from this cone snail venom.(AU)

α-Conotoxin Bt1.8 from Conus betulinus selectively inhibits α6/α3ß2ß3 and α3ß2 nicotinic acetylcholine receptor subtypes.

α-Conotoxins are small disulfide-rich peptides found in the venom of marine cone snails and are potent antagonists of nicotinic acetylcholine receptors (nAChRs). They are valuable pharmacological tools and have potential therapeutic applications for the treatment of chronic pain or neurological diseases and disorders. In the present study, we synthesized and functionally characterized a novel α-conotoxin Bt1.8, which was cloned from Conus betulinus. Bt1.8 selectively inhibited ACh-evoked currents in Xenopus oocytes expressing rat(r) α6/α3ß2ß3 and rα3ß2 nAChRs with an IC50 of 2.1 nM and 9.4 nM, respectively, and similar potency for human (h) α6/α3ß2ß3 and hα3ß2 nAChRs. Additionally, Bt1.8 had higher binding affinity with a slower dissociation rate for the rα6/α3ß2ß3 subtype compared to rα3ß2. The amino acid sequence of Bt1.8 is significantly different from other reported α-conotoxins targeting the two nAChR subtypes. Further Alanine scanning analyses demonstrated that residues Ile9, Leu10, Asn11, Asn12 and Asn14 are critical for its inhibitory activity at the α6/α3ß2ß3 and α3ß2 subtypes. Moreover, the NMR structure of Bt1.8 indicated the presence of a relatively larger hydrophobic zone than other α4/7-conotoxins which may explain its potent inhibition at α6/α3ß2ß3 nAChRs.

Synthesis, Structural and Pharmacological Characterizations of CIC, a Novel α-Conotoxin with an Extended N-Terminal Tail.

Cone snails are venomous marine predators that rely on fast-acting venom to subdue their prey and defend against aggressors. The conotoxins produced in the venom gland are small disulfide-rich peptides with high affinity and selectivity for their pharmacological targets. A dominant group comprises α-conotoxins, targeting nicotinic acetylcholine receptors. Here, we report on the synthesis, structure determination and biological activity of a novel α-conotoxin, CIC, found in the predatory venom of the piscivorous species Conus catus and its truncated mutant Δ-CIC. CIC is a 4/7 α-conotoxin with an unusual extended N-terminal tail. High-resolution NMR spectroscopy shows a major influence of the N-terminal tail on the apparent rigidity of the three-dimensional structure of CIC compared to the more flexible Δ-CIC. Surprisingly, this effect on the structure does not alter the biological activity, since both peptides selectively inhibit α3ß2 and α6/α3ß2ß3 nAChRs with almost identical sub- to low micromolar inhibition constants. Our results suggest that the N-terminal part of α-conotoxins can accommodate chemical modifications without affecting their pharmacology.

Structure-Function Elucidation of a New α-Conotoxin, MilIA, from Conus milneedwardsi.

The a-Conotoxins are peptide toxins that are found in the venom of marine cone snails and they are potent antagonists of various subtypes of nicotinic acetylcholine receptors (nAChRs). Because nAChRs have an important role in regulating transmitter release, cell excitability, and neuronal integration, nAChR dysfunctions have been implicated in a variety of severe pathologies. We describe the isolation and characterization of α-conotoxin MilIA, the first conopeptide from the venom of Conus milneedwardsi. The peptide was characterized by electrophysiological screening against several types of cloned nAChRs that were expressed in Xenopus laevis oocytes. MilIA, which is a member of the α3/5 family, is an antagonist of muscle type nAChRs with a high selectivity for muscle versus neuronal subtype nAChRs. Several analogues were designed and investigated for their activity in order to determine the key epitopes of MilIA. Native MilIA and analogues both showed activity at the fetal muscle type nAChR. Two single mutations (Met9 and Asn10) allowed for MilIA to strongly discriminate between the two types of muscle nAChRs. Moreover, one analogue, MilIA [∆1,M2R, M9G, N10K, H11K], displayed a remarkable enhanced potency when compared to native peptide. The key residues that are responsible for switching between muscle and neuronal nAChRs preference were elucidated. Interestingly, the same analogue showed a preference for α9α10 nAChRs among the neuronal types.

Conomarphins cause paralysis in mollusk: Critical and tunable structural elements for bioactivity.

