Educational guide
Conotoxin - an overview
Chapters and Articles You might find these chapters and articles relevant to this topic. Abstract Conotoxins are disulfide-rich peptides from the venoms of marine cone snails that are used in prey capture. Due to their exquisite potency and selectivity for dif
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Chapters and Articles
You might find these chapters and articles relevant to this topic.
Abstract
Conotoxins are disulfide-rich peptides from the venoms of marine cone snails that are used in prey capture. Due to their exquisite potency and selectivity for different ion channels, receptors and transporters they have attracted much interest as leads in drug design. This article gives a brief background on conotoxins, describes their structures and highlights methods for synthetic cyclization to improve their biopharmaceutical properties. The proximity of the N and C termini of many conotoxins makes them particularly suitable for cyclization with linkers of on average five to seven amino acids. By linking the ends of conotoxins it is possible to significantly decrease their susceptibility to proteolysis without loss of their intrinsic biological activity. Here, the principles of conotoxin cyclization are illustrated with applications to the α- and χ- conotoxin classes, which have been implicated as leads for the treatment of pain and a range of other disorders including neuroprotection, schizophrenia, depression and cancer.
URL: https://www.sciencedirect.com/science/article/pii/S0041010110004204
Highlights
•Conotoxins are novel analgesics that alleviate pain without obvious side effects.
•There is substantial evidence supporting α-conotoxins analgesic effect mediated by GABABRs inhibition of CaV channels.
•Conotoxins interacting with GPCRs are potential lead analgesic compounds.
URL: https://www.sciencedirect.com/science/article/pii/S0028390817302320
5 Conotoxin families
Conotoxins are a diverse set of bioactive peptides isolated from Conus. Numbering conservatively somewhere between 50,000 and 100,000 individual peptide compounds, conotoxins have been assigned to one of six superfamilies (A-, M-, O-, P-, S- and T-) based on their cysteine framework, pre-pro-sequence homology, folding motif, and physiological effects ([22,23] see Table 2). Known to have antinoceptive, antiepileptic, as well as cardio- and neuro-protective activity, conotoxins have also become useful tools for research into to cancer, neuromuscular, and psychiatric disorders [24,25].
Table 2. Conotoxin and conopeptide superfamilies.
| Super family | Disulfide framework | Family | Target (receptor/channel) | Example | Conus origin | References |
|---|---|---|---|---|---|---|
| A | CC-C-C | α | nACh | α-Iml | C. imperialis | [178] |
| ρ | nACh | ρ-TIA | C. tulipa | [179] | ||
| CC-C-C-C-C | αA | nACh | αA-PIVA | C. purpurascens | [14] | |
| κA | K+ | κA-SIVA | C. striatus | [180] | ||
| M | CC-C-C-CC | μ | Na+ | μ-GIIIA | C. geographus | [181] |
| ψ | nACh | ψ-PIIIE | C. purpurascens | [84] | ||
| κM | K+ | κM-RIIIK | C. radiatus | [182] | ||
| O | C-C-CC-C-C | μO | Na+ | μO-MrVIA | C. marmoreus | [31] |
| δ | Na+ | δ-TxVIA | C. textile | [32] | ||
| ω | Ca2+ | ω-MVIIA | C. magus | [63] | ||
| κ | K+ | κ-PVIIA | C. purpurascens | [85] | ||
| γ | Voltage-gated pacemaker | γ-PnVIIA | C. pennaceus | [183] | ||
| P | C-C-C-C-C-C | Spastics | Undetermined | Tx9a | C. textile | [34] |
| S | C-C-C-C-C-C-C-C | σ | 5-HT3 | σ-GVIIIA | C. geographus | [36] |
| T | CC-CC | τ | Ca2+ | τ-TxIX | C. textile | [184] |
| χ | Norepinephrine transporter | χ-MrIA | C. marmoreus | [185] | ||
| I | C-C-CC-CC-C-C | Excitatory | Undetermined | Sr11a | C. spurius | [186] |
| No assignment | C-C | Conopressins | Vasopressin | Lys-conopressin-G | C. geographus | [37] |
| Contryphans | L-type Ca2+ channel | Am975 | C. amadis | [187] | ||
| Linear | No cysteines | Conantokins | Glutamate (NMDA) | Pr1 | C. parius | [106] |
| Conorfamide | RF amide | CNF-Sr2 | C. spurius | [188] | ||
| O-linked | Contulakins | Neurotensin | Contulakin-G | C. geographus | [189] |
The A-Superfamily consists of four toxin families, α-, αA-, κA- and ρ-conotoxins ([22]; Table 2). Alpha (α)-, and αA-conotoxins are nAChR antagonists, κA-conotoxins are thought to target K+ channels, while Rho (ρ)-conotoxins are preferential a1B-adrenoceptor antagonists [26,27]. Of these families, α-conotoxins block nicotinic acetylcholine receptor (nAChR) function in muscle contraction and neurotransmission [28]. Typically, α-conotoxins contain anywhere from 12 to 25 amino acids (αα) and consist of 4 cysteines, forming 2 disulfide bonds (α-conotoxin SII has 6 cysteines and 3 disulfide bonds; [29]). Inhibition of receptor subtypes is shown to be directly involved in learning, antinoception, nicotine addiction, and the neurological disorders Parkinson's and Alzheimer's [28].
