Conotoxins: Chemistry and Biology.

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

Recent Advances in Conotoxin Classification by Using Machine Learning Methods.

Conotoxins are disulfide-rich small peptides, which are invaluable peptides that target ion channel and neuronal receptors. Conotoxins have been demonstrated as potent pharmaceuticals in the treatment of a series of diseases, such as Alzheimer's disease, Parkinson's disease, and epilepsy. In addition, conotoxins are also ideal molecular templates for the development of new drug lead compounds and play important roles in neurobiological research as well. Thus, the accurate identification of conotoxin types will provide key clues for the biological research and clinical medicine. Generally, conotoxin types are confirmed when their sequence, structure, and function are experimentally validated. However, it is time-consuming and costly to acquire the structure and function information by using biochemical experiments. Therefore, it is important to develop computational tools for efficiently and effectively recognizing conotoxin types based on sequence information. In this work, we reviewed the current progress in computational identification of conotoxins in the following aspects: (i) construction of benchmark dataset; (ii) strategies for extracting sequence features; (iii) feature selection techniques; (iv) machine learning methods for classifying conotoxins; (v) the results obtained by these methods and the published tools; and (vi) future perspectives on conotoxin classification. The paper provides the basis for in-depth study of conotoxins and drug therapy research.

iCTX-type: a sequence-based predictor for identifying the types of conotoxins in targeting ion channels.

Conotoxins are small disulfide-rich neurotoxic peptides, which can bind to ion channels with very high specificity and modulate their activities. Over the last few decades, conotoxins have been the drug candidates for treating chronic pain, epilepsy, spasticity, and cardiovascular diseases. According to their functions and targets, conotoxins are generally categorized into three types: potassium-channel type, sodium-channel type, and calcium-channel types. With the avalanche of peptide sequences generated in the postgenomic age, it is urgent and challenging to develop an automated method for rapidly and accurately identifying the types of conotoxins based on their sequence information alone. To address this challenge, a new predictor, called iCTX-Type, was developed by incorporating the dipeptide occurrence frequencies of a conotoxin sequence into a 400-D (dimensional) general pseudoamino acid composition, followed by the feature optimization procedure to reduce the sample representation from 400-D to 50-D vector. The overall success rate achieved by iCTX-Type via a rigorous cross-validation was over 91%, outperforming its counterpart (RBF network). Besides, iCTX-Type is so far the only predictor in this area with its web-server available, and hence is particularly useful for most experimental scientists to get their desired results without the need to follow the complicated mathematics involved.

Conotoxins and their regulatory considerations.

Venom derived peptides from marine cone snails, conotoxins, have demonstrated unique pharmacological targeting properties that have been pivotal in advancing medical research. The awareness of their true toxic origins and potent pharmacological nature is emphasized by their 'select agent' classification by the US Centers for Disease Control and Prevention. We briefly introduce the biochemical and pharmacological aspects of conotoxins, highlighting current advancements into their biological engineering, and provide details to the present regulations that govern their use in research.

Application of community phylogenetic approaches to understand gene expression: differential exploration of venom gene space in predatory marine gastropods.

BACKGROUND: Predatory marine gastropods of the genus Conus exhibit substantial variation in venom composition both within and among species. Apart from mechanisms associated with extensive turnover of gene families and rapid evolution of genes that encode venom components ('conotoxins'), the evolution of distinct conotoxin expression patterns is an additional source of variation that may drive interspecific differences in the utilization of species' 'venom gene space'. To determine the evolution of expression patterns of venom genes of Conus species, we evaluated the expression of A-superfamily conotoxin genes of a set of closely related Conus species by comparing recovered transcripts of A-superfamily genes that were previously identified from the genomes of these species. We modified community phylogenetics approaches to incorporate phylogenetic history and disparity of genes and their expression profiles to determine patterns of venom gene space utilization. RESULTS: Less than half of the A-superfamily gene repertoire of these species is expressed, and only a few orthologous genes are coexpressed among species. Species exhibit substantially distinct expression strategies, with some expressing sets of closely related loci ('under-dispersed' expression of available genes) while others express sets of more disparate genes ('over-dispersed' expression). In addition, expressed genes show higher dN/dS values than either unexpressed or ancestral genes; this implies that expression exposes genes to selection and facilitates rapid evolution of these genes. Few recent lineage-specific gene duplicates are expressed simultaneously, suggesting that expression divergence among redundant gene copies may be established shortly after gene duplication. CONCLUSIONS: Our study demonstrates that venom gene space is explored differentially by Conus species, a process that effectively permits the independent and rapid evolution of venoms in these species.