Two conomarphins were purified as the major component of the venom of Conus eburneus. Conomarphins Eb1 and Eb2 showed biological activity in the mollusk Pomacea padulosa, causing sluggishness and retraction of siphon, foot, and cephalic tentacles. To further probe the effects of conserved amino acids and posttranslational modifications in conomarphins, we prepared four synthetic analogues: conomarphin Eb1 Hyp10Pro, Hyp10Ala, d-Phe13Ala, and l-Phe13 variants. Structure-activity relationship analysis indicated that d-Phe13 is critical to the biological activity of conomarphins. In contrast, amino acid changes at position 10 and removal of posttranslational modification in Hyp10Pro can be tolerated. The high expression level and observed mollusk activity of conomarphins may suggest their potential role as defensive arsenal of Conoidean snails against other predatory gastropods.

A rink-amide soluble support: high purity conotoxins and other peptides accessed with minimal reagents.

The use of peptides as therapeutics has been growing due to their biocompatibility. Solid phase peptide synthesis typically used to access these peptides requires excess reagents and/or microwave irradiation to drive reactions to completion because the reaction medium is heterogeneous. Reported herein is a soluble polynorbornene support containing rink amide attached sites for synthesizing oligopeptides and conotoxins in high purity using only 1.2 to 2 equivalents of coupling reagents. The support can be isolated as a precipitate from the reaction medium by adding ether. The loading capacity of the support can be easily determined by spectroscopy and can also be tuned by varying the monomer ratio. This support is promising for accessing peptides as the methodology uses minimal reagents and by-products can be easily separated.

Discovery Methodology of Novel Conotoxins from Conus Species.

Cone snail venoms provide an ideal resource for neuropharmacological tools and drug candidates discovery, which have become a research hotspot in neuroscience and new drug development. More than 1,000,000 natural peptides are produced by cone snails, but less than 0.1% of the estimated conotoxins has been characterized to date. Hence, the discovery of novel conotoxins from the huge conotoxin resources with high-throughput and sensitive methods becomes a crucial key for the conotoxin-based drug development. In this review, we introduce the discovery methodology of new conotoxins from various Conus species. It focuses on obtaining full N- to C-terminal sequences, regardless of disulfide bond connectivity through crude venom purification, conotoxin precusor gene cloning, venom duct transcriptomics, venom proteomics and multi-omic methods. The protocols, advantages, disadvantages, and developments of different approaches during the last decade are summarized and the promising prospects are discussed as well.

Bioactive Compounds Isolated from Neglected Predatory Marine Gastropods.

A diverse range of predatory marine gastropods produce toxins, yet most of these molecules remain uncharacterized. Conus species have received the most attention from researchers, leading to several conopeptides reaching clinical trials. This review aims to summarize what is known about bioactive compounds isolated from species of neglected marine gastropods, especially in the Turridae, Terebridae, Babyloniidae, Muricidae, Buccinidae, Colubrariidae, Nassariidae, Cassidae, and Ranellidae families. Multiple species have been reported to contain bioactive compounds with potential toxic activity, but most of these compounds have not been characterized or even clearly identified. The bioactive properties and potential applications of echotoxins and related porins from the Ranellidae family are discussed in more detail. Finally, the review concludes with a call for research on understudied species.

A novel α-conopeptide Eu1.6 inhibits N-type (CaV2.2) calcium channels and exhibits potent analgesic activity.

We here describe a novel α-conopeptide, Eu1.6 from Conus eburneus, which exhibits strong anti-nociceptive activity by an unexpected mechanism of action. Unlike other α-conopeptides that largely target nicotinic acetylcholine receptors (nAChRs), Eu1.6 displayed only weak inhibitory activity at the α3ß4 and α7 nAChR subtypes and TTX-resistant sodium channels, and no activity at TTX-sensitive sodium channels in rat dorsal root ganglion (DRG) neurons, or opiate receptors, VR1, KCNQ1, L- and T-type calcium channels expressed in HEK293 cells. However, Eu1.6 inhibited high voltage-activated N-type calcium channel currents in isolated mouse DRG neurons which was independent of GABAB receptor activation. In HEK293 cells expressing CaV2.2 channels alone, Eu1.6 reversibly inhibited depolarization-activated Ba2+ currents in a voltage- and state-dependent manner. Inhibition of CaV2.2 by Eu1.6 was concentration-dependent (IC50 ~1 nM). Significantly, systemic administration of Eu1.6 at doses of 2.5-5.0 µg/kg exhibited potent analgesic activities in rat partial sciatic nerve injury and chronic constriction injury pain models. Furthermore, Eu1.6 had no significant side-effect on spontaneous locomotor activity, cardiac and respiratory function, and drug dependence in mice. These findings suggest α-conopeptide Eu1.6 is a potent analgesic for the treatment of neuropathic and chronic pain and opens a novel option for future analgesic drug design.