The M-Superfamily is smaller in family number at two, μ- and ψ-conotoxin groups ([22]; Table 2). Psi (ψ)-conotoxins target nAChRs, non-competitively, while the mu (μ)-family block voltage-dependent sodium (Nav) channels in excitable cells such as muscle, heart, skeletal and nerve tissues [30]. Average sized (∼22 residues) μ-conotoxins have 6 cysteines, forming 3 disulfide bonds, and elicit their effects through direct pore occlusion [30].
The O-Superfamily is the largest of the group and encompasses five distinct families, μO-, δ-, ω-, κ-, and γ-conotoxins ([26]; Table 2). Both μO-, and δ-conotoxin families target Nav channel function (different site to the μ-conotoxins above), while the others target Ca2+ channels, K+ channels, and Pacemaker channels, respectively [26]. Delta (δ)-conotoxins usually consists of about 30 ααs, and typically demonstrates a strong hydrophobic chromatographic nature [31]. These specifically function to slow inactivation of Nav channels [32]. Consisting of four homologous membrane domains, Nav channels modulate rapid electrical signaling in both neuronal and muscle cell types [33].
The P-Superfamily contains a family of conotoxins that conform to an inhibitor cysteine knot motif, known as the ‘spastics’. These conotoxins cause spastic paralysis, but their molecular target remains to be identified ([22]; Table 2). A representative example of a spastic-conotoxin is Tx9a, a gamma-carboxy glutamic acid containing peptide found in the venom of mollusicvorous Conus textile ([34]; Table 2). Post-translational modifications (PTMs) within conotoxins are common and varied; this feature increases toxin chemical diversity and potential targeting ability [35].
The S-Superfamily also consists of only one family, derived from C. geographus, and remains relatively unexplored. The Sigma (σ)-conotoxin family is represented by a large 41 αα peptide containing eight cysteines forming four disulfide bonds in its bioactive form ([36]; Table 2). These chemical features provide strong differentiation from the aforementioned conotoxin families. σ-Conotoxins are 5HT3 receptor antagonists, which elicit excitatory responses in neurons when serotonin is bound (5-hydroxytryptamine).
Finally, the T-Superfamily includes both Tau (τ)- and Chi (χ)- conotoxin families. The peptides in these families have 4 cysteines, which in turn produce two disulfide bonds ([26]; Table 2). τ-Conotoxins functionally block pre-synaptic Ca2+ channels, while χ-conotoxins inhibit NETs [26].
Smaller classes of conopeptides exist that are not classified within the conotoxin superfamily structure. These either possess a single disulfide bond or are linear in nature containing no cysteines. Conopressins are vasopressin receptor antagonists [37], contryphans target unknown receptor types [38], conantokins antagonize N-methyl-d-asparate (NMDA) receptors [39], and lastly, contulakins, which display neurotensin (NT) receptor antagonism [40,41]. It is highly probable that these families are far from completing the pharmacological repertoire of ion channel and receptor targeting capabilities within Conus. Access to novel screening methods and defined biological targets may result in increased pharmacological classification of conotoxins, yet this is one of the many bottlenecks faced in conotoxin discovery, compounded by the limited access to the natural resource.
