New Conotoxin SO-3 Targeting N-type Voltage-Sensitive Calcium Channels

Selective blockers of the N-type voltage-sensitive calcium (Ca_V) channels are useful in the management of severe chronic pain. Here, the structure and function characteristics of a novel N-type Ca_V channel blocker, SO-3, are reviewed. SO-3 is a 25- amino acid conopeptide originally derived from the venom of Conus striatus, and contains the same 4-loop, 6-cysteine framework (C-C-CC-C-C) as O-superfamily conotoxins. The synthetic SO-3 has high analgesic activity similar to ω-conotoxin MVIIA (MVIIA), a selective N-type Ca_V channel blocker approved in the USA and Europe for the alleviation of persistent pain states. In electrophysiological studies, SO-3 shows more selectivity towards the N-type Ca_V channels than MVIIA. The dissimilarity between SO-3 and MVIIA in the primary and tertiary structures is further discussed in an attempt to illustrate the difference in selectivity of SO-3 and MVIIA towards N-type Ca_V channels.     

Conotoxins that confer therapeutic possibilities

Cone snails produce a distinctive repertoire of venom peptides that are used both as a defense mechanism and also to facilitate the immobilization and digestion of prey. These peptides target a wide variety of voltage- and ligand-gated ion channels, which make them an invaluable resource for studying the properties of these ion channels in normal and diseased states, as well as being a collection of compounds of potential pharmacological use in their own right. Examples include the United States Food and Drug Administration (FDA) approved pharmaceutical drug, Ziconotide (Prialt^®; Elan Pharmaceuticals, Inc.) that is the synthetic equivalent of the naturally occurring ω-conotoxin MVIIA, whilst several other conotoxins are currently being used as standard research tools and screened as potential therapeutic drugs in pre-clinical or clinical trials. These developments highlight the importance of driving conotoxin-related research. A PubMed query from 1 January 2007 to 31 August 2011 combined with hand-curation of the retrieved articles allowed for the collation of 98 recently identified conotoxins with therapeutic potential which are selectively discussed in this review. Protein sequence similarity analysis tentatively assigned uncharacterized conotoxins to predicted functional classes. Furthermore, conotoxin therapeutic potential for neurodegenerative disorders (NDD) was also inferred.     

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.     

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 α3β2 and α6/α3β2β3 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.     

Marine Toxins Targeting Ion Channels

This introductory minireview points out the importance of ion channels for cell communication. The basic concepts on the structure and function of ion channels triggered by membrane voltage changes, the so-called voltage-gated ion channels (VGICs), as well as those activated by neurotransmitters, the so-called ligand-gated ion channel (LGICs), are introduced. Among the most important VGIC superfamiles, we can name the voltage-gated Na⁺ (Na_V), Ca²⁺ (Ca_V), and K⁺ (K_V) channels. Among the most important LGIC super families, we can include the Cys-loop or nicotinicoid, the glutamate-activated (GluR), and the ATP-activated (P2X_nR) receptor superfamilies. Ion channels are transmembrane proteins that allow the passage of different ions in a specific or unspecific manner. For instance, the activation of Na_V, Ca_V, or K_V channels opens a pore that is specific for Na⁺, Ca²⁺, or K⁺, respectively. On the other hand, the activation of certain LGICs such as nicotinic acetylcholine receptors, GluRs, and P2X_nRs allows the passage of cations (e.g., Na⁺, K⁺, and/or Ca²⁺), whereas the activation of other LGICs such as type A γ-butyric acid and glycine receptors allows the passage of anions (e.g., Cl⁻ and/or HCO₃⁻). In this regard, the activation of Na_V and Ca_V as well as ligand-gated cation channels produce membrane depolarization, which finally leads to stimulatory effects in the cell, whereas the activation of K_V as well as ligand-gated anion channels induce membrane hyperpolarization that finally leads to inhibitory effects in the cell. The importance of these ion channel superfamilies is emphasized by considering their physiological functions throughout the body as well as their pathophysiological implicance in several neuronal diseases. In this regard, natural molecules, and especially marine toxins, can be potentially used as modulators (e.g., inhibitors or prolongers) of ion channel functions to treat or to alleviate a specific ion channel-linked disease (e.g., channelopaties).     

