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Non-opioid therapeutics for the treatment of neuropathic pain are urgently needed to address the ongoing opioid crisis. Peptides from cone snail venoms have served as invaluable molecules to target key pain-related receptors but can suffer from unfavorable physicochemical properties, which limit their therapeutic potential. In this work, we developed conformationally constrained α-RgIA analogues with high potency, receptor selectivity, and enhanced human serum stability to target the human α9α10 nicotinic acetylcholine receptor. The key lactam linkage introduced in α-RgIA fixed the favored globular conformation and suppressed disulfide scrambling. The NMR structure of the macrocyclic peptide overlays well with that of α-RgIA4, demonstrating that the cyclization does not perturb the overall conformation of backbone and key side-chain residues. Finally, a molecular docking model was used to rationalize the selective binding between a macrocyclic analogue and the α9α10 nicotinic acetylcholine receptor. These conformationally constrained antagonists are therefore promising candidates for antinociceptive therapeutic intervention.

Marine gastropods of the genus Conus, comprising more than 800 species, have the characteristic of injecting worms and other prey with venom. These conopeptide toxins, highly diverse in structure and action, are highly potent and specific for their molecular targets (ion channels, receptors, and transporters of the prey’s nervous system), and thus are important research tools and source for drug discovery. Next-generation sequencing technologies are speeding up the discovery of novel conopeptides in many of these species, but only limited information is available for Conus spurius, which inhabits sandy mud. To search for new precursor conopeptides, we analyzed the transcriptome of the venous ducts of C. spurius and identified 55 putative conotoxins. Seven were selected for further study and confirmed by Sanger sequencing to belong to the M-superfamily (Sr3.M01 and Sr3.M02), A-superfamily (Sr1.A01 and Sr1.A02), O-superfamily (Sr15.O01), and Con-ikot-ikot (Sr21.CII01 and Sr22.CII02). Six of these have never been reported. To our knowledge, this report is the first to use high-throughput RNA sequencing for the study of the diversity of C. spurius conotoxins.

Marine gastropods of the genusConusare renowned for their remarkable diversity and deadly venoms. WhileConusvenoms are increasingly well studied for their biomedical applications, we know surprisingly little about venom composition in other lineages of Conidae. We performed comprehensive venom transcriptomic profiling forConasprella coriolisiandPygmaeconus traillii, first time for both respective genera. We complemented reference-based transcriptome annotation by ade novotoxin prediction guided by phylogeny, which involved transcriptomic data on two additional ‘divergent’ cone snail lineages,Profundiconus, andCaliforniconus. We identified toxin clusters (SSCs) shared among all or some of the four analysed genera based on the identity of the signal region—a molecular tag present in toxins. In total, 116 and 98 putative toxins represent 29 and 28 toxin gene superfamilies inConasprellaandPygmaeconus, respectively; about quarter of these only found by semi-manual annotation of the SSCs. Two rare gene superfamilies, originally identified from fish-hunting cone snails, were detected outsideConusrather unexpectedly, so we further investigated their distribution across Conidae radiation. We demonstrate that both these, in fact, are ubiquitous in Conidae, sometimes with extremely high expression. Our findings demonstrate how a phylogeny-aware approach circumvents methodological caveats of similarity-based transcriptome annotation.

Conorfamides are a poorly studied family of cone snail venom peptides with broad biological activities, including inhibition of glutamate receptors, acid-sensing ion channels, and voltage-gated potassium channels. The aim of this study was to characterize the pharmacological activity of two novel linear conorfamides (conorfamide_As1a and conorfamide_As2a) and their non-amidated counterparts (conopeptide_As1b and conopeptide_As2b) that were isolated from the venom of the Mexican cone snail Conus austini. Although As1a, As2a, As1b and As2b were identified by activity-guided fractionation using a high-throughput fluorescence imaging plate reader (FLIPR) assay assessing α7 nAChR activity, sequence determination revealed activity associated with four linear peptides of the conorfamide rather than the anticipated α-conotoxin family. Pharmacological testing revealed that the amidated peptide variants altered desensitization of acid-sensing ion channels (ASICs) 1a and 3, and the native lysine to arginine mutation differentiating As1a and As1b from As2a and As2b introduced ASIC1a peak current potentiation. Surprisingly, these conorfamides also inhibited α7 and muscle-type nicotinic acetylcholine receptors (nAChR) at nanomolar concentrations. This is the first report of conorfamides with dual activity, with the nAChR activity being the most potent molecular target of any conorfamide discovered to date.