URL: https://www.sciencedirect.com/science/article/pii/S0009279709004244
Abstract
Conotoxins (CnTX) are bioactive peptides produced by marine molluscs belonging to Conus genus. The biochemical structure of these venomous peptides is characterized by a low number of amino acids linked with disulfide bonds formed by a high degree of post-translational modifications and glycosylation steps which increase the diversity and rate of evolution of these molecules. CnTX different isoforms are known to target ion channels and, in particular, voltage-gated sodium (Na+) channels (Nav channels). These are transmembrane proteins fundamental in excitable cells for generating the depolarization of plasma membrane potential known as action potential which propagates electrical signals in muscles and nerves for physiological functions. Disorders in Nav channel activity have been shown to induce neurological pathologies and pain states. Here, we describe the current knowledge of CnTX isoform modulation of the Nav channel activity, the mechanism of action and the potential therapeutic use of these toxins in counteracting neurological dysfunctions.
URL: https://www.sciencedirect.com/science/article/pii/S0041010120303688
7 Perspectives
Conotoxins are an invaluable source of novel pharmacoactive molecules with the potential to become analgesic drugs. ω-Conotoxin MVIIA (Ziconotide or Prialt), a non-opioid analgesic drug, directly inhibits the N-type CaV2.2 calcium channel, and was the first FDA approved analgesic currently administered intrathecally for the management of intractable chronic pain. MVIIA has a narrow therapeutic index given the required mode of administration, however, new dosing strategies can increase its efficacy (McDowell and Pope, 2016). The detrimental secondary effects of opioid analgesic drugs are well recognised and thus there is an urgent need for potent and selective, systemically acting analgesics.
Targeting specific, peripherally expressed membrane receptors and ion channels that are involved in nociception and pain transmission is a strategy worth pursuing. The role of GPCRs such as GABAB (see Malcangio, 2017), neurotensin, κ-opioid, as well as VGCCs involved in pain pathways is well established and therefore, targeting specific GPCRs mediating VGCC inhibition peripherally may reduce the side effects associated with direct blockade of VGCCs in general.
In several animal pain models, α-conotoxins produce analgesia upon systemic administration without overt deleterious side effects. Therefore, there is considerable interest in studying these peptides as novel analgesics. GABABR mediated inhibition of VGCCs is a primary analgesic mechanism of action of these conopeptides, however, the α-conotoxins also inhibit nAChRs and appear to modulate some pain pathways. Further studies are required to determine the molecular determinants of activity and selectivity of the α-conotoxins at their targets.
In general, Conus peptides targeting various GPCRs are promising alternatives for the treatment of chronic visceral pain in irritable bowel syndrome (IBS) patients that needs further investigation. Further studies involving derivatization and modification of those conopeptides affecting GPCRs will likely increase their potency, selectivity and bioavailability to expand the analgesic pharmacopeia available.
URL: https://www.sciencedirect.com/science/article/pii/S0028390817302320
13.09.4.1.2 μ-Conotoxins
μ-Conotoxins are pore-blocking conotoxins isolated from the venoms of the predatory marine cone snails (Heinemann and Leipold 2007). They include the μ-III conotoxins from Conus geographus (GIIIA, GIIIB, GIIIC), Conus purpurascens (PIIIA), and Conus stercusmuscarum (SmIIIA) (French and Terlau 2004). These toxins act by physically occluding the ion-conducting pore of the voltage-gated sodium channels to inhibit sodium channel conductance. They compete with TTX and STX for binding to site-1 in the extracellular entry to the pore of certain sodium channels (Ekberg et al. 2008). However, owing to their larger physical size, they bind more superficially in the pore (Li and Tomaselli 2004). A degree of subtype selectivity is reported in their blocking of sodium channels. For example, GIIIA, GIIIB, and GIIIC have little or no effect on neuronal sodium channels, but they are potent blockers of certain skeletal muscle sodium channel. PIIIA blocks select skeletal muscle sodium channels and to a less extent, it preferentially blocks neuronal TTX-S sodium channels in hippocampal CA1 neurons. Recently, a novel μ-conotoxin TIIIA, which exhibits unusual sodium channel subtype selectivity, has been isolated from Conus tulipa. It inhibits select skeletal muscle sodium channels with high potency as do other μ-conotoxins but as it preferentially binds to select neuronal sodium channels, it can discriminate between different neuronal sodium channels (Ekberg et al. 2008).