Conus regius-Derived Conotoxins: Novel Therapeutic Opportunities from a Marine Organism

Conus regius is a marine venomous mollusk of the Conus genus that captures its prey by injecting a rich cocktail of bioactive disulfide bond rich peptides called conotoxins. These peptides selectively target a broad range of ion channels, membrane receptors, transporters, and enzymes, making them valuable pharmacological tools and potential drug leads. C. regius-derived conotoxins are particularly attractive due to their marked potency and selectivity against specific nicotinic acetylcholine receptor subtypes, whose signalling is involved in pain, cognitive disorders, drug addiction, and cancer. However, the species-specific differences in sensitivity and the low stability and bioavailability of these conotoxins limit their clinical development as novel therapeutic agents for these disorders. Here, we give an overview of the main pharmacological features of the C. regius-derived conotoxins described so far, focusing on the molecular mechanisms underlying their potential therapeutic effects. Additionally, we describe adoptable chemical engineering solutions to improve their pharmacological properties for future potential clinical translation.     

Computational Design of High-Affinity Blockers for Sodium Channel NaV1.2 from μ-Conotoxin KIIIA

The voltage-gated sodium channel subtype 1.2 (Na_V1.2) is instrumental in the initiation of action potentials in the nervous system, making it a natural drug target for neurological diseases. Therefore, there is much pharmacological interest in finding blockers of Na_V1.2 and improving their affinity and selectivity properties. An extensive family of peptide toxins from cone snails (conotoxins) block Na_V channels, thus they provide natural templates for the design of drugs targeting Na_V channels. Unfortunately, progress was hampered due to the absence of any Na_V structures. The recent determination of cryo-EM structures for Na_V channels has finally broken this impasse. Here, we use the Na_V1.2 structure in complex with μ-conotoxin KIIIA (KIIIA) in computational studies with the aim of improving KIIIA’s affinity and blocking capacity for Na_V1.2. Only three KIIIA amino acid residues are available for mutation (S5, S6, and S13). After performing molecular modeling and simulations on Na_V1.2–KIIIA complex, we have identified the S5R, S6D, and S13K mutations as the most promising for additional contacts. We estimate these contacts to boost the affinity of KIIIA for Na_V1.2 from nanomole to picomole domain. Moreover, the KIIIA[S5R, S6D, S13K] analogue makes contacts with all four channel domains, thus enabling the complete blocking of the channel (KIIIA partially blocks as it has contacts with three domains). The proposed KIIIA analogue, once confirmed experimentally, may lead to novel anti-epileptic drugs.     

AsKC11, a Kunitz Peptide from Anemonia sulcata,Is a Novel Activator of G Protein-Coupled Inward-Rectifier Potassium Channels

(1) Background: G protein-coupled inward-rectifier potassium (GIRK) channels, especially neuronal GIRK1/2 channels, have been the focus of intense research interest for developing drugs against brain diseases. In this context, venom peptides that selectively activate GIRK channels can be seen as a new source for drug development. Here, we report on the identification and electrophysiological characterization of a novel activator of GIRK1/2 channels, AsKC11, found in the venom of the sea anemone Anemonia sulcata. (2) Methods: AsKC11 was purified from the sea anemone venom by reverse-phase chromatography and the sequence was identified by mass spectrometry. Using the two-electrode voltage-clamp technique, the activity of AsKC11 on GIRK1/2 channels was studied and its selectivity for other potassium channels was investigated. (3) Results: AsKC11, a Kunitz peptide found in the venom of A. sulcata, is the first peptide shown to directly activate neuronal GIRK1/2 channels independent from Gi/o protein activity, without affecting the inward-rectifier potassium channel (IRK1) and with only a minor effect on K_V1.6 channels. Thus, AsKC11 is a novel activator of GIRK channels resulting in larger K⁺ currents because of an increased chord conductance. (4) Conclusions: These discoveries provide new insights into a novel class of GIRK activators.     

