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α-Conotoxins that target muscle nicotinic acetylcholine receptors (nAChRs) commonly fall into two structural classes, frameworks I and II containing two and three disulfide bonds, respectively. Conotoxin SII is the sole member of the cysteine-rich framework II with ill-defined interactions at the nAChRs. Following directed synthesis of α-SII, NMR analysis revealed a well-defined structure containing a 3 10 -helix frequently employed by framework I α-conotoxins; α-SII acted at the muscle nAChR with half-maximal inhibitory concentrations (IC 50 ) of 120 nM (adult) and 370 nM (fetal) though weakly at neuronal nAChRs. Truncation of α-SII to a two disulfide bond amidated peptide with framework I disulfide connectivity led to similar activity. Surprisingly, the more constrained α-SII was less stable under mild reducing conditions and displayed a unique docking mode at the nAChR.

The defensive use of cone snail venom is hypothesised to have first arisen in ancestral worm-hunting snails and later repurposed in a compartmentalised venom duct to facilitate the dietary shift to molluscivory and piscivory. Consistent with its placement in a basal lineage, we demonstrate that the C. distans venom gland lacked distinct compartmentalisation. Transcriptomics revealed C. distans expressed a wide range of structural classes, with inhibitory cysteine knot (ICK)-containing peptides dominating. To better understand the evolution of the venom gland compartmentalisation, we compared C. distans to C. planorbis , the earliest diverging species from which a defence-evoked venom has been obtained, and fish-hunting C. geographus from the Gastridium subgenus that injects distinct defensive and predatory venoms. These comparisons support the hypothesis that venom gland compartmentalisation arose in worm-hunting species and enabled repurposing of venom peptides to facilitate the dietary shift from vermivory to molluscivory and piscivory in more recently diverged cone snail lineages.

α-Conotoxins are small disulfide-rich peptides targeting nicotinic acetylcholine receptors (nAChRs) characterised by a C I C II -X m -C III -X n -C IV framework that invariably adopt the native globular conformations which is typically most potent. α-Conotoxins are divided into several structural subgroups based on the number of residues within the two loops braced by the disulfide bonds (m/n), with the 4/7 and 4/3 subgroups dominating. AusIA is a relatively rare α5/5-conotoxin isolated from the venom of Conus australis. Surprisingly, the ribbon isomer displayed equipotency to the wild-type globular AusIA at human α7-containing nAChR. To understand the molecular basis for equipotency, we determined the co-crystal structures of both isomers at Lymnea stagnalis acetylcholine binding protein. The additional residue in the first loop of AusIA was found to be a critical determinant of equipotency, with 11-fold and 86-fold shifts in potency in favour of globular AusIA over ribbon AusIA observed following deletion of Ala4 or Arg5, respectively. This divergence in the potency between globular AusIA and ribbon AusIA was further enhanced upon truncation of the non-conserved Val at the C-termini. Conversely, equipotency could be replicated in LsIA and TxIA [A10L] following insertion of an Ala in the first loop. These findings provide a new understanding of the role the first loop in ribbon and globular α-conotoxins can play in directing α-conotoxin nAChR pharmacology., (© 2021. The Author(s).)

The venom duct origins of predatory and defensive venoms has not been studied for hook-and-line fish hunting cone snails despite the pharmacological importance of their venoms. To better understand the biochemistry and evolution of injected predatory and defensive venoms, we compared distal, central and proximal venom duct sections across three specimens of C. striatus (Pionoconus) using proteomic and transcriptomic approaches. A total of 370 conotoxin precursors were identified from the whole venom duct transcriptome. Milked defensive venom was enriched with a potent cocktail of proximally expressed inhibitory α-, ω- and μ-conotoxins compared to milked predatory venom. In contrast, excitatory κA-conotoxins dominated both the predatory and defensive venoms despite their distal expression, suggesting this class of conotoxin can be selectively expressed from the same duct segment in response to either a predatory or defensive stimuli. Given the high abundance of κA-conotoxins in the Pionoconus clade, we hypothesise that the κA-conotoxins have evolved through adaptive evolution following their repurposing from ancestral inhibitory A superfamily conotoxins to facilitate the dietary shift to fish hunting and species radiation in this clade.

The 27-amino acid (aa)-long d-conotoxin TxVIA, originally isolated from the mollusc-hunting cone snail Conus textile , slows voltage-gated sodium (Na V ) channel inactivation in molluscan neurons, but its mammalian ion channel targets remain undetermined. In this study, we confirmed that TxVIA was inactive on mammalian Na V 1.2 and Na V 1.7 even at high concentrations (10 µM). Given the fact that invertebrate Na V channel and T-type calcium channels (Ca V 3.x) are evolutionarily related, we examined the possibility that TxVIA may act on Ca V 3.x. Electrophysiological characterisation of the native TxVIA on Ca V 3.1, 3.2 and 3.3 revealed that TxVIA preferentially inhibits Ca V 3.2 current (IC 50 = 0.24 mM) and enhances Ca V 3.1 current at higher concentrations. In fish bioassays TxVIA showed little effect on zebrafish behaviours when injected intramuscular at 250 ng/100 mg fish. The binding sites for TxVIA at Na V 1.7 and Ca V 3.1 revealed that their channel binding sites contained a common epitope.

Cone snails produce a fast-acting and often paralyzing venom, largely dominated by disulfide-rich conotoxins targeting ion channels. Although disulfide-poor conopeptides are usually minor components of cone snail venoms, their ability to target key membrane receptors such as GPCRs make them highly valuable as drug lead compounds. From the venom gland transcriptome of Conus miliaris , we report here on the discovery and characterization of two conopressins, which are nonapeptide ligands of the vasopressin/oxytocin receptor family. These novel sequence variants show unusual features, including a charge inversion at the critical position 8, with an aspartate instead of a highly conserved lysine or arginine residue. Both the amidated and acid C-terminal analogues were synthesized, followed by pharmacological characterization on human and zebrafish receptors and structural investigation by NMR. Whereas conopressin-M1 showed weak and only partial agonist activity at hV1bR (amidated form only) and ZFV1a1R (both amidated and acid form), both conopressin-M2 analogues acted as full agonists at the ZFV2 receptor with low micromolar affinity. Together with the NMR structures of amidated conopressins-M1, -M2 and -G, this study provides novel structure-activity relationship information that may help in the design of more selective ligands.

