Synthesis and Biological Activity of Novel α-Conotoxins Derived from Endemic Polynesian Cone Snails.

α-Conotoxins are well-known probes for the characterization of the various subtypes of nicotinic acetylcholine receptors (nAChRs). Identifying new α-conotoxins with different pharmacological profiles can provide further insights into the physiological or pathological roles of the numerous nAChR isoforms found at the neuromuscular junction, the central and peripheral nervous systems, and other cells such as immune cells. This study focuses on the synthesis and characterization of two novel α-conotoxins obtained from two species endemic to the Marquesas Islands, namely Conus gauguini and Conus adamsonii. Both species prey on fish, and their venom is considered a rich source of bioactive peptides that can target a wide range of pharmacological receptors in vertebrates. Here, we demonstrate the versatile use of a one-pot disulfide bond synthesis to achieve the α-conotoxin fold [Cys 1-3; 2-4] for GaIA and AdIA, using the 2-nitrobenzyl (NBzl) protecting group of cysteines for effective regioselective oxidation. The potency and selectivity of GaIA and AdIA against rat nicotinic acetylcholine receptors were investigated electrophysiologically and revealed potent inhibitory activities. GaIA was most active at the muscle nAChR (IC50 = 38 nM), whereas AdIA was most potent at the neuronal α6/3 ß2ß3 subtype (IC50 = 177 nM). Overall, this study contributes to a better understanding of the structure-activity relationships of α-conotoxins, which may help in the design of more selective tools.

Biomedical Potential of the Neglected Molluscivorous and Vermivorous Conus Species.

Within the Conidae family, the piscivorous Conus species have been a hotspot target for drug discovery. Here, we assess the relevance of Conus and their other feeding habits, and thus under distinctive evolutionary constraints, to highlight the potential of neglected molluscivorous and vermivorous species in biomedical research and pharmaceutical industry. By singling out the areas with inadequate Conus disquisition, such as the Tamil Nadu Coast and the Andaman Islands, research resources can be expanded and better protected through awareness. In this study, 728 Conus species and 190 species from three other genera (1 from Californiconus, 159 from Conasprella and 30 from Profundiconus) in the Conidae family are assessed. The phylogenetic relationships of the Conidae species are determined and their known feeding habits superimposed. The worm-hunting species appeared first, and later the mollusc- and fish-hunting species were derived independently in the Neogene period (around 23 million years ago). Interestingly, many Conus species in the warm and shallow waters become polyphagous, allowing them to hunt both fish and worms, given the opportunities. Such newly gained trait is multi originated. This is controversial, given the traditional idea that most Conus species are specialized to hunt certain prey categories. However, it shows the functional complexity and great potential of conopeptides from some worm-eating species. Pharmaceutical attempts and relevant omics data have been differentially obtained. Indeed, data from the fish-hunting species receive strong preference over the worm-hunting ones. Expectedly, conopeptides from the fish-hunting species are believed to include the most potential candidates for biomedical research. Our work revisits major findings throughout the Conus evolution and emphasizes the importance of increasing omics surveys complemented with further behavior observation studies. Hence, we claim that Conus species and their feeding habits are equally important, highlighting many places left for Conus exploration worldwide. We also discuss the Conotoxin drug discovery potentials and the urgency of protecting the bioresources of Conus species. In particular, some vermivorous species have demonstrated great potential in malaria therapy, while other conotoxins from several worm- and mollusc-eating species exhibited explicit correlation with SARS-CoV-2. Reclaiming idle data with new perspectives could also promote interdisciplinary studies in both virological and toxicological fields.

Venom duct origins of prey capture and defensive conotoxins in piscivorous Conus striatus.

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.

Globular and ribbon isomers of Conus geographus α-conotoxins antagonize human nicotinic acetylcholine receptors.

The short disulfide-rich α-conotoxins derived from the venom of Conus snails comprise a conserved CICII(m)CIII(n)CIV cysteine framework (m and n, number of amino acids) and the majority antagonize nicotinic acetylcholine receptors (nAChRs). Depending on disulfide connectivity, α-conotoxins can exist as either globular (CI-CIII, CII-CIV), ribbon (CI-CIV, CII-CIII) or bead (CI-CII, CIII-CIV) isomers. In the present study, C. geographus α-conotoxins GI, GIB, G1.5 and G1.9 were chemically synthesized as globular and ribbon isomers and their activity investigated at human nAChRs expressed in Xenopus oocytes using the two-electrode voltage clamp recording technique. Both the globular and ribbon isomers of the 3/5 (m/n) α-conotoxins GI and GIB selectively inhibit heterologous human muscle-type α1ß1δε nAChRs, whereas G1.5, a 4/7 α-conotoxin, selectively antagonizes neuronal (non-muscle) nAChR subtypes particularly human α3ß2, α7 and α9α10 nAChRs. In contrast, globular and ribbon isomers of G1.9, a novel C-terminal elongated 4/8 α-conotoxin exhibited no activity at the human nAChR subtypes studied. This study reinforces earlier observations that 3/5 α-conotoxins selectively target the muscle nAChR subtypes, although interestingly, GIB is also active at α7 and α9 α10 nAChRs. The 4/7 α-conotoxins target human neuronal nAChR subtypes whereas the pharmacology of the 4/8 α-conotoxin remains unknown.