Putative prolyl-4-hydroxylase (P4H) α-subunit sequences have been extracted by mining transcriptomic data obtained from seven cone snail species C. amadis, C. monile, C. araneosus, C. miles, C. litteratus, C. frigidus, and C. ebraeus. Sequences ranging from 518 to 559 residues have been compared with representative animal P4H sequences. The α-subunit consists of an N-terminus double domain, involved in dimerization and substrate binding, while the C-terminus contains the catalytic domain. Definitive functional annotation of the cone snail sequences has been achieved by an analysis of conserved residues responsible for catalytic function, specific conformational features, and subunit interactions, using two independent structures of the double domain, and the catalytic domain, previously reported in the literature. The variability of proline hydroxylation in conotoxins is illustrated by a mass spectrometric analysis of C. amadis venom. Site specific hydroxylation and the presence of peptides with multiple proline residues, resistant to modification, suggests that sequence and conformational effects may determine the substrate specificity of the Conus prolyl-4-hydroxylases. SIGNIFICANCE: Proline hydroxylation is a widely observed post translational modification, with collagen being the pre-eminent example. Hydroxylation of proline is also widely observed in conotoxins, which are a major component of marine cone snail venom. This paper describes newly identified prolyl-4-hydroxylase sequences, using transcriptome data from seven Conus species. The predicted functional annotation of prolyl-4-hydroxylase sequences was carried out using two available crystal structures of independent domains. The mass spectrometric characterisation of proline/hydroxyproline containing peptides in C. amadis venom confirms sequence specific hydroxylation in Conus venom as shown previously by others.

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 α1β1δe nAChRs, whereas G1.5, a 4/7 α-conotoxin, selectively antagonizes neuronal (non-muscle) nAChR subtypes particularly human α3β2, 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.

Marine cone snails are predatory gastropods characterized by a well-developed venom apparatus and highly evolved hunting strategies that utilize toxins to paralyze prey and defend against predators. The venom of each species of cone snail has a large number of pharmacologically active peptides known as conopeptides or conotoxins that are usually unique in each species. Nevertheless, venoms of only very few species have been characterized so far by transcriptomic approaches. In this study, we used transcriptome sequencing technologies and mass spectrometric methods to describe the diversity of venom components expressed by a worm-hunting species, Conus bayani. A total of 82 conotoxin sequences were retrieved from transcriptomic data that contain 54 validated conotoxin sequences clustered into 21 gene superfamilies including divergent gene family, 17 sequences clustered to 6 different conotoxin classes, and 11 conotoxins classified as unassigned gene family. Seven new conotoxin sequences showed unusual cysteine patterns. We were also able to identify 19 peptide sequences using mass spectrometry that completely overlapped with the conotoxin sequences obtained from transcriptome analysis. Importantly, herein we document the presence of 16 proteins that include five post-translational modifying enzymes obtained from transcriptomic data. Our results revealed diverse and novel conopeptides of an unexplored species that could be used extensively in biomedical research due to their therapeutic potentials.

Conotoxins are disulfide-rich peptides found in the venom of cone snails. Due to their exquisite potency and high selectivity for a wide range of voltage and ligand gated ion channels they are attractive drug leads in neuropharmacology. Recently, cone snails were found to have the capability to rapidly switch between venom types with different proteome profiles in response to predatory or defensive stimuli. A novel conotoxin, GXIA (original name G117), belonging to the I3-subfamily was identified as the major component of the predatory venom of piscivorous Conus geographus. Using 2D solution NMR spectroscopy techniques, we resolved the 3D structure for GXIA, the first structure reported for the I3-subfamily and framework XI family. The 32 amino acid peptide is comprised of eight cysteine residues with the resultant disulfide connectivity forming an ICK+1 motif. With a triple stranded β-sheet, the GXIA backbone shows striking similarity to several tarantula toxins targeting the voltage sensor of voltage gated potassium and sodium channels. Supported by an amphipathic surface, the structural evidence suggests that GXIA is able to embed in the membrane and bind to the voltage sensor domain of a putative ion channel target.