The venom of the marine predatory cone snails (genus Conus ) has evolved for prey capture and defense, providing the basis for survival and rapid diversification of the now estimated 750+ species. A typical Conus venom contains hundreds to thousands of bioactive peptides known as conotoxins. These mostly disulfide-rich and well-structured peptides act on a wide range of targets such as ion channels, G protein-coupled receptors, transporters, and enzymes. Conotoxins are of interest to neuroscientists as well as drug developers due to their exquisite potency and selectivity, not just against prey but also mammalian targets, thereby providing a rich source of molecular probes and therapeutic leads. The rise of integrated venomics has accelerated conotoxin discovery with now well over 10,000 conotoxin sequences published. However, their structural and pharmacological characterization lags considerably behind. In this review, we highlight the diversity of new conotoxins uncovered since 2014, their three-dimensional structures and folds, novel chemical approaches to their syntheses, and their value as pharmacological tools to unravel complex biology. Additionally, we discuss challenges and future directions for the field.

Individual variation in animal venom has been linked to geographical location, feeding habit, season, size, and gender. Uniquely, cone snails possess the remarkable ability to change venom composition in response to predatory or defensive stimuli. To date, correlations between the venom gland transcriptome and proteome within and between individual cone snails have not been reported. In this study, we use 454 pyrosequencing and mass spectrometry to decipher the transcriptomes and proteomes of the venom gland and corresponding predation-evoked venom of two specimens of Conus imperialis . Transcriptomic analyses revealed 17 conotoxin gene superfamilies common to both animals, including 5 novel superfamilies and two novel cysteine frameworks. While highly expressed transcripts were common to both specimens, variation of moderately and weakly expressed precursor sequences was surprisingly diverse, with one specimen expressing two unique gene superfamilies and consistently producing more paralogs within each conotoxin gene superfamily. Using a quantitative labelling method, conotoxin variability was compared quantitatively, with highly expressed peptides showing a strong correlation between transcription and translation, whereas peptides expressed at lower levels showed a poor correlation. These results suggest that major transcripts are subject to stabilizing selection, while minor transcripts are subject to diversifying selection.

Integrated venomics techniques have shown that variable processing of conotoxins from Conus marmoreus resulted in a dramatic expansion in the number of expressed conotoxins. One conotoxin from C. marmoreus , the χ-conotoxin MrIA, is a selective inhibitor of human norepinephrine transporters (hNET) and therefore a drug candidate for attenuating chronic neuropathic pain. It has been found that "messy" processing of the MrIA transcripts results in the expression of MrIA analogs with different truncations of the pro-peptide that contains portions of the MrIA molecule. The aim of this study was to investigate if variable processing of the expressed peptides results in modulation of the existing hNET pharmacology or creates new pharmacologies. To this end, a number of MrIA analogs found in C. marmoreus venom were synthesized and evaluated for their activity at hNET receptors. While several of the analogs exhibited norepinephrine transporter inhibitory activity comparable to that of MrIA, none significantly improved on the potency of conotoxin MrIA, and those analogs with disrupted pharmacophores produced greatly reduced NET inhibition, confirming previous structure-activity relationships seen on χ-class conopeptides. Additionally, analogs were screened for new activities on ion channels using calcium influx assays, although no major new pharmacology was revealed.

Marine cone snails are a large family of gastropods that have evolved highly potent venoms for predation and defense. The cone snail venom has exceptional molecular diversity in neuropharmacologically active compounds, targeting a range of receptors, ion channels, and transporters. These conotoxins have helped to dissect the structure and function of many of these therapeutically significant targets in the central and peripheral nervous systems, as well as unravelling the complex cellular mechanisms modulated by these receptors and ion channels. This review provides an overview of α-conotoxins targeting neuronal nicotinic acetylcholine receptors. The structure and activity of both classical and non-classical α-conotoxins are discussed, along with their contributions towards understanding nicotinic acetylcholine receptor (nAChR) structure and function.

Cone snail venoms are considered a treasure trove of bioactive peptides. Despite over 800 species of cone snails being known, each producing over 1000 venom peptides, only about 150 unique venom peptides are structurally and functionally characterized. To overcome the limitations of the traditional low-throughput bio-discovery approaches, multi-omics systems approaches have been introduced to accelerate venom peptide discovery and characterisation. This "venomic" approach is starting to unravel the full complexity of cone snail venoms and to provide new insights into their biology and evolution. The main challenge for venomics is the effective integration of transcriptomics, proteomics, and pharmacological data and the efficient analysis of big datasets. Novel database search tools and visualisation techniques are now being introduced that facilitate data exploration, with ongoing advances in related omics fields being expected to further enhance venomics studies. Despite these challenges and future opportunities, cone snail venomics has already exponentially expanded the number of novel venom peptide sequences identified from the species investigated, although most novel conotoxins remain to be pharmacologically characterised. Therefore, efficient high-throughput peptide production systems and/or banks of miniaturized discovery assays are required to overcome this bottleneck and thus enhance cone snail venom bioprospecting and accelerate the identification of novel drug leads., Competing Interests: The authors declare no conflict of interest.

Conotoxins are a large family of disulfide-rich peptides that contain unique cysteine frameworks that target a broad range of ion channels and receptors. We recently discovered the 33-residue conotoxin Φ-MiXXVIIA from Conus miles with a novel cysteine framework comprising three consecutive cysteine residues and four disulfide bonds. Regioselective chemical synthesis helped decipher the disulfide bond connectivity and the structure of Φ-MiXXVIIA was determined by NMR spectroscopy. The 3D structure displays a unique topology containing two β-hairpins that resemble the N-terminal domain of granulin. Similar to granulin, Φ-MiXXVIIA promotes cell proliferation (EC 50 17.85 μm) while inhibiting apoptosis (EC 50 2.2 μm). Additional framework XXVII sequences were discovered with homologous signal peptides that define the new conotoxin superfamily G2. The novel structure and biological activity of Φ-MiXXVIIA expands the repertoire of disulfide-rich conotoxins that recognize mammalian receptors., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Nicotinic acetylcholine receptors (nAChR) are therapeutic targets for a range of human diseases. α-Conotoxins are naturally occurring peptide antagonists of nAChRs that have been used as pharmacological probes and investigated as drug leads for nAChR related disorders. However, α-conotoxin interactions have been mostly characterised at the α7 and α3β2 nAChRs, with interactions at other subtypes poorly understood. This study provides novel structural insights into the molecular basis for α-conotoxin activity at α3β4 nAChR, a therapeutic target where subtype specific antagonists have potential to treat nicotine addiction and lung cancer. A co-crystal structure of α-conotoxin LsIA with Lymnaea stagnalis acetylcholine binding protein guided the design and functional characterisations of LsIA analogues that identified the minimum pharmacophore regulating α3β4 antagonism. Interactions of the LsIA R10F with β4 K57 and the conserved -NN- α-conotoxin motif with β4 I77 and I109 conferred α3β4 activity to the otherwise inactive LsIA. Using these structural insights, we designed LsIA analogues with α3β4 activity. This new understanding of the structural basis of protein-protein interactions between α-conotoxins and α3β4 may help rationally guide the development of α3β4 selective antagonists with therapeutic potential.