URL: https://www.sciencedirect.com/science/article/pii/B9780080468846013105
1.5.2.5 μO-Conotoxins
The μO-conotoxins are a small family of hydrophobic peptides stabilized by an inhibitory cysteine knot motif that also cause net NaV channel inhibition. However, they belong to the O- rather than M-superfamily of conotoxins and are structurally and functionally quite dissimilar from the μ-conotoxins, as exemplified by the pharmacology of MrVIA, MrVIB, and more recently, MfVIA (Deuis, Dekan, et al., 2016; Vetter et al., 2012; Wilson, Zhang, et al., 2011). Specifically, the μO-conotoxins act as gating modifiers at a poorly defined binding site that overlaps at least partially with the binding sites of the δ-conotoxins and β-scorpion toxins, as depolarization-induced relief of inhibition was mapped to the β-scorpion toxin binding site on the domain II voltage-sensor, while in binding experiments, MrVIA was able to displace δ-conotoxin TxVIA (Ekberg et al., 2006; Leipold et al., 2007). Apart from their extraordinarily high hydrophobicity, making them difficult to synthesize or express recombinantly, they are remarkable for a number of reasons. The μO-conotoxins most potently affect NaV1.8, a TTX-R isoform that is affected by very few venom peptides (Table 10) (Deuis, Dekan, et al., 2016; Vetter et al., 2012). Intriguingly, despite small but significant hyperpolarizing shifts in the voltage dependence of activation, typically associated with NaV activators, they decrease peak Na+ current and act as analgesics in vivo (Fig. 2E) (Deuis, Dekan, et al., 2016; Ekberg et al., 2006; Vetter et al., 2012). In addition, their pharmacology is heavily influenced by the presence of auxiliary β subunits, which affects the on-rate of block in particular (Wilson, Zhang, et al., 2011). Like several μ-theraphotoxins, the μO-conotoxins also interact with lipid membranes, with mutation of two glutamic acid residues (E5/E8) in MfVIA to positively charged arginines improving lipid interactions and potency at NaV1.8 (Deuis, Dekan, et al., 2016).
Table 10. Potency of Selected μO-Conotoxins Toxins at NaV1.1–1.8
| Toxin | Mechanism of Action | NaV1.1 | NaV1.2 | NaV1.3 | NaV1.4 | NaV1.5 | NaV1.6 | NaV1.7 | NaV1.8 | References |
|---|---|---|---|---|---|---|---|---|---|---|
The NaV subtype that each toxin is most potent at and any subtype with less than 10-fold selectivity is highlighted in gray.
URL: https://www.sciencedirect.com/science/article/pii/S1054358917300042
Conotoxin subtypes
Each of the estimated 500 different species of cone Conus synthesizes and injects its own ‘repertoire’ of about 100 peptide toxins in their venom, giving rise to about 50,000 biologically active compounds. The peptide toxins are initially translated as prepropeptide precursors. Proteolytic cleavage and extensive posttranslational modification yields the diversity of final products (Olivera 1997). In addition to peptides, the venom of a small number of species also contains serotonin (5-HT), arachidonic acid, polypeptides, or enzymes (e.g., Lirazan et al. 2002; McIntosh et al. 1995).
For purposes of correlation to their biological activity, it is convenient to categorize conotoxins according to their physiologic actions on biological tissues. The majority of known components of conotoxins target specific voltage-gated and ligand-gated ion channels. Other known toxin components target GPCRs (Craig et al. 1998; Cruz et al. 1987; Sharpe et al. 2001) and possibly Ca2+ channels (Rigby et al. 1999; Walker et al. 1999), or norepinephrine transmitter (Sharpe et al. 2001). Widely conserved peptide families target voltage-gated Na+-channels (both TTX-sensitive and TTX-insensitive types), including: μ -conotoxins (block channels), μO-conotoxins (inhibit conductance), and δ-conotoxins (inhibit inactivation steps). The widely studied ω-conotoxin targets voltage-gated Ca2+-channels, and structurally divergent peptides target voltage-gated K+-channels, including κ-conotoxin, κM-conotoxin, and κA-conotoxin. Each of these has multiple subtypes, designated by the snail of origin. For example (Terlau and Olivera 2004): μ–GIIIA, GIIIB, PIIIA, and SmIIIA; μO–MrVIA (where O = trans-4-hydroxyproline) and MrVIB; δ–PVIA, SVIE, TxVIA, and GmVIA; κ–PVIIA, κM–RIIIK, κA–SIVA; and ω–GVIA, MVIIA, MVIIC, and CVID. Ligand-gated targets include the nicotinic acetylcholine (nACh) receptor (e.g., by α -, αA-, and ψ -conotoxins), 5-HT3 receptor (σ-conotoxins), and the NMDA receptor (conantokins). Other putative conopeptide targets include the vasopressin receptor (conopressin-G), neurotensin receptor (contulakin-G), α1-adrenoceptor (ρ-conotoxin), neuronal norepinephrine transporter (χ-conotoxin), and other sites (Table 2).