Binding of κ-Conotoxin-PVIIA to Open and Closed Shaker K-Channels Are Differentially Affected by the Ionic Strength

κ-Conotoxin-PVIIA (κ-PVIIA) is a potassium-channel blocking peptide from the venom of the fish-hunting snail, Conus purpurascens, which is essential for quick prey’s excitotoxic immobilization. Binding of one κ-PVIIA to Shaker K-channels occludes the K⁺-conduction pore without additional conformational effects. Because this 27-residue toxin is +4-charged at neutral pH, we asked if electrostatic interactions play a role in binding. With Voltage-Clamp electrophysiology, we tested how ionic strength (IS) affects κ-PVIIA blockade to Shaker. When IS varied from ~0.06 to ~0.16 M, the dissociation constant for open and closed channels increased by ~5- and ~16-fold, respectively. While the association rates decreased equally, by ~4-fold, in open and closed channels, the dissociation rates increased 4–5-fold in closed channels but was IS-insensitive in open channels. To explain this differential IS-dependency, we propose that the bound κ-PVIIA wobbles, so that in open channels the intracellular environment, via ion-conduction pore, buffers the imposed IS-changes in the toxin-channel interface. A Brønsted-Bjerrum analysis on the rates predicts that if, instead of fish, the snail preyed on organisms with seawater-like lymph ionic composition, a severely harmless toxin, with >100-fold diminished affinity, would result. Thus, considerations of the native ionic environment are essential for conotoxins evaluation as pharmacological leads.     

Interactions of Nereistoxin and Its Analogs with Vertebrate Nicotinic Acetylcholine Receptors and Molluscan ACh Binding Proteins

Nereistoxin (NTX) is a marine toxin isolated from an annelid worm that lives along the coasts of Japan. Its insecticidal properties were discovered decades ago and this stimulated the development of a variety of insecticides such as Cartap that are readily transformed into NTX. One unusual feature of NTX is that it is a small cyclic molecule that contains a disulfide bond. In spite of its size, it acts as an antagonist at insect and mammalian nicotinic acetylcholine receptors (nAChRs). The functional importance of the disulfide bond was assessed by determining the effects of inserting a methylene group between the two sulfur atoms, creating dimethylaminodithiane (DMA-DT). We also assessed the effect of methylating the NTX and DMA-DT dimethylamino groups on binding to three vertebrate nAChRs. Radioligand receptor binding experiments were carried out using washed membranes from rat brain and fish (Torpedo) electric organ; [³H]-cytisine displacement was used to assess binding to the predominantly high affinity alpha4beta2 nAChRs and [¹²⁵I]-alpha-bungarotoxin displacement was used to measure binding of NTX and analogs to the alpha7 and skeletal muscle type nAChRs. While the two quaternary nitrogen analogs, relative to their respective tertiary amines, displayed lower α4β2 nAChR binding affinities, both displayed much higher affinities for the Torpedo muscle nAChR and rat alpha7 brain receptors than their respective tertiary amine forms. The binding affinities of DMA-DT for the three nAChRs were lower than those of NTX and MeNTX. An AChBP mutant lacking the C loop disulfide bond that would potentially react with the NTX disulfide bond displayed an NTX affinity very similar to the parent AChBP. Inhibition of [³H]-epibatidine binding to the AChBPs was not affected by exposure to NTX or MeNTX for up to 24 hr prior to addition of the radioligand. Thus, the disulfide bond of NTX is not required to react with the vicinal disulfide in the AChBP C loop for inhibition of [³H]-epibatidine binding. However, a reversible disulfide interchange reaction of NTX with nAChRs might still occur, especially under reducing conditions. Labeled MeNTX, because it can be readily prepared with high specific radioactivity and possesses relatively high affinity for the nAChR-rich Torpedo nAChR, would be a useful probe to detect and identify any nereistoxin adducts.     