The μO-conotoxins MrVIA, MrVIB, and MfVIA inhibit the voltage-gated sodium channel NaV1.8, a well described target for the treatment of pain; however, little is known about the residues or structural elements that define this activity. In this study, we determined the three-dimensional structure of MfVIA, examined its membrane binding properties, performed alanine-scanning mutagenesis, and identified residues important for its activity at human NaV1.8. A second round of mutations resulted in (E5K,E8K)MfVIA, a double mutant with greater positive surface charge and greater affinity for lipid membranes compared with MfVIA. This analogue had increased potency at NaV1.8 and was analgesic in the mouse formalin assay., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Peptide dendrimers are a novel class of macromolecules of emerging interest with the potential of delayed renal clearance due to their molecular size and enhanced activity due to the multivalency effect. In this work, an active analogue of the disulfide-rich χ-conotoxin χ-MrIA (χ-MrIA), a norepinephrine reuptake (norepinephrine transporter) inhibitor, was grafted onto a polylysine dendron. Dendron decoration was achieved by employing copper-catalyzed alkyne-azide cycloaddition with azido-PEG chain-modified χ-MrIA analogues, leading to homogenous 4-mer and 8-mer χ-MrIA dendrimers with molecular weights ranging from 8 to 22 kDa. These dendrimers were investigated for their impact on peptide secondary structure, in vitro functional activity, and potential anti-allodynia in vivo. NMR studies showed that the χ-MrIA tertiary structure was maintained in the χ-MrIA dendrimers. In a functional norepinephrine transporter reuptake assay, χ-MrIA dendrimers showed slightly increased potency relative to the azido-PEGylated χ-MrIA analogues with similar potency to the parent peptide. In contrast to χ-MrIA, no anti-allodynic action was observed when the χ-MrIA dendrimers were administered intrathecally in a rat model of neuropathic pain, suggesting that the larger dendrimer structures are unable to diffuse through the spinal column tissue and reach the norepinephrine transporter. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd., (Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.)

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.

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.

Some venomous cone snails feed on small fishes using an immobilizing combination of synergistic venom peptides that target Kv and Nav channels. As part of this envenomation strategy, δ-conotoxins are potent ichtyotoxins that enhance Nav channel function. δ-Conotoxins belong to an ancient and widely distributed gene superfamily, but any evolutionary link from ancestral worm-eating cone snails to modern piscivorous species has not been elucidated. Here, we report the discovery of SuVIA, a potent vertebrate-active δ-conotoxin characterized from a vermivorous cone snail (Conus suturatus). SuVIA is equipotent at hNaV1.3, hNaV1.4 and hNaV1.6 with EC50s in the low nanomolar range. SuVIA also increased peak hNaV1.7 current by approximately 75% and shifted the voltage-dependence of activation to more hyperpolarized potentials from -15 mV to -25 mV, with little effect on the voltage-dependence of inactivation. Interestingly, the proximal venom gland expression and pain-inducing effect of SuVIA in mammals suggest that δ-conotoxins in vermivorous cone snails play a defensive role against higher order vertebrates. We propose that δ-conotoxins originally evolved in ancestral vermivorous cones to defend against larger predators including fishes have been repurposed to facilitate a shift to piscivorous behaviour, suggesting an unexpected underlying mechanism for this remarkable evolutionary transition., (© 2015 The Author(s) Published by the Royal Society. All rights reserved.)

MrIC is a recently described selective agonist of endogenously expressed α7 nAChR. In this study, we further characterize the pharmacological activity of MrIC using Ca(2+) imaging approaches in SH-SY5Y cells endogenously expressing α7 nAChR and demonstrate that MrIC exclusively activates α7 nAChR modulated by type II positive allosteric modulators, including PNU120596. MrIC was a full agonist at PNU120596-modulated α7 nAChR compared with choline, albeit with slower kinetics, but failed to elicit a Ca(2+) response in the absence of PNU120596. Interestingly, the NMR structure of MrIC showed a typical 4/7 α-conotoxin fold, indicating that its unusual pharmacological activity is likely sequence-dependent. Overall, our results suggest that MrIC acts as a biased agonist that can only activate α7 nAChR modified by type II positive allosteric modulators, and thus represents a valuable tool to probe the pharmacological properties of this important ion channel., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Covalently attached peptide dendrimers can enhance binding affinity and functional activity. Homogenous di- and tetravalent dendrimers incorporating the α7-nicotinic receptor blocker α-conotoxin ImI (α-ImI) with polyethylene glycol spacers were designed and synthesized via a copper-catalyzed azide-alkyne cycloaddition of azide-modified α-ImI to an alkyne-modified polylysine dendron. NMR and CD structural analysis confirmed that each α-ImI moiety in the dendrimers had the same 3D structure as native α-ImI. The binding of the α-ImI dendrimers to binding protein Ac-AChBP was measured by surface plasmon resonance and revealed enhanced affinity. Quantitative electrophysiology showed that α-ImI dendrimers had ∼100-fold enhanced potency at hα7 nAChRs (IC50 = 4 nM) compared to native α-ImI (IC50 = 440 nM). In contrast, no significant potency enhancement was observed at heteromeric hα3β2 and hα9α10 nAChRs. These findings indicate that multimeric ligands can significantly enhance conotoxin potency and selectivity at homomeric nicotinic ion channels.