Table 2. Classification and postulated targets of some cone snail (Conus) peptide toxins (T&O; Muttenthaler et al. 2010). γ = γ-Carboxyglutamate; T§ = O-glycosylated Thr.
| Family | Subtype | Sequence | Target |
|---|---|---|---|
| α-Conotoxin | α-MI | GRCCHPACGKNYSC-NH2 | Nicotinic |
| α-EI | RDOCCYHPTCNMSNPQIC-NH2 | cholinergic | |
| α-AuIB | GCCSYPPCFATNPDC-NH2 | receptor | |
| α-BuIA | GCCSTPPCAVLYC-NH2 | (nAChR) | |
| α-MII | GCCSNPVCHLEHSNLC-NH2 | ||
| α-PIA | RDPCCSNPVCTVHNPQIC-NH2 | ||
| α-ImI | GCCSDPRCAWRC-NH2 | ||
| α-ImII | ACCSDRRCRWRC-NH2 | ||
| α-PnIA | GCCSLPPCAANNPDYC-NH2 | ||
| α-PnIB | GCCSLPPCALSNPDYC-NH2 | ||
| Vc1.1 | GCCSDPRCNYDHPEIC-NH2 | ||
| αA-Conotoxin | αA-PIVA | GCCGSYONAACHOCSCKDROSYCGQ-NH2 | nAChR |
| αA-EIVA | GCCPYONAACHOCGCKVGROOYCDROSGG-NH2 | ||
| δ-Conotoxin | δ-PVIA | EACYAOGTFCGIKOGLCCSEFCLPGVCFG | Na+ channel |
| δ-SVIE | DGCSSGGTFCGIHOGLCCSEFCFLWCITFID | ||
| δ-TxVIA | WCKQSGEMCNLLDQNCCDGYCIVLVCT | ||
| δ-GmVIA | VKPCRKEGQLCDPIFQNCCRGWNCVLFCV | ||
| κ-Conotoxin | κ-PVIIA | CRIONQKCFQHLDDCCSRKCNRFNKCV | K+ channel |
| κM-RIIIK | LOSCCSLNLRLCOVOACKRNOCCT-NH2 | ||
| κA-SIVA | ZKSLVPSVITTCCGYDOGTMCOOCRCTNSC-NH2 | ||
| μ-Conotoxin | μ-GIIIA | RDCCTOOKKCKDRQCKOQRCCA-NH2 | Na+ channel |
| μ-GIIIB | RDCCTOORKCKDRRCKOMKCCA-NH2 | ||
| μ-PIIIA | ZRLCCGFOKSCRSRQCKOHRCC-NH2 | ||
| μ-SmIIIA | ZRCCNGRRGCSSRWCRDHSRCC-NH2 | ||
| μO-Conotoxin | μO-MrVIA | ACRKKWEYCIVPIIGFIYCCPGLICGPFVCV | Na+ channel |
| μO-MrVIB | ACSKKWEYCIVPILGFVYCCPGLICGPFVCV | ||
| ψ-Conotoxin | ψ-PIIIE | HOOCCLYGKCRRYOGCSSASCCQR-NH2 | nAChR |
| σ-Conotoxin | σ-GVIIIA | GCTRTCGGOKCTGTCTCTNSSKCGCRYNVHPSGBGCGCACS-NH2 | 5-HT3 receptor |
| ρ-Conotoxin | ρ-TIA | FNWRCCLIPACRRNHKKFC-NH2 | α1-adrenoceptor |
| ω-Conotoxin | ω-GVIA | CKSOGSSCSOTSYNCCRSCNOYTKRCY-NH2 | Ca2+ channel |
| ω-MVIIA | CKGKGAKCSRLMYDCCTGSCRSGKC-NH2 | ||
| ω-MVIIC | CKGKGAPCRKTMYDCCSGSCGRRGKC-NH2 | ||
| ω-CVID | CKSKGAKCSKLMYDCCTGSCSGTVGTC-NH2 | ||
| Conantokin | G | GEγγLQγNQγLIRγKSN-NH2 | NMDA receptor |
| T | GEγγVQKMLγNLRγAEVKKNA-NH2 | ||
| R | GEγγVAKMAAγLARγNIAKGCKVNCYP | ||
| L | GEγγVAKMAAγLARγDAVN-NH2 | ||
| Conopressin | G | CFIRNCPLG-NH2 | Vasopressin receptor |
| Contulakin | G | ZSEEGGSNAT§KKPYIL | Neurotensin receptor |
URL: https://www.sciencedirect.com/science/article/pii/S0024320510003188
2.15.6.7 Conopeptide Druggability and other Uses
As with most bioactive peptides, the delivery of these compounds to the site of action is a major concern when considering the druggability of a conopeptide lead. Improvement in the bioavailability of conotoxins can be achieved with modified peptides with improved potency and lipid solubility. The synthesis of lipo- and liposaccharide conjugates of α-conotoxin, such as MII,143 may improve the oral availability and stability of the peptide and facilitate the crossing of the blood-brain barrier, thus improving the pharmaceutical suitability of these compounds.