Identification of Conomarphin Variants in the Conus eburneus Venom and the Effect of Sequence and PTM Variations on Conomarphin Conformations.

Marine cone snails belonging to the Conidae family make use of neuroactive peptides in their venom to capture prey. Here we report the proteome profile of the venom duct of Conus eburneus, a cone snail belonging to the Tesseliconus clade. Through tandem mass spectrometry and database searching against the C. eburneus transcriptome and the ConoServer database, we identified 24 unique conopeptide sequences in the venom duct. The majority of these peptides belong to the T and M gene superfamilies and are disulfide-bonded, with cysteine frameworks V, XIV, VI/VII, and III being the most abundant. All seven of the Cys-free peptides are conomarphin variants belonging to the M superfamily that eluted out as dominant peaks in the chromatogram. These conomarphins vary not only in amino acid residues in select positions along the backbone but also have one or more post-translational modifications (PTMs) such as proline hydroxylation, C-term amidation, and γ-carboxylation of glutamic acid. Using molecular dynamics simulations, the conomarphin variants were predicted to predominantly have hairpin-like or elongated structures in acidic pH. These two structures were found to have significant differences in electrostatic properties and the inclusion of PTMs seems to complement this disparity. The presence of polar PTMs (hydroxyproline and γ-carboxyglutamic acid) also appear to stabilize hydrogen bond networks in these conformations. Furthermore, these predicted structures are pH sensitive, becoming more spherical and compact at higher pH. The subtle conformational variations observed here might play an important role in the selection and binding of the peptides to their molecular targets.

The α1-adrenoceptor inhibitor ρ-TIA facilitates net hunting in piscivorous Conus tulipa.

Cone snails use separately evolved venoms for prey capture and defence. While most use a harpoon for prey capture, the Gastridium clade that includes the well-studied Conus geographus and Conus tulipa, have developed a net hunting strategy to catch fish. This unique feeding behaviour requires secretion of "nirvana cabal" peptides to dampen the escape response of targeted fish allowing for their capture directly by mouth. However, the active components of the nirvana cabal remain poorly defined. In this study, we evaluated the behavioural effects of likely nirvana cabal peptides on the teleost model, Danio rerio (zebrafish). Surprisingly, the conantokins (NMDA receptor antagonists) and/or conopressins (vasopressin receptor agonists and antagonists) found in C. geographus and C. tulipa venom failed to produce a nirvana cabal-like effect in zebrafish. In contrast, low concentrations of the non-competitive adrenoceptor antagonist ρ-TIA found in C. tulipa venom (EC50 = 190 nM) dramatically reduced the escape response of zebrafish larvae when added directly to aquarium water. ρ-TIA inhibited the zebrafish α1-adrenoceptor, confirming ρ-TIA has the potential to reverse the known stimulating effects of norepinephrine on fish behaviour. ρ-TIA may act alone and not as part of a cabal, since it did not synergise with conopressins and/or conantokins. This study highlights the importance of using ecologically relevant animal behaviour models to decipher the complex neurobiology underlying the prey capture and defensive strategies of cone snails.

The high speed radular prey strike of a fish-hunting cone snail.

Cone snails are venomous marine gastropods that hydraulically propel a hollow, chitinous radular harpoon into prey [1,2]. This radular harpoon serves both as projectile and conduit for venom delivery. In the fish-hunting cone snail Conus catus, the radular harpoon is also utilized to tether the snail to its prey, rapidly paralyzed by neuroexcitatory peptides [2,3]. Effective prey capture in C. catus requires both fast-acting neurotoxins and a delivery system quick enough to exceed the prey fish's rapid escape responses [4]. We report here that the cone snail's prey strike is one of the fastest in the animal kingdom. A unique cellular latch mechanism prevents harpoon release until sufficient pressure builds and overcomes the forces of the latch, resulting in rapid acceleration into prey [2]. The radular harpoon then rapidly decelerates as its bulbous base reaches the end of the proboscis, a distensible hydrostatic skeleton extended toward the prey [2], with little slowing during prey impalement. The velocities achieved are the fastest movements of any mollusk and exceed previous estimates by over an order of magnitude [1].