The design of disulfide bond mimetics is an important strategy for optimising cysteine-rich peptides in drug development. Mimetics of the drug lead conotoxin MrIA, in which one disulfide bond is selectively replaced of by a 1,4-disubstituted-1,2,3-triazole bridge, are described. Sequential copper-catalyzed azide-alkyne cycloaddition (CuAAC; click reaction) followed by disulfide formation resulted in the regioselective syntheses of triazole-disulfide hybrid MrIA analogues. Mimetics with a triazole replacing the Cys4-Cys13 disulfide bond retained tertiary structure and full in vitro and in vivo activity as norepinephrine reuptake inhibitors. Importantly, these mimetics are resistant to reduction in the presence of glutathione, thus resulting in improved plasma stability and increased suitability for drug development., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Neuronal nicotinic acetylcholine receptors (nAChRs) are a diverse class of ligand-gated ion channels involved in neurological conditions such as neuropathic pain and Alzheimer's disease. α-Conotoxin [A10L]PnIA is a potent and selective antagonist of the mammalian α7 nAChR with a key binding interaction at position 10. We now describe a molecular analysis of the receptor-ligand interactions that determine the role of position 10 in determining potency and selectivity for the α7 and α3β2 nAChR subtypes. Using electrophysiological and radioligand binding methods on a suite of [A10L]PnIA analogs we observed that hydrophobic residues in position 10 maintained potency at both subtypes whereas charged or polar residues abolished α7 binding. Molecular docking revealed dominant hydrophobic interactions with several α7 and α3β2 receptor residues via a hydrophobic funnel. Incorporation of norleucine (Nle) caused the largest (8-fold) increase in affinity for the α7 subtype (Ki=44nM) though selectivity reverted to α3β2 (IC50=0.7nM). It appears that the placement of a single methyl group determines selectivity between α7 and α3β2 nAChRs via different molecular determinants., (Crown Copyright © 2014. Published by Elsevier Inc. All rights reserved.)

α-Conotoxins are competitive antagonists of nicotinic acetylcholine receptors (nAChRs). Their high selectivity and affinity for the various subtypes of nAChRs have led to significant advances in our understanding of the structure and function of these key ion channels. Here we report the discovery of a novel 4/7 α-conotoxin, MrIC from the venom duct of Conus marmoreus, which acts as an agonist at the endogenous human α7 nAChR in SH-SY5Y cells pretreated with PNU120596 (PNU). This unique agonist activity of MrIC at α7 nAChRs may guide the development of novel α7 nAChR modulators.

Marine cone snails have developed sophisticated chemical strategies to capture prey and defend themselves against predators. Among the vast array of bioactive molecules in their venom, peptide components called conotoxins or conopeptides dominate, with many binding with high affinity and selectivity to a broad range of cellular targets, including receptors and transporters of the nervous system. Whereas the conopeptide gene precursor organization has a conserved topology, the peptides in the venom duct are highly processed. Indeed, deep sequencing transcriptomics has uncovered on average fewer than 100 toxin gene precursors per species, whereas advanced proteomics has revealed >10-fold greater diversity at the peptide level. In the present study, second-generation sequencing technologies coupled to highly sensitive mass spectrometry methods were applied to rapidly uncover the conopeptide diversity in the venom of a worm-hunting species, Conus miles. A total of 662 putative conopeptide encoded sequences were retrieved from transcriptomic data, comprising 48 validated conotoxin sequences that clustered into 10 gene superfamilies, including 3 novel superfamilies and a novel cysteine framework (C-C-C-CCC-C-C) identified at both transcript and peptide levels. A surprisingly large number of conopeptide gene sequences were expressed at low levels, including a series of single amino acid variants, as well as sequences containing deletions and frame and stop codon shifts. Some of the toxin variants generate alternative cleavage sites, interrupted or elongated cysteine frameworks, and highly variable isoforms within families that could be identified at the peptide level. Together with the variable peptide processing identified previously, background genetic and phenotypic levels of biological messiness in venoms contribute to the hypervariability of venom peptides and their ability to evolve rapidly.

Background: Conopeptides, often generically referred to as conotoxins, are small neurotoxins found in the venom of predatory marine cone snails. These molecules are highly stable and are able to efficiently and selectively interact with a wide variety of heterologous receptors and channels, making them valuable pharmacological probes and potential drug leads. Recent advances in next-generation RNA sequencing and high-throughput proteomics have led to the generation of large data sets that require purpose-built and dedicated bioinformatics tools for efficient data mining., Results: Here we describe ConoSorter, an algorithm that categorizes cDNA or protein sequences into conopeptide superfamilies and classes based on their signal, pro- and mature region sequence composition. ConoSorter also catalogues key sequence characteristics (including relative sequence frequency, length, number of cysteines, N-terminal hydrophobicity, sequence similarity score) and automatically searches the ConoServer database for known precursor sequences, facilitating identification of known and novel conopeptides. When applied to ConoServer and UniProtKB/Swiss-Prot databases, ConoSorter is able to recognize 100% of known conotoxin superfamilies and classes with a minimum species specificity of 99%. As a proof of concept, we performed a reanalysis of Conus marmoreus venom duct transcriptome and (i) correctly classified all sequences previously annotated, (ii) identified 158 novel precursor conopeptide transcripts, 106 of which were confirmed by protein mass spectrometry, and (iii) identified another 13 novel conotoxin gene superfamilies., Conclusions: Taken together, these findings indicate that ConoSorter is not only capable of robust classification of known conopeptides from large RNA data sets, but can also facilitate de novo identification of conopeptides which may have pharmaceutical importance.

A new α-conotoxin LsIA was isolated from the crude venom of Conus limpusi using assay-guided RP-HPLC fractionation. Synthetic LsIA was a potent antagonist of α3β2, α3α5β2 and α7 nAChRs, with half-maximal inhibitory concentrations of 10, 31 and 10 nM, respectively. The structure of LsIA determined by NMR spectroscopy comprised a characteristic disulfide bond-stabilized α-helical structure and disordered N-terminal region. Potency reductions of up to 9-fold were observed for N-terminally truncated analogues of LsIA at α7 and α3β2 nAChRs, whereas C-terminal carboxylation enhanced potency 3-fold at α3β2 nAChRs but reduced potency 3-fold at α7 nAChRs. This study gives further insight into α-conotoxin pharmacology and the molecular basis of nAChR selectivity, highlighting the influence of N-terminal residues and C-terminal amidation on conotoxin pharmacology., (Copyright © 2013. Published by Elsevier Inc.)