Besides their therapeutic applications, conotoxins have exhibited great potential for the development of neurological probes.73 Conotoxins are currently being used in hundreds of research laboratories for a wide variety of physiological investigations. Some conotoxins have become well-established neurobiological tools. They can also be used as antagonists of specific subtypes in functional studies; for example, ω-conotoxin MVIIA is used as a specific N-type calcium channel blocker. Conotoxins are also utilized in research to provide information on the role and distribution of different receptor subtypes. Undoubtedly, these research applications will continue to expand.
URL: https://www.sciencedirect.com/science/article/pii/B9780080453828006389
7 ω-Conotoxins
ω-Conotoxins have been established as a valuable class of pharmacological research tools with over 1500 scientific references pertaining to their discovery, characterization, synthesis, three-dimensional structural analysis, pharmacological properties as well as medical applications ([59]; Table 1). Searching the US Patent database, ω-conotoxins are the subject of several patents and have been either mentioned or directly cited as having potential applications in over two hundred patent claims. More recently, the ω-conotoxins have garnered public attention, patent protection and FDA approval as potential therapeutic candidates in the treatment of chronic pain [60].
Nineteen individual peptides presently comprise the piscivorous ω-conotoxin family (see Table 4); these represent some of the first conotoxins fully characterized from Conus. Originally designated as the ‘shaker-peptides’ due to the induced tremors observed after intracranial injections in mice [16,61], the ω-conotoxins now represent some of the most widely used peptide toxins in neuroscience. The expanse of the ω-conotoxin family was realized with the sequencing and pharmacological classification of ω-conotoxin GVIA from C. geographus[62], then later with ω-conotoxins MVIIA and MVIIB being characterized from C. magus in 1987 ([63]; see Table 4). Sequence analysis provided the first indication of a potentially larger group of VGCC antagonists within cone shell venoms. Over the next 17 years ω-conotoxin isolation from other piscivorous snails continued to strengthen and confirm their unique neuronal-specific relationship. Pharmacological investigations further demonstrated selective subtype or isoform targeting within VGCCs [64,65], which fuelled both academic and commercial research interests.
Table 4. ω-Conotoxin sequences aligned by disulfide framework.
∞Cone shell pictures from www.conchology.be; *C-terminus amidation; O:4-trans-hydroxyproline.
Presently the piscivorous ω-conotoxin family consists of peptides ranging from 24 to 27 ααs in length that retain a highly conserved cysteine framework pattern (C-C-CC-C-C; Table 4). This stabilized framework allows for a four-loop structure, with most of the loop length variability confined to loops 3 and 4.