Characterizing the evolution and functions of the M-superfamily conotoxins.

Conotoxins from cone snails are valuable in physiology research and therapeutic applications. Evolutionary mechanisms of conotoxins have been investigated in several superfamilies, but there is no phylogenetic analysis on M-superfamily conotoxins. In this study, we characterized identical sequences, gene structure, novel cysteine frameworks, functions and evolutionary mechanisms of M-superfamily conotoxins. Identical M-superfamily conotoxins can be found in different Conus species from the analysis of novel 467 M-superfamily conotoxin sequences and other published M-superfamily conotoxins sequences. M-superfamily conotoxin genes consist of two introns and three exons from the results of genome walking. Eighteen cysteine frameworks were identified from the M-superfamily conotoxins, and 10 of the 18 may be generated from framework III. An analysis between diet types and phylogeny of the M-superfamily conotoxins indicate that M-superfamily conotoxins might not evolve in a concerted manner but were subject to birth-and-death evolution. Codon usage analysis shows that position-specific codon conservation is not restricted to cysteines, but also to other conserved residues. By analysing primary structures and physiological functions of M-superfamily conotoxins, we proposed a hypothesis that insertions and deletions, especially insertions in the third cysteine loop, are involved in the creation of new functions and structures of the M-superfamily conotoxins.

Molecular phylogeny, classification and evolution of conopeptides.

Conopeptides are toxins expressed in the venom duct of cone snails (Conoidea, Conus). These are mostly well-structured peptides and mini-proteins with high potency and selectivity for a broad range of cellular targets. In view of these properties, they are widely used as pharmacological tools and many are candidates for innovative drugs. The conopeptides are primarily classified into superfamilies according to their peptide signal sequence, a classification that is thought to reflect the evolution of the multigenic system. However, this hypothesis has never been thoroughly tested. Here we present a phylogenetic analysis of 1,364 conopeptide signal sequences extracted from GenBank. The results validate the current conopeptide superfamily classification, but also reveal several important new features. The so-called "cysteine-poor" conopeptides are revealed to be closely related to "cysteine-rich" conopeptides; with some of them sharing very similar signal sequences, suggesting that a distinction based on cysteine content and configuration is not phylogenetically relevant and does not reflect the evolutionary history of conopeptides. A given cysteine pattern or pharmacological activity can be found across different superfamilies. Furthermore, a few conopeptides from GenBank do not cluster in any of the known superfamilies, and could represent yet-undefined superfamilies. A clear phylogenetically based classification should help to disentangle the diversity of conopeptides, and could also serve as a rationale to understand the evolution of the toxins in the numerous other species of conoideans and venomous animals at large.

ConoDictor: a tool for prediction of conopeptide superfamilies.

ConoDictor is a tool that enables fast and accurate classification of conopeptides into superfamilies based on their amino acid sequence. ConoDictor combines predictions from two complementary approaches-profile hidden Markov models and generalized profiles. Results appear in a browser as tables that can be downloaded in various formats. This application is particularly valuable in view of the exponentially increasing number of conopeptides that are being identified. ConoDictor was written in Perl using the common gateway interface module with a php submission page. Sequence matching is performed with hmmsearch from HMMER 3 and ps_scan.pl from the pftools 2.3 package. ConoDictor is freely accessible at http://conco.ebc.ee.

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.

Conotoxin superfamily prediction using diffusion maps dimensionality reduction and subspace classifier.

Conotoxins are disulfide-rich small peptides that are invaluable channel-targeted peptides and target neuronal receptors, which have been demonstrated to be potent pharmaceuticals in the treatment of Alzheimer's disease, Parkinson's disease, and epilepsy. Accurate prediction of conotoxin superfamily would have many important applications towards the understanding of its biological and pharmacological functions. In this study, a novel method, named dHKNN, is developed to predict conotoxin superfamily. Firstly, we extract the protein's sequential features composed of physicochemical properties, evolutionary information, predicted secondary structures and amino acid composition. Secondly, we use the diffusion maps for dimensionality reduction, which interpret the eigenfunctions of Markov matrices as a system of coordinates on the original data set in order to obtain efficient representation of data geometric descriptions. Finally, an improved K-local hyperplane distance nearest neighbor subspace classifier method called dHKNN is proposed for predicting conotoxin superfamilies by considering the local density information in the diffusion space. The overall accuracy of 91.90% is obtained through the jackknife cross-validation test on a benchmark dataset, indicating the proposed dHKNN is promising.