Cone snails produce highly complex venom comprising mostly small biologically active peptides known as conotoxins or conopeptides. Early estimates that suggested 50-200 venom peptides are produced per species have been recently increased at least 10-fold using advanced mass spectrometry. To uncover the mechanism(s) responsible for generating this impressive diversity, we used an integrated approach combining second-generation transcriptome sequencing with high sensitivity proteomics. From the venom gland transcriptome of Conus marmoreus, a total of 105 conopeptide precursor sequences from 13 gene superfamilies were identified. Over 60% of these precursors belonged to the three gene superfamilies O1, T, and M, consistent with their high levels of expression, which suggests these conotoxins play an important role in prey capture and/or defense. Seven gene superfamilies not previously identified in C. marmoreus, including five novel superfamilies, were also discovered. To confirm the expression of toxins identified at the transcript level, the injected venom of C. marmoreus was comprehensively analyzed by mass spectrometry, revealing 2710 and 3172 peptides using MALDI and ESI-MS, respectively, and 6254 peptides using an ESI-MS TripleTOF 5600 instrument. All conopeptides derived from transcriptomic sequences could be matched to masses obtained on the TripleTOF within 100 ppm accuracy, with 66 (63%) providing MS/MS coverage that unambiguously confirmed these matches. Comprehensive integration of transcriptomic and proteomic data revealed for the first time that the vast majority of the conopeptide diversity arises from a more limited set of genes through a process of variable peptide processing, which generates conopeptides with alternative cleavage sites, heterogeneous post-translational modifications, and highly variable N- and C-terminal truncations. Variable peptide processing is expected to contribute to the evolution of venoms, and explains how a limited set of ∼ 100 gene transcripts can generate thousands of conopeptides in a single species of cone snail.

Conopeptides and conotoxins are small peptides produced by cone snails as a part of their predatory/defense strategies that target key ion channels and receptors in the nervous system. Some of these peptides also potently target mammalian ion channels involved in pain pathways. As a result, these venoms are a source of valuable pharmacological and therapeutic agents. The traditional approach towards conopeptide discovery relied on activity-guided fractionation, which is time consuming and resource-intensive. In this review, we discuss the advances in the fields of transcriptomics, proteomics and bioinformatics that now allow researchers to integrate these three platforms towards a more efficient discovery strategy. In this review, we also highlight the challenges associated with the wealth of data generated with this integrated approach and briefly discuss the impact these methods could have on the field of toxinology., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The μO-conotoxins are notable for their unique selectivity for Na(v)1.8 over other sodium channel isoforms, making them attractive drug leads for the treatment of neuropathic pain. We describe the discovery of a novel μO-conotoxin, MfVIA, from the venom of Conus magnificus using high-throughput screening approaches. MfVIA was found to be a hydrophobic 32-residue peptide (amino acid sequence RDCQEKWEYCIVPILGFVYCCPGLICGPFVCV) with highest sequence homology to μO-conotoxin MrVIB. To overcome the synthetic challenges posed by μO-conotoxins due to their hydrophobic nature and difficult folding, we developed a novel regioselective approach for the synthesis of μO-conotoxins. Performing selective oxidative deprotections of the cysteine side-chain protecting groups of the fully protected peptide allowed manipulations in organic solvents with no chromatography required between steps. Using this approach, we obtained correctly folded MfVIA with increased synthetic yields. Biological activity of MfVIA was assessed using membrane potential-sensitive dyes and electrophysiological recording techniques. MfVIA preferentially inhibits Na(v)1.8 (IC₅₀ 95.9±74.3 nM) and Na(v)1.4 (IC₅₀ 81±16 nM), with significantly lower affinity for other Na(v) subtypes (IC₅₀ 431-6203 nM; Na(v)1.5>1.6∼1.7∼1.3∼1.1∼1.2). This improved approach to μO-conotoxin synthesis will facilitate the optimization of μO-conotoxins as novel analgesic molecules to improve pain management., (Crown Copyright © 2012. Published by Elsevier Inc. All rights reserved.)

A dual-pharmacophoric peptide was engineered by grafting the integrin binding RGD motif between the C- and N-termini of a disulfide-rich noradrenaline transporter inhibiting χ-conotoxin resulting in a stable backbone cyclized peptide. The construct maintained two independent biological activities and showed increased plasma stability with no adverse effects observed following administration to rats, highlighting the potential value of pharmacophore grafting into constrained peptide scaffolds.

Conopeptides are a diverse group of recently evolved venom peptides used for prey capture and/or defense. Each species of cone snails produces in excess of 1000 conopeptides, with those pharmacologically characterized (≈ 0.1%) targeting a diverse range of membrane proteins typically with high potency and specificity. The majority of conopeptides inhibit voltage- or ligand-gated ion channels, providing valuable research tools for the dissection of the role played by specific ion channels in excitable cells. It is noteworthy that many of these targets are found to be expressed in pain pathways, with several conopeptides having entered the clinic as potential treatments for pain [e.g., pyroglutamate1-MrIA (Xen2174)] and one now marketed for intrathecal treatment of severe pain [ziconotide (Prialt)]. This review discusses the diversity, pharmacology, structure-activity relationships, and therapeutic potential of cone snail venom peptide families acting at voltage-gated ion channels (ω-, μ-, μO-, δ-, ι-, and κ-conotoxins), ligand-gated ion channels (α-conotoxins, σ-conotoxin, ikot-ikot, and conantokins), G-protein-coupled receptors (ρ-conopeptides, conopressins, and contulakins), and neurotransmitter transporters (χ-conopeptides), with expanded discussion on the clinical potential of sodium and calcium channel inhibitors and α-conotoxins. Expanding the discovery of new bioactives using proteomic/transcriptomic approaches combined with high-throughput platforms and better defining conopeptide structure-activity relationships using relevant membrane protein crystal structures are expected to grow the already significant impact conopeptides have had as both research probes and leads to new therapies.