Effects of Predator-Prey Interactions on Predator Traits: Differentiation of Diets and Venoms of a Marine Snail.

Species interactions are fundamental ecological forces that can have significant impacts on the evolutionary trajectories of species. Nonetheless, the contribution of predator-prey interactions to genetic and phenotypic divergence remains largely unknown. Predatory marine snails of the family Conidae exhibit specializations for different prey items and intraspecific variation in prey utilization patterns at geographic scales. Because cone snails utilize venom to capture prey and venom peptides are direct gene products, it is feasible to examine the evolution of genes associated with changes in resource utilization. Here, we compared feeding ecologies and venom duct transcriptomes of individuals from three populations of Conus miliaris, a species that exhibits geographic variation in prey utilization and dietary breadth, in order to determine the extent to which dietary differences are correlated with differences in venom composition, and if expanded niche breadth is associated with increased variation in venom composition. While populations showed little to no overlap in resource utilization, taxonomic richness of prey was greatest at Easter Island. Changes in dietary breadth were associated with differences in expression patterns and increased genetic differentiation of toxin-related genes. The Easter Island population also exhibited greater diversity of toxin-related transcripts, but did not show increased variance in expression of these transcripts. These results imply that differences in dietary breadth contribute more to the structural and regulatory differentiation of venoms than differences in diet.

Transcriptomic-Proteomic Correlation in the Predation-Evoked Venom of the Cone Snail, Conus imperialis.

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.

Venomics Reveals Venom Complexity of the Piscivorous Cone Snail, Conus tulipa.

The piscivorous cone snail Conus tulipa has evolved a net-hunting strategy, akin to the deadly Conus geographus, and is considered the second most dangerous cone snail to humans. Here, we present the first venomics study of C. tulipa venom using integrated transcriptomic and proteomic approaches. Parallel transcriptomic analysis of two C. tulipa specimens revealed striking differences in conopeptide expression levels (2.5-fold) between individuals, identifying 522 and 328 conotoxin precursors from 18 known gene superfamilies. Despite broad overlap at the superfamily level, only 86 precursors (11%) were common to both specimens. Conantokins (NMDA antagonists) from the superfamily B1 dominated the transcriptome and proteome of C. tulipa venom, along with superfamilies B2, A, O1, O3, con-ikot-ikot and conopressins, plus novel putative conotoxins precursors T1.3, T6.2, T6.3, T6.4 and T8.1. Thus, C. tulipa venom comprised both paralytic (putative ion channel modulating α-, ω-, µ-, δ-) and non-paralytic (conantokins, con-ikot-ikots, conopressins) conotoxins. This venomic study confirms the potential for non-paralytic conotoxins to contribute to the net-hunting strategy of C. tulipa.

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.

Venomics-Accelerated Cone Snail Venom Peptide Discovery.

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.

Accelerated proteomic visualization of individual predatory venoms of Conus purpurascens reveals separately evolved predation-evoked venom cabals.

Cone snail venoms have separately evolved for predation and defense. Despite remarkable inter- and intra-species variability, defined sets of synergistic venom peptides (cabals) are considered essential for prey capture by cone snails. To better understand the role of predatory cabals in cone snails, we used a high-throughput proteomic data mining and visualisation approach. Using this approach, the relationship between the predatory venom peptides from nine C. purpurascens was systematically analysed. Surprisingly, potentially synergistic levels of κ-PVIIA and δ-PVIA were only identified in five of nine specimens. In contrast, the remaining four specimens lacked significant levels of these known excitotoxins and instead contained high levels of the muscle nAChR blockers ψ-PIIIE and αA-PIVA. Interestingly, one of nine specimens expressed both cabals, suggesting that these sub-groups might represent inter-breeding sub-species of C. purpurascens. High throughput cluster analysis also revealed these two cabals clustered with distinct groups of venom peptides that are presently uncharacterised. This is the first report showing that the cone snails of the same species can deploy two separate and distinct predatory cabals for prey capture and shows that the cabals deployed by this species can be more complex than presently realized. Our semi-automated proteomic analysis facilitates the deconvolution of complex venoms to identify co-evolved families of peptides and help unravel their evolutionary relationships in complex venoms.

