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

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

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

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

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

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

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

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

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

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

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

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

Discovery Methodology of Novel Conotoxins from Conus Species.

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

Bioactive Compounds Isolated from Neglected Predatory Marine Gastropods.

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

Structure and Biological Activity of a Turripeptide from Unedogemmula bisaya Venom.

The turripeptide ubi3a was isolated from the venom of the marine gastropod Unedogemmula bisaya, family Turridae, by bioassay-guided purification; both native and synthetic ubi3a elicited prolonged tremors when injected intracranially into mice. The sequence of the peptide, DCCOCOAGAVRCRFACC-NH2 (O = 4-hydroxyproline) follows the framework III pattern for cysteines (CC-C-C-CC) in the M-superfamily of conopeptides. The three-dimensional structure determined by NMR spectroscopy indicated a disulfide connectivity that is not found in conopeptides with the cysteine framework III: C1-C4, C2-C6, C3-C5. The peptide inhibited the activity of the α9α10 nicotinic acetylcholine receptor with relatively low affinity (IC50, 10.2 µM). Initial Constellation Pharmacology data revealed an excitatory activity of ubi3a on a specific subset of mouse dorsal root ganglion neurons.

Various conotoxin diversifications revealed by a venomic study of Conus flavidus.

Conotoxins are peptide neurotoxins produced by predatory cone snails. They are mostly cysteine-rich short peptides with remarkable structural diversity. The conserved signal peptide sequences of their mRNA-encoded precursors have enabled the grouping of known conotoxins into a limited number of superfamilies. However, the conotoxins within each superfamily often present variable sequences, cysteine frameworks, and post-translational modifications. To understand better how conotoxins are diversified, we performed a venomic study with C. flavidus, an uninvestigated vermivorous Conus species, by combining transcriptomic and proteomic analyses. In order to obtain the full-length conotoxin sequences, protease digestion was not performed with the venom extraction prior to spectra acquisition via tandem mass spectrometry (MS/MS). Because conotoxins are produced from mRNA-encoded precursors by means of proteolytic cleavage, nonspecific digestion of precursors was applied during the database search. Special attention was also paid in interpreting the MS/MS spectra. All together, these analyses identified 69 nonredundant cDNA sequences and 31 conotoxin components with confident MS/MS spectra. A new Q-superfamily was also identified. More importantly, this study revealed that conotoxin-encoding transcripts are diversified by hypermutation, fragment insertion/deletion, and mutation-induced premature termination, and that a single mRNA species can produce multiple toxin products through alternative post-translational modifications and alternative cleavages of the translated precursor. These multiple diversification strategies at different levels may explain, at least in part, the diversity of conotoxins, and provide the basis for further investigation.

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.

Structure-function elucidation of a new α-conotoxin, Lo1a, from Conus longurionis.

α-Conotoxins are peptide toxins found in the venom of marine cone snails and potent antagonists of various subtypes of nicotinic acetylcholine receptors (nAChRs). nAChRs are cholinergic receptors forming ligand-gated ion channels in the plasma membranes of certain neurons and the neuromuscular junction. Because nAChRs have an important role in regulating transmitter release, cell excitability, and neuronal integration, nAChR dysfunctions have been implicated in a variety of severe pathologies such as epilepsy, myasthenic syndromes, schizophrenia, Parkinson disease, and Alzheimer disease. To expand the knowledge concerning cone snail toxins, we examined the venom of Conus longurionis. We isolated an 18-amino acid peptide named α-conotoxin Lo1a, which is active on nAChRs. To the best of our knowledge, this is the first characterization of a conotoxin from this species. The peptide was characterized by electrophysiological screening against several types of cloned nAChRs expressed in Xenopus laevis oocytes. The three-dimensional solution structure of the α-conotoxin Lo1a was determined by NMR spectroscopy. Lo1a, a member of the α4/7 family, blocks the response to acetylcholine in oocytes expressing α7 nAChRs with an IC50 of 3.24 ± 0.7 µM. Furthermore, Lo1a shows a high selectivity for neuronal versus muscle subtype nAChRs. Because Lo1a has an unusual C terminus, we designed two mutants, Lo1a-ΔD and Lo1a-RRR, to investigate the influence of the C-terminal residue. Lo1a-ΔD has a C-terminal Asp deletion, whereas in Lo1a-RRR, a triple-Arg tail replaces the Asp. They blocked the neuronal nAChR α7 with a lower IC50 value, but remarkably, both adopted affinity for the muscle subtype α1ß1δε.

Novel M-Superfamily and T-Superfamily conotoxins and contryphans from the vermivorous snail Conus figulinus.

The venom of Conus figulinus, a vermivorous cone snail, found in the south east coast of India, has been studied in an effort to identify novel peptide toxins. The amino acid sequences of seven peptides have been established using de novo mass spectrometric based sequencing methods. Among these, three peptides belong to the M-Superfamily conotoxins, namely, Fi3a, Fi3b, and Fi3c, and one that belongs to the T-Superfamily, namely, Fi5a. The other three peptides are contryphans, namely, contryphans fib, fic, and fid. Of these Fi3b, Fi3c, Fi5a, and contryphan fib are novel and are reported for the first time from venom of C. figulinus. The details of the sequencing methods and the relationship of these peptides with other 'M'-Superfamily conotoxins from the fish hunting and mollusk hunting clades are discussed. These novel peptides could serve as a lead compounds for the development of neuropharmacologically important drugs.

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

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

Transcriptomic messiness in the venom duct of Conus miles contributes to conotoxin diversity.

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.

Influence of disulfide connectivity on structure and bioactivity of α-conotoxin TxIA.