The venom of various Conus species is composed of a rich variety of unique bioactive peptides, commonly referred to as conotoxins (conopeptides). Most conopeptides have specific receptors or ion channels as physiologically relevant targets. In this paper, high-throughput transcriptome sequencing was performed to analyze putative conotoxin transcripts from the venom duct of a vermivorous cone snail species, Conus litteratus native to the South China Sea. A total of 128 putative conotoxins were identified, most of them belonging to 22 known superfamilies, with 43 conotoxins being regarded as belonging to new superfamilies. Notably, the M superfamily was the most abundant in conotoxins among the known superfamilies. A total of 15 known cysteine frameworks were also described. The largest proportion of cysteine frameworks were VI/VII (C-C-CC-C-C), IX (C-C-C-C-C-C) and XIV (C-C-C-C). In addition, five novel cysteine patterns were also discovered. Simple sequence repeat detection results showed that di-nucleotide was the major type of repetition, and the codon usage bias results indicated that the codon usage bias of the conotoxin genes was weak, but the M, O1, O2 superfamilies differed in codon preference. Gene cloning indicated that there was no intron in conotoxins of the B1- or J superfamily, one intron with 1273&ndash, 1339 bp existed in a mature region of the F superfamily, which is different from the previously reported gene structure of conotoxins from other superfamilies. This study will enhance our understanding of conotoxin diversity, and the new conotoxins discovered in this paper will provide more potential candidates for the development of pharmacological probes and marine peptide drugs.

Previous studies have indicated that each conotoxin precursor has a hyperconserved signal region, a rather conserved pro region and a hypervariable mature region, and nucleotide mutations are the main driven factor. However, in this study, we made an in-depth analysis on the M-superfamily conotoxin precursors and found that the diversity of the signal, pro and mature regions are more complicated than previous findings. Different conotoxin precursors can have same signal, pro and/or mature regions, especially different conotoxin precursors with same mature region but different signal and pro regions. In addition, insertions and deletions (indels) were detected in conotoxin precursors. Indels are infrequent in the signal region but frequent in the pro and mature regions. In contrast to deletions that dominate in the pro region, insertions dominate in the mature region. The number of amino acids is crucial for the physiological functions of mature conotoxins, therefore indels, especially insertions in the mature region, play an important role in the sequence and function diversity of conotoxins.

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.

Transcriptomic analysis of cone snail venom duct tissue has permitted the identification of diverse conopressin/conophysin precursor sequences from seven distinct Conus species. Multiple precursor isoforms are present in C.monile, C.lividus and C.loroisii. Aqueous extracts of the venom duct tissue from C.monile yield a band, at ~ 15-20 kDa on SDS-PAGE. In-gel trypsin digestion, followed by mass spectrometry establishes the presence of two distinct conopressin/conophysin isoforms that differ at position 8 in the predicted conopressin nonapeptide sequence. Mass spectrometric analysis of aqueous extracts revealed the presence of four conopressin related peptides, whose sequences could be deduced from MS/MS fragmentation patterns. The four sequences determined in this study are CFIRNCPKG*, CFIRNCPEG*, CFIRNCPK* and CFIRNCPE* (∗ indicates amide), which were further confirmed by comparison with chemically synthesized peptides. A conophysin with a mass of 9419.7 Da was also detected, corresponding to one of the isoforms revealed by the transcriptome data. Complete conservation of fourteen Cys residues and the key residues involved in peptide hormone binding is established by comparison of conophysin sequences, with the crystallographically characterized sequence of bovine neurophysin, in complex with vasopressin. A survey of available sequences for oxytocin/vasopressin peptides in both vertebrates and invertebrates establishes the conopressins as a distinct group in this family. C-terminal amidated, truncated conopressin analogs may arise by alternate post-translational processing.