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

The marine cone snail Conus gloriamaris is an iconic species. For over two centuries, its shell was one of the most prized and valuable natural history objects in the world. Today, cone snails have attracted attention for their remarkable venom components. Many conotoxins are proving valuable as research tools, drug leads, and drugs. In this article, we present the venom gland transcriptome of C. gloriamaris, revealing this species' conotoxin repertoire. More than 100 conotoxin sequences were identified, representing a valuable resource for future drug discovery efforts.

Linking neuroethology to the chemical biology of natural products: interactions between cone snails and their fish prey, a case study.

From a biological perspective, a natural product can be defined as a compound evolved by an organism for chemical interactions with another organism including prey, predator, competitor, pathogen, symbiont or host. Natural products hold tremendous potential as drug leads and have been extensively studied by chemists and biochemists in the pharmaceutical industry. However, the biological purpose for which a natural product evolved is rarely addressed. By focusing on a well-studied group of natural products-venom components from predatory marine cone snails-this review provides a rationale for why a better understanding of the evolution, biology and biochemistry of natural products will facilitate both neuroscience and the potential for drug leads. The larger goal is to establish a new sub-discipline in the broader field of neuroethology that we refer to as "Chemical Neuroethology", linking the substantial work carried out by chemists on natural products with accelerating advances in neuroethology.

Ocean acidification alters predator behaviour and reduces predation rate.

Ocean acidification poses a range of threats to marine invertebrates; however, the emerging and likely widespread effects of rising carbon dioxide (CO2) levels on marine invertebrate behaviour are still little understood. Here, we show that ocean acidification alters and impairs key ecological behaviours of the predatory cone snail Conus marmoreus Projected near-future seawater CO2 levels (975 µatm) increased activity in this coral reef molluscivore more than threefold (from less than 4 to more than 12 mm min-1) and decreased the time spent buried to less than one-third when compared with the present-day control conditions (390 µatm). Despite increasing activity, elevated CO2 reduced predation rate during predator-prey interactions with control-treated humpbacked conch, Gibberulus gibberulus gibbosus; 60% of control predators successfully captured and consumed their prey, compared with only 10% of elevated CO2 predators. The alteration of key ecological behaviours of predatory invertebrates by near-future ocean acidification could have potentially far-reaching implications for predator-prey interactions and trophic dynamics in marine ecosystems. Combined evidence that the behaviours of both species in this predator-prey relationship are altered by elevated CO2 suggests food web interactions and ecosystem structure will become increasingly difficult to predict as ocean acidification advances over coming decades.

Insulin as a weapon.

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

Age-related association of venom gene expression and diet of predatory gastropods.

BACKGROUND: Venomous organisms serve as wonderful systems to study the evolution and expression of genes that are directly associated with prey capture. To evaluate the relationship between venom gene expression and prey utilization, we examined these features among individuals of different ages of the venomous, worm-eating marine snail Conus ebraeus. We determined expression levels of six genes that encode venom components, used a DNA-based approach to evaluate the identity of prey items, and compared patterns of venom gene expression and dietary specialization. RESULTS: C. ebraeus exhibits two major shifts in diet with age-an initial transition from a relatively broad dietary breadth to a narrower one and then a return to a broader diet. Venom gene expression patterns also change with growth. All six venom genes are up-regulated in small individuals, down-regulated in medium-sized individuals, and then either up-regulated or continued to be down-regulated in members of the largest size class. Venom gene expression is not significantly different among individuals consuming different types of prey, but instead is coupled and slightly delayed with shifts in prey diversity. CONCLUSION: These results imply that changes in gene expression contribute to intraspecific variation of venom composition and that gene expression patterns respond to changes in the diversity of food resources during different growth stages.

Prey-Capture Strategies of Fish-Hunting Cone Snails: Behavior, Neurobiology and Evolution.

The venomous fish-hunting cone snails (Conus) comprise eight distinct lineages evolved from ancestors that preyed on worms. In this article, we attempt to reconstruct events resulting in this shift in food resource by closely examining patterns of behavior, biochemical agents (toxins) that facilitate prey capture and the combinations of toxins present in extant species. The first sections introduce three different hunting behaviors associated with piscivory: 'taser-and-tether', 'net-engulfment' and 'strike-and-stalk'. The first two fish-hunting behaviors are clearly associated with distinct groups of venom components, called cabals, which act in concert to modify the behavior of prey in a specific manner. Derived fish-hunting behavior clearly also correlates with physical features of the radular tooth, the device that injects these biochemical components. Mapping behavior, biochemical components and radular tooth features onto phylogenetic trees shows that fish-hunting behavior emerged at least twice during evolution. The system presented here may be one of the best examples where diversity in structure, physiology and molecular features were initially driven by particular pathways selected through behavior.

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.