Selective Blocking Effects of 4,9-Anhydrotetrodotoxin,Purified from a Crude Mixture of Tetrodotoxin Analogues,on NaV1.6 Channels and Its Chemical Aspects

Tetrodotoxin (TTX) is a potent neurotoxin found in a number of marine creatures including the pufferfish, where it is synthesized by bacteria and accumulated through the food chain. It is a potent and selective blocker of some types of voltage-gated Na⁺ channel (Na_V channel). 4,9-Anhydrotetrodotoxin (4,9-anhydroTTX) was purified from a crude mixture of TTX analogues (such as TTX, 4-epiTTX, 6-epiTTX, 11-oxoTTX and 11-deoxyTTX) by the use of liquid chromatography-fluorescence detection (LC-FLD) techniques. Recently, it has been reported that 4,9-anhydroTTX selectively blocks the activity of Na_V1.6 channels with a blocking efficacy 40–160 times higher than that for other TTX-sensitive Na_V1.x channel isoforms. However, little attention has been paid to the molecular properties of the α-subunit in Na_V1.6 channels and the characteristics of binding of 4,9-anhydroTTX. From a functional point of view, it is important to determine the relative expression of Na_V1.6 channels in a wide variety of tissues. The aim of this review is to discuss briefly current knowledge about the pharmacology of 4,9-anhydroTTX, and provide an analysis of the molecular structure of native Na_V1.6 channels. In addition, chemical aspects of 4,9-anhydroTTX are briefly covered.     

Chemical Synthesis and NMR Solution Structure of Conotoxin GXIA from Conus geographus

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.     

Characterization of a Novel Conus bandanus Conopeptide Belonging to the M-Superfamily Containing Bromotryptophan

A novel conotoxin (conopeptide) was biochemically characterized from the crude venom of the molluscivorous marine snail, Conus bandanus (Hwass in Bruguière, 1792), collected in the south-central coast of Vietnam. The peptide was identified by screening bromotryptophan from chromatographic fractions of the crude venom. Tandem mass spectrometry techniques were used to detect and localize different post-translational modifications (PTMs) present in the BnIIID conopeptide. The sequence was confirmed by Edman’s degradation and mass spectrometry revealing that the purified BnIIID conopeptide had 15 amino acid residues, with six cysteines at positions 1, 2, 7, 11, 13, and 14, and three PTMs: bromotryptophan, γ-carboxy glutamate, and amidated aspartic acid, at positions “4”, “5”, and “15”, respectively. The BnIIID peptide was synthesized for comparison with the native peptide. Homology comparison with conopeptides having the III-cysteine framework (–CCx₁x₂x₃x₄Cx₁x₂x₃Cx₁CC–) revealed that BnIIID belongs to the M-1 family of conotoxins. This is the first report of a member of the M-superfamily containing bromotryptophan as PTM.     

Conotoxins: Therapeutic Potential and Application

The pharmacological variety of conotoxins, diverse peptides found in the venoms of marine cone snails, is well recognized. Venoms from each of the estimated 500 species of cone snails contain 50 to 200 distinct biologically active peptides. Most conotoxins characterized to date target receptors and ion channels of excitable tissues, such as ligand-gated nicotinic acetylcholine, N-methyl-D-aspartate, and type 3 serotonin receptors, as well as voltage-gated calcium, sodium, and potassium channels, and G-protein-coupled receptors including α-adrenergic, neurotensin, and vasopressin receptors, and the norepinephrine transporter. Several conotoxins have shown promise in preclinical models of pain, convulsive disorders, stroke, neuromuscular block, and cardioprotection. The pharmacological selectivity of the conotoxins, coupled with the safety and efficacy demonstrated in preclinical models, has led to their investigation as human therapeutic agents. In the following review, we will survey the pharmacology and therapeutic rationale of those conotoxins with potential clinical application, and discuss the unique challenges that each will face in the course of their transition from venom component to human therapeutic.     