Profundiconus is the most divergent cone snail genus and its unique phylogenetic position, sister to the rest of the family Conidae, makes it a key taxon for examining venom evolution and diversity. Venom gland and foot transcriptomes of Profundiconus cf. vaubani and Profundiconus neocaledonicus were de novo assembled, annotated, and analyzed for differential expression. One hundred and thirty-seven venom components were identified from P. cf. vaubani and 82 from P. neocaledonicus, with only four shared by both species. The majority of the transcript diversity was composed of putative peptides, including conotoxins, profunditoxins, turripeptides, insulin, and prohormone-4. However, there were also a significant percentage of other putative venom components such as chymotrypsin and L-rhamnose-binding lectin. The large majority of conotoxins appeared to be from new gene superfamilies, three of which are highly different from previously reported venom peptide toxins. Their low conotoxin diversity and the type of insulin found suggested that these species, for which no ecological information are available, have a worm or molluscan diet associated with a narrow dietary breadth. Our results indicate that Profundiconus venom is highly distinct from that of other cone snails, and therefore important for examining venom evolution in the Conidae family.

To expand our capacity to discover venom sequences from the genomes of venomous organisms, we applied targeted sequencing techniques to selectively recover venom gene superfamilies and nontoxin loci from the genomes of 32 cone snail species (family, Conidae), a diverse group of marine gastropods that capture their prey using a cocktail of neurotoxic peptides (conotoxins). We were able to successfully recover conotoxin gene superfamilies across all species with high confidence (> 100× coverage) and used these data to provide new insights into conotoxin evolution. First, we found that conotoxin gene superfamilies are composed of one to six exons and are typically short in length (mean = ∼85 bp). Second, we expanded our understanding of the following genetic features of conotoxin evolution: 1) positive selection, where exons coding the mature toxin region were often three times more divergent than their adjacent noncoding regions, 2) expression regulation, with comparisons to transcriptome data showing that cone snails only express a fraction of the genes available in their genome (24–63%), and 3) extensive gene turnover, where Conidae species varied from 120 to 859 conotoxin gene copies. Finally, using comparative phylogenetic methods, we found that while diet specificity did not predict patterns of conotoxin evolution, dietary breadth was positively correlated with total conotoxin gene diversity. Overall, the targeted sequencing technique demonstrated here has the potential to radically increase the pace at which venom gene families are sequenced and studied, reshaping our ability to understand the impact of genetic changes on ecologically relevant phenotypes and subsequent diversification.

The mini-M conotoxins are peptidic scaffolds found in the venom of cones snails. These scaffolds are tightly folded structures held together by three disulfide bonds with a CC-C-C-CC arrangement (conotoxin framework III) and belong to the M Superfamily of conotoxins. Here, we describe mini-M conotoxins from the venom of Conus regius, a Western Atlantic worm-hunting cone snail species using transcriptomic and peptidomic analyses. These C. regius conotoxins belong to three different subtypes: M1, M2, and M3. The subtypes show little sequence homology, and their loop sizes (intercysteine amino acid chains) vary significantly. The mini-Ms isolated from dissected venom contains preferentially hydroxylated proline residues, thus augmenting the structural reach of this conotoxin class. Using 2D-NMR methods, we have determined the 3D structure of reg3b, an M2 subtype conotoxin, which shows a constrained multi-turn scaffold. The structural diversity found within mini-M conotoxin scaffolds of C. regius is indicative of structural hypervariability of the conotoxin M superfamily that is not seen in other superfamilies. These stable minimalistic scaffolds may be investigated for the development of engineered peptides for therapeutic applications. DATABASES Sequences are available in GenBank under accession numbers MF588935-MF588952. Structural data are available in the RCSB protein database under the accession code 6BX9.

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.