Cone snail venoms continue to provide a rich source of bioactive peptides useful as research tools and leads to new therapeutics. We isolated two closely related conopeptides, MrIA and MrIB, which defined the χ-conopeptide class of bioactive peptides based on their unique ability to highly selectively and non-competitively inhibit the norepinephrine transporter (NET). An alanine scan of χ-MrIA revealed this class of peptides had a unusual cysteine-stabilised scaffold that presented a γ-turn in an optimised conformation for high affinity interactions with NET. χ-MrIA reversed the behavioural signs of mechanical allodynia in a chronic constriction injury rat model but its chemically unstable N-terminal asparagine precluded long-term use in implanted pumps. An extensive analoguing program identified Xen2174 to have improved stability and extended duration of analgaesia, without compromising efficacy versus side effects window observed forχ-MrIA. An open label, single IT bolus, dose-escalating study in cancer patients suffering severe chronic pain found Xen2174 relieved pain quickly over an extended period across a wide range of well-tolerated doses. Currently, Xen2174 is entering a Phase IIb double-blind study to determine safety and efficacy in bunionectomy pain., (Crown Copyright © 2011. Published by Elsevier Ltd. All rights reserved.)

μ-Conotoxins are peptide blockers of voltage-gated sodium channels (sodium channels), inhibiting tetrodotoxin-sensitive neuronal (Na(v) 1.2) and skeletal (Na(v) 1.4) subtypes with highest affinity. Structure-activity relationship studies of μ-conotoxins SIIIA, TIIIA, and KIIIA have shown that it is mainly the C-terminal part of the three-loop peptide that is involved in binding to the sodium channel. In this study, we characterize the effect of N- and C-terminal extensions of μ-conotoxins SIIIA, SIIIB, and TIIIA on their potency and selectivity for neuronal versus muscle sodium channels. Interestingly, extending the N- or C-terminal of the peptide by introducing neutral, positive, and/or negatively charged residues, the selectivity of the native peptide can be altered from neuronal to skeletal and the other way around. The results from this study provide further insight into the binding profile of μ-conotoxins at voltage-gated sodium channels, revealing that binding interactions outside the cysteine-stablilized loops can contribute to μ-conotoxin affinity and sodium channel selectivity., (Copyright © 2012 Wiley Periodicals, Inc.)

Cone snails have evolved many 1000s of small, structurally stable venom peptides (conopeptides) for prey capture and defense. Whilst < 0.1% have been pharmacologically characterised, those with known function typically target membrane proteins of therapeutic importance, including ion channels, transporters and GPCRs. Several conopeptides reduce pain in animals models, with one in clinical development (χ-conopeptide analogue Xen2174) and one marketed (ω- conotoxin MVIIA or Prialt) for the treatment of severe pain. In addition to their therapeutic potential, conopeptides have been valuable probes for studying the role of a number of key membrane proteins in normal and disease physiology.

The two disulfide bonds of α-conotoxin ImI, a peptide antagonist of the α7 nicotinic acetylcholine receptor (nAChR), were systematically replaced with isosteric redox-stable cystathionine thioethers. Regioselective thioether formation was accomplished on solid support through substitution of a γ-chlorohomoalanine by an intramolecular cysteine thiol to produce hybrid thioether/disulfide analogues (2 and 3) as well as a dual cystathionine analogue (4) that were found to be structurally homologous to α-conotoxin ImI by (1)H NMR. The antagonistic activity at the α7 nAChR of cystathionine analogue 3 (pIC(50) = 6.41 ± 0.09) was identical to that of α-conotoxin ImI (1, pIC(50) = 6.41 ± 0.09), whereas those of 2 (pIC(50) = 5.96 ± 0.09) and 4 (pIC(50) = 5.89 ± 0.09) showed a modest decrease. The effect of oxidation of the thioethers to sulfoxides was also investigated, with significant changes in the biological activities observed ranging from a >30-fold reduction (2S═O) to a 3-fold increase (3S═O(B)) in potencies.

Venomous animals produce a diverse range of peptides and small molecules that are of both therapeutic and pharmacologic value. One such animal, the cone snail, produces peptides known as conotoxins, which may be of interest to those studying the mammalian immune system. Conotoxins are a family of venom peptides that display extraordinary diversity and often exquisite specificity for membrane protein targets, especially voltage and ligand activated ion channels. Conopeptides are proving to be important pharmacological tools to probe human physiology, with some showing promise as therapeutics for conditions such as neuropathic pain. The potential of these peptides to interact and modulate the human immune system has not been investigated despite literature suggesting that conotoxins could be valuable research tools and potential therapeutics in area of immunology. Known pharmacological targets of conopeptides expressed by immunocompetent cells include voltage-gated potassium channel (Kv), voltage-gated calcium channel (Cav), nicotinic and acetylcholine receptors. In addition, the 5-HT3, GABAB and NMDA receptors that are not considered classic immunomodulators but may play a secondary role in modulating immune responses. This review highlights venom peptides with potential to act at immunological targets within the mammalian immune system.

Non-native disulfide isomers of alpha-conotoxins are generally inactive although some unexpectedly demonstrate comparable or enhanced bioactivity. The actions of "globular" and "ribbon" isomers of alpha-conotoxin AuIB have been characterized on alpha3beta4 nicotinic acetylcholine receptors (nAChRs) heterologously expressed in Xenopus oocytes. Using two-electrode voltage clamp recording, we showed that the inhibitory efficacy of the ribbon isomer of AuIB is limited to approximately 50%. The maximal inhibition was stoichiometry-dependent because altering alpha3:beta4 RNA injection ratios either increased AuIB(ribbon) efficacy (10alpha:1beta) or completely abolished blockade (1alpha:10beta). In contrast, inhibition by AuIB(globular) was independent of injection ratios. ACh-evoked current amplitude was largest for 1:10 injected oocytes and smallest for the 10:1 ratio. ACh concentration-response curves revealed high (HS, 1:10) and low (LS, 10:1) sensitivity alpha3beta4 nAChRs with corresponding EC(50) values of 22.6 and 176.9 microM, respectively. Increasing the agonist concentration antagonized the inhibition of LS alpha3beta4 nAChRs by AuIB(ribbon), whereas inhibition of HS and LS alpha3beta4 nAChRs by AuIB(globular) was unaffected. Inhibition of LS and HS alpha3beta4 nAChRs by AuIB(globular) was insurmountable and independent of membrane potential. Molecular docking simulation suggested that AuIB(globular) is likely to bind to both alpha3beta4 nAChR stoichiometries outside of the ACh-binding pocket, whereas AuIB(ribbon) binds to the classical agonist-binding site of the LS alpha3beta4 nAChR only. In conclusion, the two isomers of AuIB differ in their inhibitory mechanisms such that AuIB(ribbon) inhibits only LS alpha3beta4 nAChRs competitively, whereas AuIB(globular) inhibits alpha3beta4 nAChRs irrespective of receptor stoichiometry, primarily by a non-competitive mechanism.