Like the majority of Conus peptides, the ω-conotoxins contain a high density of disulfide bonds that contribute to tertiary structure stabilization. All ω-conotoxins possess a C-terminal amide that may limit potential degradative carboxylase activity, which would otherwise render the peptide biologically inactive. The relative abundance of basic residues within the ω-conotoxins gives them an overall net positive charge. This characteristic is seen in other peptide toxin families, which assists in complementary binding to the target channel. An interesting feature of the ω-conotoxins is the relative low diversity of post-translationally modified (PTM) ααs in comparison to other conotoxin families [35,66]. When present, PTM ααs are restricted to 4-trans-hydroxyproline, with hydroxylation being mandatory for all proline moieties present, the one exception being ω-conotoxin MVIIC (see Table 4); however, being a sequence derived from genetic material,2 the potential for the modification of native ω-conotoxin MVIIC is apparent [67] and proven [43].
The differential pharmacological selectivity of VGCCs is reflected in the complex and diverse array of αα sequences amongst the members of the ω-conotoxin family. Phenogram representation of peptide lineage based on αα homology is illustrated in Fig. 2, indicating the presence of five basic sequence enclaves, or peptide sequence homologous classes A–E. Factors that define ω-conotoxin VGCC isoform selectivity have been a major focal point of researchers [68]. Most of these efforts have been unable to define the precise individual molecular determinants for VGCC isoform pharmaco-selectivity, as demonstrated by overlapping pharmacological targets between distant enclaves. Illustrative examples are the peptides isolated from C. magus. ω-Conotoxin MVIIA (enclave C, Fig. 2) shares an approximately 72% homology to ω-Conotoxin MVIIC (enclave A, Fig. 2), each has high preferential binding affinity to the N- and P/Q-type VGCCs, respectively. Comparison of two N-type VGCC-selective peptides, ω-conotoxin MVIIA (enclave C) and ω-conotoxin GVIA (enclave D, Fig. 2), illustrates a strong structural divergence in the number of ααs in loops 3 and 4, and the occurrence of PTMs (see Table 4). The two conotoxins share low sequence identity with less than 33% of their ααs being conserved, after excluding the disulfide bridge cysteine framework (confirmed by the distal phenogram clustering in Fig. 2; see Table 4), both peptides retain strong N-type VGCC selectivity. It is the unique coverage of the N- and P/Q-type of VGCCs that make ω-conotoxins a powerful pharmacological research tool. They may serve as a conduit for understanding receptor isoform targeting and the structure–activity relationships between those isoforms. This relationship will become more evident as additional ω-conotoxin sequences and activities are reported.
Fig. 2. Phylogenetic tree showing Conus sequence relationships based on sequence homology and cysteine residue alignment. Pharmacological selective toward VGCCs is indicated. The tree was created using ClustalX2 and MEGA 4 software.
Other ω-conotoxin constituents have been purified from the duct venom of C. striatus[29]. ω-Conotoxin SVIA (enclave D, Fig. 2; Table 4) demonstrates poor specificity to mammalian VGCCs while causing rapid paralysis and death in fish. This illustrates how a defined and common peptide toxin structure does not always bring about the same pharmacological interactions [29,31]. These apparent pharmacological divergences can be equated to the minor structural differences between conserved ion channel and receptors in different phyla, which impact their selective abilities [69,70]. The majority of piscovorous ω-conotoxins have successfully targeted conserved elements across phyla that normally allow for action potential propagation. Similar conserved receptor targeting strategies within complex venoms for Nav, Kv and Cav-channels can be seen with other venomous predators such as scorpions [71] and spiders [72].
The duct venom of C. striatus also contains ω-conotoxin SVIB (enclave A, Fig. 2; Table 4). This toxin targets the N- and P/Q-types of mammalian VGCC [73] and demonstrates a higher level of αα homology than ω-conotoxin SVIA to the bulk of piscivorous ω-conotoxin members (enclave A-C, Fig. 2; Table 4). Minor structural changes within ω-conotoxin SVIB are observed with increased length variability in loop 4 [74]. Within pooled milked venom of C. striatus ω-conotoxin SVIA is dominantly expressed with a near absence of ω-conotoxin SVIB. This is pharmacologically advantageous to the active piscivorous predator [43,44,75].