Hyaluronidases are ubiquitous enzymes commonly found in venom and their main function is to degrade hyaluran, which is the major glycosaminoglycan of the extracellular matrix in animal tissues. Here we describe the purification and characterization of a 60 kDa hyaluronidase found in the injected venom from Conus purpurascens , Conohyal-P1. Using a combined strategy based on transcriptomic and proteomic analysis, we determined the Conohyal-P1 sequence. Conohyal-P1 has conserved consensus catalytic and positioning domain residues characteristic of hyaluronidases and a C-terminus EGF-like domain. Additionally, the enzyme is expressed as a mixture of glycosylated isoforms at five asparagine sites. The activity of the native Conohyal-P1 was assess MS-based methods and confirmed by classical turbidimetric methods. The MS-based assay is particularly sensitive and provides the first detailed analysis of a venom hyaluronidase activity monitored with this method. The discovery of new hyaluronidases and the development of techniques to evaluate their performance can advance several therapeutic procedures, as these enzymes are widely used for enhanced drug delivery applications. Biological significance Cone snail venom is a remarkable source of therapeutically important molecules, as is the case of conotoxins, which have undergone extensive clinical trials for several applications. In addition to the conotoxins, a large array of proteins have been reported in the venom of several species of cone snails, including enzymes that were found in dissected and injected Conus venom. Here we describe the isolation and characterization of the hyaluronidase Conohyal-P1 from the injected venom of C. purpurascens . We employed a combined transcriptomic and proteomic analysis to obtain the full sequence of this hyaluronidase. The activity of Conohyal-P1 was assessed by a mass spectrometry-based method, which provide the first detailed venom hyaluronidase activity analysis monitored by mass spectrometry allowing the visualization of the substrate degradation by the enzyme.

Venomous animals have developed a huge arsenal of reticulated peptides for defense and predation. Based on various scaffolds, they represent a colossal pharmacological diversity, making them top candidates for the development of innovative drugs. Instead of relying on the classical, low-throughput bioassay-guided approach to identify innovative bioactive peptides, this work exploits a recent paradigm to access to venom diversity. This strategy bypasses the classical approach by combining high-throughput transcriptomics, proteomics and bioinformatics cutting-edge technologies to generate reliable peptide sequences. The strategy employed to generate hundreds of reliable sequences from Conus venoms is deeply described. The study led to the discovery of (i) conotoxins that belong to known pharmacological families targeting various GPCRs or ion-gated channels, and (ii) new families of conotoxins, never described to date. It also focusses on the diversity of genes, sequences, folds, and PTM's provided by such species.

Conus ateralbus is a cone snail endemic to the west side of the island of Sal, in the Cabo Verde Archipelago off West Africa. We describe the isolation and characterization of the first bioactive peptide from the venom of this species. This 30AA venom peptide is named conotoxin AtVIA (&delta, conotoxin-like). An excitatory activity was manifested by the peptide on a majority of mouse lumbar dorsal root ganglion neurons. An analog of AtVIA with conservative changes on three amino acid residues at the C-terminal region was synthesized and this analog produced an identical effect on the mouse neurons. AtVIA has homology with &delta, conotoxins from other worm-hunters, which include conserved sequence elements that are shared with &delta, conotoxins from fish-hunting Conus. In contrast, there is no comparable sequence similarity with &delta, conotoxins from the venoms of molluscivorous Conus species. A rationale for the potential presence of &delta, conotoxins, that are potent in vertebrate systems in two different lineages of worm-hunting cone snails, is discussed.