Cone Snails: A Big Store of Conotoxins for Novel Drug Discovery.

Marine drugs have developed rapidly in recent decades. Cone snails, a group of more than 700 species, have always been one of the focuses for new drug discovery. These venomous snails capture prey using a diverse array of unique bioactive neurotoxins, usually named as conotoxins or conopeptides. These conotoxins have proven to be valuable pharmacological probes and potential drugs due to their high specificity and affinity to ion channels, receptors, and transporters in the nervous systems of target prey and humans. Several research groups, including ours, have examined the venom gland of cone snails using a combination of transcriptomic and proteomic sequencing, and revealed the existence of hundreds of conotoxin transcripts and thousands of conopeptides in each Conus species. Over 2000 nucleotide and 8000 peptide sequences of conotoxins have been published, and the number is still increasing quickly. However, more than 98% of these sequences still lack 3D structural and functional information. With the rapid development of genomics and bioinformatics in recent years, functional predictions and investigations on conotoxins are making great progress in promoting the discovery of novel drugs. For example, ω-MVIIA was approved by the U.S. Food and Drug Administration in 2004 to treat chronic pain, and nine more conotoxins are at various stages of preclinical or clinical evaluation. In short, the genus Conus, the big family of cone snails, has become an important genetic resource for conotoxin identification and drug development.

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.

Discovery of two P-superfamily conotoxins, lt9a and lt9b, with different modifications on voltage-sensitive sodium channels.

In this work, two P-superfamily conotoxins, lt9a and lt9b, were purified and characterized from the crude venom of Conus litteratus. The amino acid sequences of lt9a and lt9b were determined by the Edman degradation method. It has been suggested that both lt9a and lt9b are produced from the precursor encoded by the gene Lt9.1. During the conotoxin maturation process, different post-translational modifications occurred between lt9a and lt9b. Conotoxin lt9b was predicted to have two prolines that underwent hydroxylation and one glutamate that underwent carboxylation, while lt9a had no hydroxyproline and carboxyglutamate residue. The calculated mass weights of two P-superfamily conotoxins with three proposed disulfide bonds were confirmed by matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry after considering corresponding post-translational modifications. These two conotoxins showed different effects on tetrodotoxin-sensitive sodium currents. Conotoxin lt9a (300 nM) resulted in marked slowing of the tetrodotoxin-sensitive sodium current decay, a notable increase in the peak current, and an alteration in reversal potential. However, lt9b inhibits tetrodotoxin-sensitive sodium currents, and the inhibition showed a concentration-dependent with the half maximal inhibitory concentration (IC50) value of 504.04 nM, but there were no change in the activation and inactivation kinetics of currents. To the best of our knowledge, this is the first investigation of two P-superfamily conotoxins identified to act on voltage-sensitive sodium channels with different modifications.

A novel µ-conotoxin from worm-hunting Conus tessulatus that selectively inhibit rat TTX-resistant sodium currents.

µ-conotoxins are a group of marine Conus peptides that inhibit sodium currents, so µ-conotoxins are valuable in sodium channel research and new analgesic drug discovery. Here, a novel µ-conotoxin TsIIIA was identified from a worm-hunting Conus tessulatus. TsIIIA was chemical synthesized according to its amino acid sequence GCCRWPCPSRCGMARCCSS and identified by mass spectrum. Patch clamp on rat dorsal root ganglion cells showed that 10 µM TsIIIA specifically inhibit TTX-resistant sodium currents but has no effect on TTX-sensitive sodium currents. TsIIIA inhibits TTX-resistant sodium currents by a dose-dependent mode with an IC50 of 2.61 µM. Further study showed 10 µM TsIIIA has no obvious effect on the current-voltage relationships, conductance-voltage relationships and voltage-dependence of steady-state inactivation of TTX-resistant sodium channels. Mice hotplate analgesic assay indicated that TsIIIA obviously increase the pain threshold at 0.5-4 h. In addition, TsIIIA has better analgesic effects than Ziconotide, indicating that TsIIIA was a valuable lead compound for development of new analgesic drug.

Discovery and characterization of EIIB, a new α-conotoxin from Conus ermineus venom by nAChRs affinity capture monitored by MALDI-TOF/TOF mass spectrometry.