Conotoxin protein classification using free scores of words and support vector machines.

BACKGROUND: Conotoxin has been proven to be effective in drug design and could be used to treat various disorders such as schizophrenia, neuromuscular disorders and chronic pain. With the rapidly growing interest in conotoxin, accurate conotoxin superfamily classification tools are desirable to systematize the increasing number of newly discovered sequences and structures. However, despite the significance and extensive experimental investigations on conotoxin, those tools have not been intensively explored. RESULTS: In this paper, we propose to consider suboptimal alignments of words with restricted length. We developed a scoring system based on local alignment partition functions, called free score. The scoring system plays the key role in the feature extraction step of support vector machine classification. In the classification of conotoxin proteins, our method, SVM-Freescore, features an improved sensitivity and specificity by approximately 5.864% and 3.76%, respectively, over previously reported methods. For the generalization purpose, SVM-Freescore was also applied to classify superfamilies from curated and high quality database such as ConoServer. The average computed sensitivity and specificity for the superfamily classification were found to be 0.9742 and 0.9917, respectively. CONCLUSIONS: The SVM-Freescore method is shown to be a useful sequence-based analysis tool for functional and structural characterization of conotoxin proteins. The datasets and the software are available at http://faculty.uaeu.ac.ae/nzaki/SVM-Freescore.htm.

NMR and protein structure in drug design: application to cyclotides and conotoxins.

Nuclear magnetic resonance spectroscopy (NMR) is a powerful technique for determining the structures, dynamics and interactions of molecules, and the derived information can be useful in drug design applications. This article gives a brief overview of the role of NMR in drug design and illustrates this role with examples studied in our laboratory in recent years on disulfide-rich peptides, including cyclotides and conotoxins. Cyclotides are head-to-tail cyclized proteins from plants that are exceptionally stable and hence make useful templates for the stabilization of bioactive peptide epitopes as well as potential leads for anti-HIV drugs. Natural cyclotides target cell membranes, so understanding cyclotide-membrane interactions is useful in applying cyclotides in drug design applications. NMR studies of these interactions are described in this article. Conotoxins are disulfide-rich peptides, from the venoms of marine cone snails, which are of pharmaceutical interest because they potently interact with a range of ion channels, transporters and other receptor sites implicated in disease states. Chemically re-engineering conotoxins to give them a cyclic backbone has been used to engender them with improved biopharmaceutical properties, such as are observed in cyclotides.

cDNA cloning of conotoxins with framework XII from several Conus species.

In our efforts for cloning novel I(2)-superfamily conotoxins using the signal peptide sequence, we identified a novel conotoxin Lt12.4 from Conus litteratus. This gene has a framework XII (-C-C-C-C-CC-C-C-), which is distinct from the cysteine pattern I(2)-superfamily conotoxin (-C-C-CC-CC-C-C-). Subsequently, we found the signal peptide sequence of Lt12.4 by 5'-RACE. Using this new sequence, we identified another five novel conotoxins with this cysteine pattern from four Conus species (Conus eburneus, Conus imperialis, Conus marmoreus, and C. litteratus). These novel conotoxins have the same cysteine pattern as the reported Gla-TxX and Gla-MII, and may contain Gla residues. Furthermore, they have the highly conserved signal peptide and hypervariable mature peptide sequences, and widely exist in Conus species. Therefore, it could be defined as a new superfamily of E-conotoxins.

Atypical alpha-conotoxin LtIA from Conus litteratus targets a novel microsite of the alpha3beta2 nicotinic receptor.

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

Identification, by molecular cloning, of a novel type of I2-superfamily conotoxin precursor and two novel I2-conotoxins from the worm-hunter snail Conus spurius from the Gulf of México.

cDNA was prepared from the venom duct of a single Conus spurius specimen collected near the coast of Campeche, México. From it, PCR products were generated aiming to clone I-conotoxin precursors. Thirty clones were sequenced and predicted to encode ten distinct precursors: seven of I(2)-conotoxins and three of I(2)-like-conotoxins. These precursors contain three different, mature toxins, sr11a, sr11b and sr11c, of which two are novel and one (sr11a) has been previously purified and characterized from the venom of this species. The precursors include a 26- (I(2)) or 23- residue signal peptide (I(2)-like), a 31-residue "pro" region (I(2)-like), and a 32-residue mature toxin region (I(2) and I(2)-like). In addition, all the precursors have a 13-residue "post" region which contains a gamma-carboxylation recognition sequence that directs the gamma-carboxylation of Glu-9 and Glu-10 of toxin sr11a and, possibly, Glu-13 of toxin sr11b and Glu-9 of toxin sr11c. This is the first time that a "post" region has been found in precursors of I-conotoxins that also contain a "pro" region. The "post" peptide is enzymatically processed to yield the amidated mature toxin sr11a, which implies that gamma-carboxylation occurs before amidation. Phylogenetic analysis at the whole precursor level indicates that the I(2)-like-conotoxins of C. spurius are more related to I(2)-conotoxins than to I(1)- and I(3)-conotoxins from other species, and that they might represent a new subgroup of the I(2)-superfamily. The three I-conotoxins from C. spurius have charge differences at seven to nine positions, suggesting that they might have different molecular target types or subtypes.

