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.

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.

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-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.

Insights into the origins of fish hunting in venomous cone snails from studies of Conus tessulatus.

Prey shifts in carnivorous predators are events that can initiate the accelerated generation of new biodiversity. However, it is seldom possible to reconstruct how the change in prey preference occurred. Here we describe an evolutionary "smoking gun" that illuminates the transition from worm hunting to fish hunting among marine cone snails, resulting in the adaptive radiation of fish-hunting lineages comprising ∼100 piscivorous Conus species. This smoking gun is δ-conotoxin TsVIA, a peptide from the venom of Conus tessulatus that delays inactivation of vertebrate voltage-gated sodium channels. C. tessulatus is a species in a worm-hunting clade, which is phylogenetically closely related to the fish-hunting cone snail specialists. The discovery of a δ-conotoxin that potently acts on vertebrate sodium channels in the venom of a worm-hunting cone snail suggests that a closely related ancestral toxin enabled the transition from worm hunting to fish hunting, as δ-conotoxins are highly conserved among fish hunters and critical to their mechanism of prey capture; this peptide, δ-conotoxin TsVIA, has striking sequence similarity to these δ-conotoxins from piscivorous cone snail venoms. Calcium-imaging studies on dissociated dorsal root ganglion (DRG) neurons revealed the peptide's putative molecular target (voltage-gated sodium channels) and mechanism of action (inhibition of channel inactivation). The results were confirmed by electrophysiology. This work demonstrates how elucidating the specific interactions between toxins and receptors from phylogenetically well-defined lineages can uncover molecular mechanisms that underlie significant evolutionary transitions.

Diversity of conotoxin types from Conus californicus reflects a diversity of prey types and a novel evolutionary history.

Most species within the genus Conus are considered to be specialists in their consumption of prey, typically feeding on molluscs, vermiform invertebrates or fish, and employ peptide toxins to immobilize prey. Conus californicus Hinds 1844 atypically utilizes a wide range of food sources from all three groups. Using DNA- and protein-based methods, we analyzed the molecular diversity of C. californicus toxins and detected a correspondingly large number of conotoxin types. We identified cDNAs corresponding to seven known cysteine-frameworks containing over 40 individual inferred peptides. Additionally, we found a new framework (22) with six predicted peptide examples, along with two forms of a new peptide type of unusual length. Analysis of leader sequences allowed assignment to known superfamilies in only half of the cases, and several of these showed a framework that was not in congruence with the identified superfamily. Mass spectrometric examination of chromatographic fractions from whole venom served to identify peptides corresponding to a number of cDNAs, in several cases differing in their degree of posttranslational modification. This suggests differential or incomplete biochemical processing of these peptides. In general, it is difficult to fit conotoxins from C. californicus into established toxin classification schemes. We hypothesize that the novel structural modifications of individual peptides and their encoding genes reflect evolutionary adaptation to prey species of an unusually wide range as well as the large phylogenetic distance between C. californicus and Indo-Pacific species.

Conopeptide characterization and classifications: an analysis using ConoServer.

Cone snails are carnivorous marine gastropods that have evolved potent venoms to capture their prey. These venoms comprise a rich and diverse cocktail of peptide toxins, or conopeptides, whose high diversity has arisen from an efficient hypermutation mechanism, combined with a high frequency of post-translational modifications. Conopeptides bind with high specificity to distinct membrane receptors, ion channels, and transporters of the central and muscular nervous system. As well as serving their natural function in prey capture, conopeptides have been utilized as versatile tools in neuroscience and have proven valuable as drug leads that target the nervous system in humans. This paper examines current knowledge on conopeptide sequences based on an analysis of gene and peptide sequences in ConoServer (http://www.conoserver.org), a specialized database of conopeptide sequences and three-dimensional structures. We describe updates to the content and organization of ConoServer and discuss correlations between gene superfamilies, cysteine frameworks, pharmacological families targeted by conopeptides, and the phylogeny, habitat, and diet of cone snails. The study identifies gaps in current knowledge of conopeptides and points to potential directions for future research.

Venom kinematics during prey capture in Conus: the biomechanics of a rapid injection system.

Cone snails use an extensile, tubular proboscis as a conduit to deliver a potent cocktail of bioactive venom peptides into their prey. Previous studies have focused mainly on understanding the venom's role in prey capture but successful prey capture requires both rapid physiological and biomechanical mechanisms. Conus catus, a fish-hunting species, uses a high-speed hydraulic mechanism to inject its hollow, spear-like radular tooth into prey. We take an integrated approach to investigating the biomechanics of this process by coupling kinematic studies with morphological analyses. Taking advantage of the opaque venom and translucent proboscis of a mollusc-hunting juvenile cone snail, Conus pennaceus, we have determined that a high-speed prey capture mechanism is not unique to cone species that hunt fish prey. Two morphological structures were found to play crucial roles in this process. A constriction of the lumen near the tip of the proboscis, composed of tall epithelial cells densely packed with microfilaments, impedes forward movement of the radular tooth prior to its propulsion. Proximal to the constriction, a muscular sphincter was found to regulate venom flow and pressurization in the proboscis. In C. pennaceus, the rapid appearance and flushing of venom within the proboscis during prey capture suggests a mechanism involving the delivery of a discrete quantity of venom. The interplay between these elements provides a unique and effective biomechanical injection system for the fast-acting cone snail venom peptides.