Different nicotinic acetylcholine receptor (nAChR) subtypes are implicated in learning, pain sensation, and disease states, including Parkinson disease and nicotine addiction. alpha-Conotoxins are among the most selective nAChR ligands. Mechanistic insights into the structure, function, and receptor interaction of alpha-conotoxins may serve as a platform for development of new therapies. Previously characterized alpha-conotoxins have a highly conserved Ser-Xaa-Pro motif that is crucial for potent nAChR interaction. This study characterized the novel alpha-conotoxin LtIA, which lacks this highly conserved motif but potently blocked alpha3beta2 nAChRs with a 9.8 nm IC(50) value. The off-rate of LtIA was rapid relative to Ser-Xaa-Pro-containing alpha-conotoxin MII. Nevertheless, pre-block of alpha3beta2 nAChRs with LtIA prevented the slowly reversible block associated with MII, suggesting overlap in their binding sites. nAChR beta subunit ligand-binding interface mutations were used to examine the >1000-fold selectivity difference of LtIA for alpha3beta2 versus alpha3beta4 nAChRs. Unlike MII, LtIA had a >900-fold increased IC(50) value on alpha3beta2(F119Q) versus wild type nAChRs, whereas T59K and V111I beta2 mutants had little effect. Molecular docking simulations suggested that LtIA had a surprisingly shallow binding site on the alpha3beta2 nAChR that includes beta2 Lys-79. The K79A mutant disrupted LtIA binding but was without effect on an LtIA analog where the Ser-Xaa-Pro motif is present, consistent with distinct binding modes.

Alpha-conotoxins are tightly folded miniproteins that antagonize nicotinic acetylcholine receptors (nAChR) with high specificity for diverse subtypes. Here we report the use of selenocysteine in a supported phase method to direct native folding and produce alpha-conotoxins efficiently with improved biophysical properties. By replacing complementary cysteine pairs with selenocysteine pairs on an amphiphilic resin, we were able to chemically direct all five structural subclasses of alpha-conotoxins exclusively into their native folds. X-ray analysis at 1.4 A resolution of alpha-selenoconotoxin PnIA confirmed the isosteric character of the diselenide bond and the integrity of the alpha-conotoxin fold. The alpha-selenoconotoxins exhibited similar or improved potency at rat diaphragm muscle and alpha3beta4, alpha7, and alpha1beta1 deltagamma nAChRs expressed in Xenopus oocytes plus improved disulfide bond scrambling stability in plasma. Together, these results underpin the development of more stable and potent nicotinic antagonists suitable for new drug therapies, and highlight the application of selenocysteine technology more broadly to disulfide-bonded peptides and proteins.

Norepinephrine (NE) amplifies the strength of descending pain inhibition, giving inhibitors of spinal NET clinical utility in the management of pain. chi-MrIA isolated from the venom of a predatory marine snail noncompetitively inhibits NET and reverses allodynia in rat models of neuropathic pain. An analogue of chi-MrIA has been found to be a suitable drug candidate. On the basis of the NMR solution structure of this related peptide, Xen2174 (3), and structure-activity relationships of analogues, a pharmacophore model for the allosteric binding of 3 to NET is proposed. It is shown that 3 interacts with NET predominantly through amino acids in the first loop, forming a tight inverse turn presenting amino acids Tyr7, Lys8, and Leu9 in an orientation allowing for high affinity interaction with NET. The second loop interacts with a large hydrophobic pocket within the transporter. Analogues based on the pharmacophore demonstrated activities that support the proposed model. On the basis of improved chemical stability and a wide therapeutic index, 3 was selected for further development and is currently in phase II clinical trials.

Cone snails have evolved an assortment of venom peptides as an evolutionary strategy for rapid prey immobilization and defence. Earlier studies estimated approximately 100 conopeptides per species. In this study we optimized liquid chromatography and electrospray ionization mass spectrometry for the detection of conopeptides in crude venom to characterize conopeptides present in the venom of individual specimens of Conus textile, C. imperialis and C. marmoreus. Using this approach, we have expanded the predicted number of venom peptides 10-fold to an estimate of 1000-1900 conopeptides per species. Our investigation has also revealed a surprisingly high level of intra-species variation that distinguishes cone snails from other venomous species including spiders and scorpions. Given this inherent diversity and variability, more sensitive bioassays and sequencing techniques will be required to fully explore conotoxin bioactivity.

AlphaD-conotoxins are peptide inhibitors of nicotinic acetylcholine receptors (nAChRs) first described from Conus vexillum (alphaD-VxXIIA-C and renamed here to alphaD-VxXXA, alphaD-VxXXB, and alphaD-VxXXC). In this study, we report cDNA sequences encoding D-superfamily conopeptides identified in the Clade XII Conidae Conus vexillum, Conus capitaneus, Conus mustelinus, and Conus miles, together with partial sequences of corresponding peptides from this family. The D-superfamily signal peptide sequences display greater heterogeneity than reported for other conotoxin superfamilies. Phylogenetic analysis of the relationships among alphaD-conotoxin precursors reveals two distinct groups containing either an EMM or AVV signal peptide sequence motif. Homodimer and heterodimer combinations of predicted mature toxin sequences likely account for the partial amino acid sequences and mass values observed for several of the native dimeric peptide components identified in C. capitaneus, C. miles, and C. mustelinus venom. The discovery of the precursors and several novel conotoxins from different species defines this large conotoxin family and expands our understanding of sequence diversification mechanisms in Conus species.

alpha-Conotoxins are small disulfide-rich peptides from the venom of the Conus species that target the nicotinic acetylcholine receptor (nAChR). They are valuable pharmacological tools and also have potential therapeutic applications particularly for the treatment of chronic pain. alpha-Conotoxin GID is isolated from the venom of Conus geographus and has an unusual N-terminal tail sequence that has been shown to be important for binding to the alpha4beta2 subtype of the nAChR. To date, only four conotoxins that inhibit the alpha4beta2 subtype have been characterized, but they are of considerable interest as it is the most abundant nAChR subtype in the mammalian brain and has been implicated in a range of diseases. In this study, analysis of alanine-scan and truncation mutants of GID reveals that a conserved proline in alpha-conotoxins is important for activity at the alpha7, alpha3beta2, and alpha4beta2 subtypes. Although the proline residue was the most critical residue for activity at the alpha3beta2 subtype, Asp(3), Arg(12), and Asn(14) are also critical at the alpha7 subtype. Interestingly, very few of the mutations tested retained activity at the alpha4beta2 subtype indicating a tightly defined binding site. This lack of tolerance to sequence variation may explain the lack of selective ligands discovered for the alpha4beta2 subtype to date. Overall, our findings contribute to the understanding of the structure-activity relationships of alpha-conotoxins and may be beneficial for the ongoing attempts to exploit modulators of the neuronal nAChRs as therapeutic agents.