Animal toxins are peptides that often bind with remarkable affinity and selectivity to membrane receptors such as nicotinic acetylcholine receptors (nAChRs). The latter are, for example, targeted by α-conotoxins, a family of peptide toxins produced by venomous cone snails. nAChRs are implicated in numerous physiological processes explaining why the design of new pharmacological tools and the discovery of potential innovative drugs targeting these receptor channels appear so important. This work describes a methodology developed to discover new ligands of nAChRs from complex mixtures of peptides. The methodology was set up by the incubation of Torpedo marmorata electrocyte membranes rich in nAChRs with BSA tryptic digests (>100 peptides) doped by small amounts of known nAChRs ligands (α-conotoxins). Peptides that bind to the receptors were purified and analyzed by MALDI-TOF/TOF mass spectrometry which revealed an enrichment of α-conotoxins in membrane-containing fractions. This result exhibits the binding of α-conotoxins to nAChRs. Negative controls were performed to demonstrate the specificity of the binding. The usefulness and the power of the methodology were also investigated for a discovery issue. The workflow was then applied to the screening of Conus ermineus crude venom, aiming at characterizing new nAChRs ligands from this venom, which has not been extensively investigated to date. The methodology validated our experiments by allowing us to bind two α-conotoxins (α-EI and α-EIIA) which have already been described as nAChRs ligands. Moreover, a new conotoxin, never described to date, was also captured, identified and sequenced from this venom. Classical pharmacology tests by radioligand binding using a synthetic homologue of the toxin confirm the activity of the new peptide, called α-EIIB. The Ki value of this peptide for Torpedo nicotinic receptors was measured at 2.2 ± 0.7 nM.

Im10A, a short conopeptide isolated from Conus imperialis and possesses two highly concentrated disulfide bridges and analgesic activity.

In the present study, we isolated, synthesized and NMR structurally characterized a novel conopeptide Im10A consisting of 11 amino acids (NTICCEGCMCY-NH2) from Conus imperialis. Unlike other conopeptides with four cysteine residues, Im10A had only two residues in loop 1 and one residue in loop 2 (CC-loop1-C-loop2-C), which formed a stable disulfide connectivity "I-IV, II- III" (framework X) with a type I ß-turn. Interestingly, Im10A exhibited 50.7% analgesic activity on rat partial sciatic nerve ligation (PNL) at 2h after Im10A administration. However, 10µM Im10A exhibited no apparent effect on neuronal nicotinic acetylcholine receptor, and it did not target DRG voltage-dependent sodium, potassium and calcium ion channels and opioid receptor. To our knowledge, Im10A had the most concentrated disulfide bridges among conopeptides with four cysteine residues. This finding provided a new motif for the future development of biomimetic compounds.

A New Member of Gamma-Conotoxin Family Isolated from Conus princeps Displays a Novel Molecular Target.

A novel conotoxin, named as PiVIIA, was isolated from the venom of Conus princeps, a marine predatory cone snail collected in the Pacific Southern Coast of Mexico. Chymotryptic digest of the S-alkylated peptide in combination with liquid chromatography coupled to tandem mass spectrometry, were used to define the sequencing of this peptide. Eleven N-terminal amino acids were verified by automated Edman degradation. PiVIIA is a 25-mer peptide (CDAOTHYCTNYWγCCSGYCγHSHCW) with six cysteine residues forming three disulphide bonds, a hydroxyproline (O) and two gamma carboxyglutamic acid (γ) residues. Based on the arrangement of six Cys residues (C-C-CC-C-C), this conotoxin might belong to the O2-superfamily. Moreover, PiVIIA has a conserved motif (-γCCS-) that characterizes γ-conotoxins from molluscivorous Conus. Peptide PiVIIA has 45% sequence identity with γ-PnVIIA-the prototype of this family. Biological activity of PiVIIA was assessed by voltage-clamp recording in rat dorsal root ganglion neurons. Perfusion of PiVIIA in the µM range produces a significant increase in the Ca(2+) currents, without significantly modifying the Na⁺, K⁺ or proton-gated acid sensing ionic currents. These results indicate that PiVIIA is a new conotoxin whose activity deserves further studies to define its potential use as a positive modulator of neuronal activity.

Comparative Venomics Reveals the Complex Prey Capture Strategy of the Piscivorous Cone Snail Conus catus.