Cone snails express a sophisticated arsenal of small bioactive peptides known as conopeptides or conotoxins (CTxs). Through evolutionary selection, these peptides have gained the ability to interact with a range of ion channels and receptors, such as nicotinic acetylcholine receptors (nAChRs). Here, we used reversed-phase high performance liquid chromatography (RP-HPLC) and electrospray ionization-mass spectrometry (ESI-MS) to explore the venom peptide diversity of Conus textile, a species of cone snail native to Hainan, China. One fraction of C. textile crude venom potently blocked α3ß2 nAChRs. Subsequent purification, synthesis, and tandem mass spectrometric analysis demonstrated that the most active compound in this fraction was identical to α-CTx TxIA, an antagonist of α3ß2 nAChRs. Then three disulfide isoforms of α-CTx TxIA were synthesized and their activities were investigated systematically for the first time. As we observed, disulfide isomerisation was particularly important for α-CTx TxIA potency. Although both globular and ribbon isomers showed similar retention times in RP-HPLC, globular TxIA potently inhibited α3ß2 nAChRs with an IC50 of 5.4 nM, while ribbon TxIA had an IC50 of 430 nM. In contrast, beads isomer had little activity towards α3ß2 nAChRs. Two-step oxidation synthesis produced the highest yield of α-CTx TxIA native globular isomer, while a one-step production process based on random oxidation folding was not suitable. In summary, this study demonstrated the relationship between conotoxin activity and disulfide connectivity on α-CTx TxIA.

Identification and classification of conopeptides using profile Hidden Markov Models.

Conopeptides are small toxins produced by predatory marine snails of the genus Conus. They are studied with increasing intensity due to their potential in neurosciences and pharmacology. The number of existing conopeptides is estimated to be 1 million, but only about 1000 have been described to date. Thanks to new high-throughput sequencing technologies the number of known conopeptides is likely to increase exponentially in the near future. There is therefore a need for a fast and accurate computational method for identification and classification of the novel conopeptides in large data sets. 62 profile Hidden Markov Models (pHMMs) were built for prediction and classification of all described conopeptide superfamilies and families, based on the different parts of the corresponding protein sequences. These models showed very high specificity in detection of new peptides. 56 out of 62 models do not give a single false positive in a test with the entire UniProtKB/Swiss-Prot protein sequence database. Our study demonstrates the usefulness of mature peptide models for automatic classification with accuracy of 96% for the mature peptide models and 100% for the pro- and signal peptide models. Our conopeptide profile HMMs can be used for finding and annotation of new conopeptides from large datasets generated by transcriptome or genome sequencing. To our knowledge this is the first time this kind of computational method has been applied to predict all known conopeptide superfamilies and some conopeptide families.

Expression, renaturation and biological activity of recombinant conotoxin GeXIVAWT.

Conotoxins are a diverse array of small peptides mostly with multiple disulfide bridges. These peptides become an increasing significant source of neuro-pharmacological probes and drugs as a result of the high selectivity for ion channels and receptors. Conotoxin GeXIVAWT (CTX-GeXIVAWT) is a 28-amino acid peptide containing five cysteines isolated from the venom of Conus generalis. Here, we present a simple and fast strategy of producing disulfide-rich conotoxins via recombinant expression. The codes of novel conotoxin gene GeXIVAWT were optimized and generated two pairs of primers by chemical synthesis for construction of expression vector. Recombinant expression vector pET22b(+)-GeXIVAWT fused with pelB leader and His-tag was successfully expressed as an insoluble body in Escherichia coli BL21(DE3) cells. Recombinant conotoxin GeXIVAWT (rCTX-GeXIVAWT) was obtained by dissolving the insoluble bodies and purifying with a Ni-NTA affinity column, which was further purified using reverse-phase high-performance liquid chromatography and identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. The rCTX-GeXIVAWT renatured in vitro could inhibited the growth of Sf9 cell with biological activity assay. This expression system may prove valuable for future structure-function studies of conotoxins.

Glycosylation of conotoxins.

Conotoxins are small peptides present in the venom of cone snails. The snail uses this venom to paralyze and capture prey. The constituent conopeptides display a high level of chemical diversity and are of particular interest for scientists as tools employed in neurological studies and for drug development, because they target with exquisite specificity membrane receptors, transporters, and various ion channels in the nervous system. However, these peptides are known to contain a high frequency and variability of post-translational modifications-including sometimes O-glycosylation-which are of importance for biological activity. The potential application of specific conotoxins as neuropharmalogical agents and chemical probes requires a full characterization of the relevant peptides, including the structure of the carbohydrate part. In this review, the currently existing knowledge of O-glycosylation of conotoxins is described.

Conopeptides from Cape Verde Conus crotchii.

Marine Cone snails of the genus Conus contain complex peptide toxins in their venom. Living in tropical habitats, they usually use the powerful venom for self-defense and prey capture. Here, we study Conus crotchii venom duct using a peptide mass-matching approach. The C. crotchii was collected on the Cape Verde archipelago in the Boa Vista Island. The venom was analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). About 488 molecular masses between 700 Da and 3000 Da were searched bymatching with known peptide sequences from UniProtKB protein sequence database. Through this method we were able to identify 12 conopeptides. For validation we considered the error between the experimental molecular mass (monoisotopic) and the calculated mass of less than 0.5 Da. All conopeptides detected belong to the A-, O1-, O2-, O3-, T- and D-superfamilies, which can block Ca²âº channels, inhibit K⁺ channels and act on nicotinic acetylcholine receptors (nAChRs). Only a few of the detected peptides have a 100% UniProtKB database similarity, suggesting that several of them could be newly discovered marine drugs.