One recent addition to the piscivorous ω-conotoxin family class is the four ω-conotoxins isolated from the crude duct venom and sequence has been deduced from the cDNA of C. catus[76]. Pharmacologically, ω-conotoxin CVIA (enclave B, Fig. 2; Table 4) is selective for N-type VGCCs, while ω-conotoxins CVIB and CVIC (enclave A) are unable to differentiate between N- and P/Q-type VGCCs (Fig. 2). ω-conotoxin CVID (enclave C; AM336/Leconotide: Commonwealth Serum Laboratories Ltd./AMRAD/Zenyth; Table 1) deserves special mention since it targets the N-type VGCC with very high potency while having a much lower P/Q-type VGCC selectivity. These pharmacological targeting properties make ω-conotoxin CVID more N-type specific then ω-conotoxin MVIIA (enclave C, Fig. 2). This has been the impetus for ω-conotoxin CVID's development as a potential drug lead candidate against it's FDA approved competitor ω-conotoxin MVIIA/Prialt®[76–78].
C. radiatus and C. tulipa have had ω-conotoxins RVIA and TVIA isolated from their respective crude duct venoms (SNX-185, Neurex; [79]; see Table 4). Both peptides belong to enclave D (Fig. 2) and demonstrate N-type and P/Q-type VGCC selectivity respectively. It is expected that other ω-conotoxin or ω-conotoxin-like sequences will be isolated from C. radiatus and C. tulipa, either within the peptide venom or expected genome. This follows the observed trend of multiple forms of ω-conotoxins within single venoms, together with the accompanying synergistic activity of the highly abundant α-conotoxins [44]. Additional ω-conotoxins will potentially reiterate the present selective targeting strategies of N- and P/Q-type VGCC isoforms, with the absence of R-, L- or T-type VGCC activities [74,76,78,80].
Phylogenetic relationships between closely related Conus species, as seen with C. geographus and C. tulipa[81,82], strengthens the underlying theme; using multiple peptide toxins to target VGCCs that are heavily weighted by sequence identity, as observed in Table 4 and Fig. 2. These pharmacological similarities could possibly be a consequence of the unique ‘passive-like’ predatory behavior adopted by both species. This is in contrast to those cone shells that engage in active ‘tag and reel’ predation, as seen in C. magus, C. catus and C. striatus[82], again apparent strong sequence homology existence amongst these species (see Table 4). The distinction of ω-conotoxins within the piscivores could reflect their intended use in respect to specific VGCC targeting strategies for rapid prey immobilization. Subsequently, we are unsure if all the designated ω-conotoxins, assigned within an individual species, are fully expressed within their duct and milked venoms [42,44].
As for other unpublished piscivorous species like C. floccatus, C. circumcisus and C. monachus, to name a few, it is highly probable that additional ω-conotoxins will be discovered, again a reflection of Conus phylogenetics, feeding and pharmacological strategies. These toxin sequences will further expand the enclaves presented in Fig. 2. In nature there are exceptions. C. purpurascens, which has undergone extensive duct and milked analysis [83–87], is presently without any published ω-conotoxins. A possible explanation is provided by its Eastern Pacific isolation, which highlights the potential impact of cone shell biodiversity and its transition to venom composition. Undoubtedly, as distant locations become accessible, revealing new Conus species, an additional dimension to the discovery of new ω-conotoxins, as well as the potential for describing divergent ion channel targeting strategies will emerge.
ω-Conotoxins are not exclusive to piscivorous cone shells; invertebrate equivalents are found in the molluscivores C. textile and Conus pennaceus ([88,89] respectively; see Table 4). It is suspected that other similar ω-like peptides exist in other ‘tented’ patterned molluscivores such as Conus aulicus, Conus omaria and Conus episcopatus, all which have been subjected to preliminary analysis and α-conotoxin isolation ([90–92] respectively). The ω-conotoxin TxVII (enclave E; Fig. 2), which is isolated from C. textile, targets L-type Ca2+ channels in molluscan neurons and demonstrates a relative abundance of hydrophobic ααs while retaining the same disulfide bridge framework as the piscivorous ω-conotoxins [88,93,94]. The majority of molluscivorous ‘ω-like-conotoxins’, either reported from crude venom or cDNA, are poorly examined with little pharmacological or structural information reported. To date no vermivorous equivalents with confirmed VGCC activity have been assigned. Duda and Palumbi [95] have provided the first account of ‘ω-like-conotoxins’ in their sequence analysis of the vermivore Conus abbreviatus, yet biological activity, and important selectivity, remains to be pharmacologically established.
URL: https://www.sciencedirect.com/science/article/pii/S0009279709004244