Conus snails produce venoms containing numerous peptides such as the &alpha, conotoxins (&alpha, CTXs), which are well-known nicotinic acetylcholine receptor (nAChR) antagonists. Thirty-eight chromatographic fractions from Conus princeps venom extract were isolated by RP-HPLC. The biological activities of 37 fractions (0.07 &micro, g/&micro, L) were assayed by two-electrode voltage clamp on human &alpha, 7 nAChRs expressed in Xenopus laevis oocytes. Fractions F7 and F16 notably inhibited the response elicited by acetylcholine by 52.7 &plusmn, 15.2% and 59.6 &plusmn, 2.5%, respectively. Fraction F7 was purified, and an active peptide (F7-3) was isolated. Using a combination of Edman degradation, mass spectrometry, and RNASeq, we determined the sequence of peptide F7-3: AVKKTCIRSTOGSNWGRCCLTKMCHTLCCARSDCTCVYRSGKGHGCSCTS, with one hydroxyproline (O) and a free C-terminus. The average mass of this peptide, 10,735.54 Da, indicates that it is a homodimer of identical subunits, with 10 disulfide bonds in total. This peptide is clearly similar to &alpha, D-CTXs from species of the Indo-Pacific. Therefore, we called it &alpha, D-PiXXA. This toxin slowly and reversibly inhibited the ACh-induced response of the h&alpha, 7 nAChR subtype, with an IC50 of 6.2 &mu, M, and it does not affect the h&alpha, 3&beta, 2 subtype at 6.5 &mu, M.

Cone snails are marine gastropod mollusks with one of the most powerful venoms in nature. The toxins, named conotoxins, must act quickly on the cone snails´ prey due to the fact that snails are extremely slow, reducing their hunting capability. Therefore, the characteristics of conotoxins have become the object of investigation, and as a result medicines have been developed or are in the trialing process. Conotoxins interact with transmembrane proteins, showing specificity and potency. They target ion channels and ionotropic receptors with greater regularity, and when interaction occurs, there is immediate physiological decompensation. In this review we aimed to evaluate the structural features of conotoxins and the relationship with their target types.

The fish-hunting marine cone snail Conus geographus uses a specialized venom insulin to induce hypoglycemic shock in its prey. We recently showed that this venom insulin, Con-Ins G1, has unique characteristics relevant to the design of new insulin therapeutics. Here, we show that fish-hunting cone snails provide a rich source of minimized ligands of the vertebrate insulin receptor. Insulins from C. geographus, Conus tulipa and Conus kinoshitai exhibit diverse sequences, yet all bind to and activate the human insulin receptor. Molecular dynamics reveal unique modes of action that are distinct from any other insulins known in nature. When tested in zebrafish and mice, venom insulins significantly lower blood glucose in the streptozotocin-induced model of diabetes. Our findings suggest that cone snails have evolved diverse strategies to activate the vertebrate insulin receptor and provide unique insight into the design of novel drugs for the treatment of diabetes.

The primary objective of this study was to realize the large-scale discovery of conotoxin sequences from different organs (including the venom duct, venom bulb and salivary gland) of the vermivorous Oak cone snail, Conus quercinus. Using high-throughput transcriptome sequencing, we identified 133 putative conotoxins that belong to 34 known superfamilies, of which nine were previously reported while the remaining 124 were novel conotoxins, with 17 in new and unassigned conotoxin groups. A-, O1-, M-, and I2- superfamilies were the most abundant, and the cysteine frameworks XIII and VIII were observed for the first time in the A- and I2-superfamilies. The transcriptome data from the venom duct, venom bulb and salivary gland showed considerable inter-organizational variations. Each organ had many exclusive conotoxins, and only seven of all the inferred mature peptides were common in the three organs. As expected, most of the identified conotoxins were synthesized in the venom duct at relatively high levels, however, a number of conotoxins were also identified in the venom bulb and the salivary gland with very low transcription levels. Therefore, various organs have different conotoxins with high diversity, suggesting greater contributions from several organs to the high-throughput discovery of new conotoxins for future drug development.