Marine molluscs known as cone snails produce beautiful shells and a complex array of over 50,000 venom peptides evolved for prey capture and defence. Many of these peptides selectively modulate ion channels and transporters, making them a valuable source of new ligands for studying the role these targets play in normal and disease physiology. A number of conopeptides reduce pain in animal models, and several are now in pre-clinical and clinical development for the treatment of severe pain often associated with diseases such as cancer. Less than 1% of cone snail venom peptides are pharmacologically characterised.

Venom peptides offer enormous opportunity for the discovery of peptide drug leads. This review focusses on the potential of cone snails that have developed arrays of small peptides as part of highly evolved venoms used for prey capture and defence. Many of these peptides selectively modulate ion channels and transporters, making them a valuable source of new ligands for studying the role these targets play in normal and disease physiology. A number of these conopeptides reduce pain in animals models and several are now in preclinical and clinical development for the treatment of severe pain often associated with diseases such as cancer.

Alpha-conotoxins are competitive antagonists of nicotinic acetylcholine receptors (nAChRs). The majority of currently characterized alpha-conotoxins have a 4/7 loop size, and the major features of neuronal alpha-conotoxins include a globular disulfide connectivity and a helical structure centered around the third of their four cysteine residues. In this study, a novel "molecular pruning" approach was undertaken to define the relationship between loop size, structure, and function of alpha-conotoxins. This involved the systematic truncation of the second loop in the alpha-conotoxin [A10L]PnIA [4/7], a potent antagonist of the alpha7 nAChR. The penalty for truncation was found to be decreased conformational stability and increased susceptibility to disulfide bond scrambling. Truncation down to 4/4[A10L]PnIA maintained helicity and did not significantly reduce electrophysiological activity at alpha7 nAChRs, whereas 4/3[A10L]PnIA lost both alpha7 nAChR activity and helicity. In contrast, all truncated analogues lost approximately 100-fold affinity at the AChBP, a model protein for the extracellular domain of the nAChR. Docking simulations identified several hydrogen bonds lost upon truncation that provide an explanation for the reduced affinities observed at the alpha7 nAChR and AChBP.

Mu-conotoxins are small peptide inhibitors of muscle and neuronal tetrodotoxin (TTX)-sensitive voltage-gated sodium channels (VGSCs). Here we report the isolation of mu-conotoxins SIIIA and SIIIB by (125)I-TIIIA-guided fractionation of milked Conus striatus venom. SIIIA and SIIIB potently displaced (125)I-TIIIA from native rat brain Na(v)1.2 (IC(50) values 10 and 5 nm, respectively) and muscle Na(v)1.4 (IC(50) values 60 and 3 nm, respectively) VGSCs, and both inhibited current through Xenopus oocyte-expressed Na(v)1.2 and Na(v)1.4. An alanine scan of SIIIA-(2-20), a pyroglutamate-truncated analogue with enhanced neuronal activity, revealed residues important for affinity and selectivity. Alanine replacement of the solvent-exposed Trp-12, Arg-14, His-16, Arg-18 resulted in large reductions in SIIIA-(2-20) affinity, with His-16 replacement affecting structure. In contrast, [D15A]SIIIA-(2-20) had significantly enhanced neuronal affinity (IC(50) 0.65 nm), while the double mutant [D15A/H16R]SIIIA-(2-20) showed greatest Na(v)1.2 versus 1.4 selectivity (136-fold). (1)H NMR studies revealed that SIIIA adopted a single conformation in solution comprising a series of turns and an alpha-helical motif across residues 11-16 that is not found in larger mu-conotoxins. The structure of SIIIA provides a new structural template for the development of neuronally selective inhibitors of TTX-sensitive VGSCs based on the smaller mu-conotoxin pharmacophore.

Conotoxins are highly constrained peptide toxins that exhibit pharmaceutically relevant biological activities. We herein report the extent of absorption and profile of distribution of a native alpha-conotoxin, MII and a lipophilic analogue of MII (N-LaaMII) after intravenous (iv) and oral administration to male Sprague-Dawley rats. N-LaaMII is formed by coupling 2-amino-D,L-dodecanoic acid (Laa) to the N-terminus of MII and has previously been shown to exhibit significantly improved permeability across Caco-2 cell monolayers compared to the native MII while maintaining the potency in inhibition of nAChRs of the parent peptide. Both peptides crossed the GI tract after oral administration (approximately 6% after 30 m). While Laa conjugation did not significantly improve absorption, it did greatly increase the accumulation of the compound in the liver after iv administration. Neither peptide crossed the blood-brain barrier to any significant extent. This is the first study of the in vivo biodistribution of an alpha-conotoxin after oral administration.

Neuronal nAChRs are a diverse family of pentameric ion channels with wide distribution throughout cells of the nervous and immune systems. However, the role of specific subtypes in normal and pathological states remains poorly understood due to the lack of selective probes. Here, we used a binding assay based on acetylcholine-binding protein (AChBP), a homolog of the nicotinic acetylcholine ligand-binding domain, to discover a novel alpha-conotoxin (alpha-TxIA) in the venom of Conus textile. Alpha-TxIA bound with high affinity to AChBPs from different species and selectively targeted the alpha(3)beta(2) nAChR subtype. A co-crystal structure of Ac-AChBP with the enhanced potency analog TxIA(A10L), revealed a 20 degrees backbone tilt compared to other AChBP-conotoxin complexes. This reorientation was coordinated by a key salt bridge formed between Arg5 (TxIA) and Asp195 (Ac-AChBP). Mutagenesis studies, biochemical assays and electrophysiological recordings directly correlated the interactions observed in the co-crystal structure to binding affinity at AChBP and different nAChR subtypes. Together, these results establish a new pharmacophore for the design of novel subtype-selective ligands with therapeutic potential in nAChR-related diseases.