The M-superfamily of conotoxins: a review.

The focus of this review is the M-superfamily of Conus venom peptides. Disulfide rich peptides belonging to the M-superfamily have three loop regions and the cysteine arrangement: CC-C-C-CC, where the dashes represent loops one, two, and three, respectively. Characterization of M-superfamily peptides has demonstrated that diversity in cystine connectivity occurs between different branches of peptides even though the cysteine pattern remains consistent. This superfamily is subdivided into five branches, M-1 through M-5, based on the number of residues in the third loop region, between the fourth and fifth cysteine residues. M-superfamily peptides appear to be ubiquitous in Conus venom. They are largely unexplained in indigenous biological function, and they represent an active area of research within the scientific community.

Conotoxins: natural product drug leads.

Venomous marine cone snails harbour a variety of small disulfide-rich peptides called conotoxins, which target a broad range of ion channels, membrane receptors, and transporters. More than 700 species of Conus are thought to exist, each expressing a wide array of different peptides. Within this large repertoire of toxins, individual conotoxins are able to discriminate between different subtypes and isoforms of ion channels, making them valuable pharmacological probes or leads for drug design. This review gives a brief background to the discovery of conotoxins and describes their sequences, biological activities, and applications in drug design.

Identification of novel I-superfamily conopeptides from several clades of Conus species found in the South China Sea.

The I-superfamily of Conus peptides represents a new class of peptides with four disulfide bridges (-C-C-CC-CC-C-C-) that falls into three (I1, I2 and I3) categories according to the different signal peptide sequences. The I-superfamily has received increasing attention because it targets K+ ion channels, a function that is relatively rare in conotoxins. Herein we report 11 novel I-superfamily conotoxins from the venom ducts of five Cone snails (Conus eburneus, Conus imperialis, Conus vitulinus, Conus emaciatus and Conus litteratus) native to the South China Sea using a primer designed according to the N-terminus of the signal sequence of I2-superfamily conotoxins. The alignment of sequences revealed that signal regions exhibited moderate conservation with the exception of Eb11.3 from C. eburneus with homologies of 21.1%, 38.5% and 30.0% to the signal peptides of I1, I2 and I3 superfamily conotoxins, respectively. The mature peptides ranged from almost identical to highly divergent between species. Analyses of the evolutionary trees of these peptides with those of reported I-superfamily conotoxins showed that nine of them fall in I2 superfamily clades, but two of them were neither I1- and I2- nor I3-superfamily clades. Notably, some peptides exhibited significantly different amino acid residues in the intercysteine loops compared with group A, B and C of I-superfamily conopeptides, suggesting that they may have different bioactivities and functions.

Novel alpha D-conopeptides and their precursors identified by cDNA cloning define the D-conotoxin superfamily.

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

Variation and evolution of toxin gene expression patterns of six closely related venomous marine snails.

Venoms of predatory marine gastropods of the genus Conus show amazing levels of interspecific diversity and are comprised of a cocktail of peptide neurotoxins, termed conotoxins, that are encoded by large gene families. Conotoxin gene family evolution is characterized by gene duplications and high rates of nonsynonymous substitution among paralogues; yet, what controls the differentiation of venoms among species is not clear. We compared four-loop conotoxin transcripts of six closely related Conus species to examine conotoxin expression patterns among species. The species examined appear to express different numbers of four-loop conotoxin loci and similarity in expression patterns does not seem to correspond with phylogenetic affinity. Moreover, several loci appear to have been independently silenced while others appear to have been revived from previously silenced states. Some loci also appear to exhibit coordinated expression patterns. These results suggest that the evolution of conotoxin expression patterns is incredibly dynamic and the differentiation of venoms of Conus is controlled in part by the evolution of unique conotoxin expression patterns.