Using high-throughput BioPlex assays, we determined that six fractions from the venom of Conus nux inhibit the adhesion of various recombinant PfEMP-1 protein domains (PF08_0106 CIDR1α3.1, PF11_0521 DBL2β3, and PFL0030c DBL3X and DBL5e) to their corresponding receptors (CD36, ICAM-1, and CSA, respectively). The protein domain-receptor interactions permit P. falciparum-infected erythrocytes (IE) to evade elimination in the spleen by adhering to the microvasculature in various organs including the placenta. The sequences for the main components of the fractions, determined by tandem mass spectrometry, yielded four T-superfamily conotoxins, one (CC-Loop-CC) with I-IV, II-III connectivity and three (CC-Loop-CXaaC) with a I-III, II-IV connectivity. The 3D structure for one of the latter, NuxVA = GCCPAPLTCHCVIY, revealed a novel scaffold defined by double turns forming a hairpin-like structure stabilized by the two disulfide bonds. Two other main fraction components were a miniM conotoxin, and a O2-superfamily conotoxin with cysteine framework VI/VII. This study is the first one of its kind suggesting the use of conotoxins for developing pharmacological tools for anti-adhesion adjunct therapy against malaria. Similarly, mitigation of emerging diseases like AIDS and COVID-19, can also benefit from conotoxins as inhibitors of protein-protein interactions as treatment. Biological significance Among the 850+ species of cone snail species there are hundreds of thousands of diverse venom exopeptides that have been selected throughout several million years of evolution to capture prey and deter predators. They do so by targeting several surface proteins present in target excitable cells. This immense biomolecular library of conopeptides can be explored for potential use as therapeutic leads against persistent and emerging diseases affecting non-excitable systems. We aim to expand the pharmacological reach of conotoxins/conopeptides by revealing their in vitro capacity to disrupt protein-protein and protein-polysaccharide interactions that directly contribute to pathology of Plasmodium falciparum malaria. This is significant for severe forms of malaria, which might be deadly even after treated with current parasite-killing drugs because of persistent cytoadhesion of P. falciparum infected erythrocytes even when parasites within red blood cells are dead. Anti-adhesion adjunct drugs would de-sequester or prevent additional sequestration of infected erythrocytes and may significantly improve survival of malaria patients. These results provide a lead for further investigations into conotoxins and other venom peptides as potential candidates for anti-adhesion or blockade-therapies. This study is the first of its kind and it suggests that conotoxins can be developed as pharmacological tools for anti-adhesion adjunct therapy against malaria. Similarly, mitigation of emerging diseases like AIDS and COVID-19, can also benefit from conotoxins as potential inhibitors of protein-protein interactions as treatment.

A specialized insulin was recently found in the venom of a fish-hunting cone snail, Conus geographus. Here we show that many worm-hunting and snail-hunting cones also express venom insulins, and that this novel gene family has diversified explosively. Cone snails express a highly conserved insulin in their nerve ring; presumably this conventional signaling insulin is finely tuned to the Conus insulin receptor, which also evolves very slowly. By contrast, the venom insulins diverge rapidly, apparently in response to biotic interactions with prey and also possibly the cones’ own predators and competitors. Thus, the inwardly directed signaling insulins appear to experience predominantly purifying sele\ction to target an internal receptor that seldom changes, while the outwardly directed venom insulins frequently experience directional selection to target heterospecific insulin receptors in a changing mix of prey, predators and competitors. Prey insulin receptors may often be constrained in ways that prevent their evolutionary escape from targeted venom insulins, if amino-acid substitutions that result in escape also degrade the receptor’s signaling functions.

Conotoxin-Ac1 and its variant conotoxin-Ac1-O6P, were isolated from the venom duct of Conus achatinus, a fish-hunting cone snail species collected in the Sea of Hainan, China. Conotoxin-Ac1 is linear peptide that contain 15 amino acids. In the present study, we synthesized and structurally and functionally characterized conotoxin-Ac1 as well as 19 variants. Electrophysiological results showed that conotoxin-Ac1 inhibited N-methyl-D-aspartate receptor subunit 2B (NR2B) with an IC50 of 8.22 &plusmn, 0.022 &mu, M. Further structure-activity studies of conotoxin-Ac demonstrated that polar amino acid residues were important for modulating its active, and the replacement of N1, O9, E10, and S12 by Ala resulted in a significant decrease in potency to NR2B. &deg, Furthermore, conotoxin-Ac1 and conotoxin-Ac1-O6P were tested in hot-plate and tail-flick assays to measure the potential analgesic activity to an acute thermal stimulus in a dose-dependent manner. Subsequently, the analgesic activity of conotoxin-Ac1 mutants was analyzed by the hot-plate method. The results show that N1, Y2, Y3, E10, N11, S12, and T15 play an important role in the analgesic activity of conotoxin-Ac1. N1 and S12 have significant effects on conotoxin-Ac1 in inhibiting NR2B and analgesic activity. In conclusion, we have discovered that conotoxin-Ac1 is an inhibitor of NMDAR and displays antinociceptive activity.