Venomous marine cone snails produce a unique and remarkably diverse range of venom peptides (conotoxins and conopeptides) that have proven to be invaluable as pharmacological probes and leads to new therapies. Conus catus is a hook-and-line fish hunter from clade I, with ∼20 conotoxins identified, including the analgesic ω-conotoxin CVID (AM336). The current study unravels the venom composition of C. catus with tandem mass spectrometry and 454 sequencing data. From the venom gland transcriptome, 104 precursors were recovered from 11 superfamilies, with superfamily A (especially κA-) conotoxins dominating (77%) their venom. Proteomic analysis confirmed that κA-conotoxins dominated the predation-evoked milked venom of each of six C. catus analyzed and revealed remarkable intraspecific variation in both the intensity and type of conotoxins. High-throughput FLIPR assays revealed that the predation-evoked venom contained a range of conotoxins targeting the nAChR, Cav, and Nav ion channels, consistent with α- and ω-conotoxins being used for predation by C. catus. However, the κA-conotoxins did not act at these targets but induced potent and rapid immobilization followed by bursts of activity and finally paralysis when injected intramuscularly in zebrafish. Our venomics approach revealed the complexity of the envenomation strategy used by C. catus, which contains a mix of both excitatory and inhibitory venom peptides.

Intraspecific variations in Conus purpurascens injected venom using LC/MALDI-TOF-MS and LC-ESI-TripleTOF-MS.

The venom of cone snails is composed of highly modified peptides (conopeptides) that target a variety of ion channels and receptors. The venom of these marine gastropods represents a largely untapped resource of bioactive compounds of potential pharmaceutical value. Here, we use a combination of bioanalytical techniques to uncover the extent of venom expression variability in Conus purpurascens, a fish-hunting cone snail species. The injected venom of nine specimens of C. purpurascens was separated by reversed-phase high-performance liquid chromatography (RP-HPLC), and fractions were analyzed using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) in parallel with liquid chromatography-electrospray ionization (LC-ESI)-TripleTOF-MS to compare standard analytical protocols used in preparative bioassay-guided fractionations with a deeper peptidomic analysis. Here, we show that C. purpurascens exhibits pronounced intraspecific venom variability. RP-HPLC fractionation followed by MALDI-TOF-MS analysis of the injected venom of these nine specimens identified 463 distinct masses, with none common to all specimens. Using LC-ESI-TripleTOF-MS, the injected venom of these nine specimens yielded a total of 5517 unique masses. We also compare the injected venom of two specimens with their corresponding dissected venom. We found 2566 and 1990 unique masses for the dissected venom compared to 941 and 1959 masses in their corresponding injected venom. Of these, 742 and 1004 masses overlapped between the dissected and injected venom, respectively. The results indicate that larger conopeptide libraries can be assessed by studying multiple individuals of a given cone snail species. This expanded library of conopeptides enhances the opportunities for discovery of molecular modulators with direct relevance to human therapeutics. Graphical Abstract The venom of cone snails are extraordinarily complex mixtures of highly modified peptides. Venom analysis requires separation through RP-HPLC followed by MALDI-TOF mass spectrometry or direct analysis using LC-ESI-TripleTOF-MS. Using these techniques, venom intraspecific variability and comparison between injected and dissected were assessed.

αS-conotoxin GVIIIB potently and selectively blocks α9α10 nicotinic acetylcholine receptors.

Although acetylcholine is widely utilized in vertebrate nervous systems, nicotinic acetylcholine receptors (nAChRs), including the α9α10 subtype, also are expressed in a wide variety of non-neuronal cells. These cell types include cochlear hair cells, adrenal chromaffin cells and immune cells. α9α10 nAChRs present in these cells may respectively play roles in protection from noise-induced hearing loss, response to stress and neuroprotection. Despite these critical functions, there are few available selective ligands to confirm mechanistic hypothesis regarding the role of α9α10 nAChRs. Conus, has been a rich source of ligands for receptors and ion channels. Here, we identified Conus geographus venom as a lead source for a novel α9α10 antagonist. The active component was isolated and the encoding gene cloned. The peptide signal sequence and cysteine arrangement had the signature of the σ-conotoxin superfamily. Previously isolated σ-conotoxin GVIIIA, also from Conus geographus, targets the 5-HT3 receptor. In contrast, αS-GVIIIB blocked the α9α10 nAChR with an IC50 of 9.8 nM, yet was inactive at the 5-HT3 receptor. Pharmacological characterization of αS-GVIIIB shows that it is over 100-fold selective for the α9α10 nAChR compared to other nAChR subtypes. Thus, the S-superfamily represents a novel conotoxin scaffold for flexibly targeting a variety of receptor subtypes. Functional competition studies utilized distinct off-rate kinetics of conotoxins to identify the α10/α9 nAChR interface as the site of αS-GVIIIB binding; this adds to the importance of the (+) face of the α10 rather than the (+) face of the α9 nAChR subunit as critical to binding of α9α10-targeted conotoxins.