A Conus regularis Conotoxin with a Novel Eight-Cysteine Framework Inhibits CaV2.2 Channels and Displays an Anti-Nociceptive Activity

A novel peptide, RsXXIVA, was isolated from the venom duct of Conus regularis, a worm-hunting species collected in the Sea of Cortez, México. Its primary structure was determined by mass spectrometry and confirmed by automated Edman degradation. This conotoxin contains 40 amino acids and exhibits a novel arrangement of eight cysteine residues (C-C-C-C-CC-CC). Surprisingly, two loops of the novel peptide are highly identical to the amino acids sequence of ω-MVIIA. The total length and disulfide pairing of both peptides are quite different, although the two most important residues for the described function of ω-MVIIA (Lys2 and Tyr13) are also present in the peptide reported here. Electrophysiological analysis using superior cervical ganglion (SCG) neurons indicates that RsXXIVA inhibits Ca_V2.2 channel current in a dose-dependent manner with an EC₅₀ of 2.8 μM, whose effect is partially reversed after washing. Furthermore, RsXXIVA was tested in hot-plate assays to measure the potential anti-nociceptive effect to an acute thermal stimulus, showing an analgesic effect in acute thermal pain at 30 and 45 min post-injection. Also, the toxin shows an anti-nociceptive effect in a formalin chronic pain test. However, the low affinity for Ca_V2.2 suggests that the primary target of the peptide could be different from that of ω-MVIIA.     

Bioactive indole derivatives from the South Pacific marine sponges Rhopaloeides odorabile and Hyrtios sp

Indole derivatives including bromoindoles have been isolated from the South Pacific marine sponges Rhopaloeides odorabile and Hyrtios sp. Their structures were established through analysis of mass spectra and 1D and 2D NMR spectroscopic data. Their potential inhibitory phospholipase A₂ (PLA₂), antioxidant and cytotoxic activities were evaluated. The new derivative 5,6-dibromo-l-hypaphorine (9) isolated from Hyrtios sp. revealed a weak bee venom PLA₂ inhibition (IC₅₀ 0.2 mM) and a significant antioxidant activity with an Oxygen Radical Absorbance Capacity (ORAC) value of 0.22. The sesquiterpene aureol (4), also isolated from Hyrtios sp., showed the most potent antioxidant activity with an ORAC value of 0.29.     

Strategies for the Development of Conotoxins as New Therapeutic Leads

Peptide toxins typically bind to their target ion channels or receptors with high potency and selectivity, making them attractive leads for therapeutic development. In some cases the native peptide as it is found in the venom from which it originates can be used directly, but in many instances it is desirable to truncate and/or stabilize the peptide to improve its therapeutic properties. A complementary strategy is to display the key residues that make up the pharmacophore of the peptide toxin on a non-peptidic scaffold, thereby creating a peptidomimetic. This review exemplifies these approaches with peptide toxins from marine organisms, with a particular focus on conotoxins.     

Marine Natural Products Acting on the Acetylcholine-Binding Protein and Nicotinic Receptors: From Computer Modeling to Binding Studies and Electrophysiology

For a small library of natural products from marine sponges and ascidians, in silico docking to the Lymnaea stagnalis acetylcholine-binding protein (AChBP), a model for the ligand-binding domains of nicotinic acetylcholine receptors (nAChRs), was carried out and the possibility of complex formation was revealed. It was further experimentally confirmed via competition with radioiodinated α-bungarotoxin ([¹²⁵I]-αBgt) for binding to AChBP of the majority of analyzed compounds. Alkaloids pibocin, varacin and makaluvamines С and G had relatively high affinities (K_i 0.5–1.3 μM). With the muscle-type nAChR from Torpedo californica ray and human neuronal α7 nAChR, heterologously expressed in the GH₄C₁ cell line, no competition with [¹²⁵I]-αBgt was detected in four compounds, while the rest showed an inhibition. Makaluvamines (K_i ~ 1.5 μM) were the most active compounds, but only makaluvamine G and crambescidine 359 revealed a weak selectivity towards muscle-type nAChR. Rhizochalin, aglycone of rhizochalin, pibocin, makaluvamine G, monanchocidin, crambescidine 359 and aaptamine showed inhibitory activities in electrophysiology experiments on the mouse muscle and human α7 nAChRs, expressed in Xenopus laevis oocytes. Thus, our results confirm the utility of the modeling studies on AChBPs in a search for natural compounds with cholinergic activity and demonstrate the presence of the latter in the analyzed marine biological sources.