Structural analysis of peptides with nuclear magnetic resonance (NMR) spectroscopy generally relies on knowledge of the primary sequence to enable assignment of the resonances prior to determination of the three-dimensional structure. Resonance assignment without knowledge of the sequence is complicated by redundancy in amino acid type, making complete de novo sequencing using NMR spectroscopy unlikely to be feasible. Despite this redundancy, we show here that NMR spectroscopy can be used to identify short sequence tags that can be used to elucidate full-length peptide sequences via database searching. In the current study, we have used this approach to identify conotoxins from the venom of the cone snail Conus geographus and determined the three-dimensional structure of a member of the I3 superfamily. This approach is most likely to be useful for the characterization of disulfide-rich peptides, such as those that were chosen for this study, as they generally have well-defined structures, which enhances the quality of the NMR spectra. In contrast to other sequencing methods, the lack of sample manipulation, such as protease digestion, allows for subsequent bioassays to be carried out using the native sample used for sequence identification.

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

The venom ducts of marine cone snails secrete highly complex mixtures of cysteine-rich active peptides, which are generally known as conotoxins or conopeptides and provide a potential fertile resource for pharmacological neuroscience research and the discovery of new drugs. Previous studies have devoted substantial effort to the identification of novel conopeptides, and the 109 cone snail species have yielded 7000 known conopeptides to date. Here, we used de novo deep transcriptome sequencing analyses combined with traditional Sanger sequencing and liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) to identify 30 distinct conopeptide precursors. Twenty of these were previously reported and the other 10 were novel conopeptide precursors. The study provides the first identification of the Con-ikot-ikot, NSF-bt05, O3 and I1 gene superfamilies in C. litteratus. A new putative superfamily was identified. In addition, the following cysteine frameworks were first identified in this study: CC-C-C-C-C-C-C-C-C-C-C-C-CC-C-C-C-C-C and C-C-C-C-C-CC-C. Several isomerases involved in post-translational modification of conopeptides were identified as well. The discovery of new conopeptides in C. litteratus will enhance our understanding of the conopeptide diversity in this particular clade of cone snails. We also found the existence of intraspecific variations in vermivorous species. Finally, the analysis strategy offers a relatively reliable workflow for screening for peptide drug candidates. SIGNIFICANCE: These novel conopeptides provide a potential resource for the development of new channel-targeting drugs. The intraspecific variation in C. litteratus enhance our understanding of the conopeptide diversity in this particular clade of cone snails. The identified three cysteine residues, which might participate in the formation of disulfide bonds, provide a clue to get the connectivity of cysteine frameworks. Finally, the analysis strategy offers a relatively reliable workflow for screening for peptide drug candidates.

Background Conotoxins are neuro-pharmacologically active cysteine rich peptides isolated from the venom complex of marine cone snails. These are usually made of even number of cysteines. Method In this study we characterised six novel conotoxin sequences from the venom of Conus leopardus collected from the Andaman Sea, namely Le907 (C-C), Le868 (C-C), Le933 (-C-CC), Le949 (-C-CC), Le1988 (C-C-CC-C) and Le1642 (CC-C-C) using de novo mass spectrometrybased sequencing methods. Astonishingly 3 of these peptides possess novel arrangements of cysteine residues with odd number of cysteines (-C-CC; C-C-CC-C), namely Le933, Le949 and Le1988. Further, a post-translational variant of peptide Le933 was identified and experimentally determined to contain hydroxyproline. Results The unusual cysteine arrangements observed suggests novel class of conotoxins. These results expand our understanding of the diversity of odd cysteine arrangements in conotoxins.