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Peptide toxins elaborated by sea anemones target various ion-channel sub-types. Recent transcriptomic studies of sea anemones have identified several novel candidate peptides, some of which have cysteine frameworks identical to those of previously reported sequences. One such peptide is AsK132958, which was identified in a transcriptomic study of Anemonia sulcata and has a cysteine framework similar to that of ShK from Stichodactyla helianthus, but is six amino acid residues shorter. We have determined the solution structure of this novel peptide using NMR spectroscopy. The disulfide connectivities and structural scaffold of AsK132958 are very similar to those of ShK but the structure is more constrained. Toxicity assays were performed using grass shrimp (Palaemonetes sp) and Artemia nauplii, and patch-clamp electrophysiology assays were performed to assess the activity of AsK132958 against a range of voltage-gated potassium (KV) channels. AsK132958 showed no activity against grass shrimp, Artemia nauplii, or any of the KV channels tested, owing partly to the absence of a functional Lys-Tyr dyad. Three AsK132958 analogues, each containing a Tyr in the vicinity of Lys19, were therefore generated in an effort to restore binding, but none showed activity against any of KV channels tested. However, AsK132958 and its analogues are less susceptible to proteolysis than that of ShK. Our structure suggests that Lys19, which might be expected to occupy the pore of the channel, is not sufficiently accessible for binding, and therefore that AsK132958 must have a distinct functional role that does not involve KV channels.

In the search for novel anticancer drugs, the potassium channel KV10.1 has emerged as an interesting cancer target. Here, we report a new group of KV10.1 inhibitors, namely the purpurealidin analogs. These alkaloids are produced by the Verongida sponges and are known for their wide variety of bioactivities. In this study, we describe the synthesis and characterization of 27 purpurealidin analogs. Structurally, bromine substituents at the central phenyl ring and a methoxy group at the distal phenyl ring seem to enhance the activity on KV10.1. The mechanism of action of the most potent analog 5 was investigated. A shift of the activation curve to more negative potentials and an apparent inactivation was observed. Since KV10.1 inhibitors can be interesting anticancer drug lead compounds, the effect of 5 was evaluated on cancerous and non-cancerous cell lines. Compound 5 showed to be cytotoxic and appeared to induce apoptosis in all the evaluated cell lines.

Cone snails are predatory creatures using venom as a weapon for prey capture and defense. Since this venom is neurotoxic, the venom gland is considered as an enormous collection of pharmacologically interesting compounds having a broad spectrum of targets. As such, cone snail peptides represent an interesting treasure for drug development. Here, we report five novel peptides isolated from the venom of Conus longurionis, Conus asiaticus and Conus australis. Lo6/7a and Lo6/7b were retrieved from C. longurionis and have a cysteine framework VI/VII. Lo6/7b has an exceptional amino acid sequence because no similar conopeptide has been described to date (similarity percentage C. asiaticus, has a typical framework III Cys arrangement, classifying the peptide in the M-superfamily. Asi14a, another peptide of C. asiaticus, belongs to framework XIV peptides and has a unique amino acid sequence. Finally, AusB is a novel conopeptide from C. australis. The peptide has only one disulfide bond, but is structurally very different as compared to other disulfide-poor peptides. The peptides were screened on nAChRs, NaV and KV channels depending on their cysteine framework and proposed classification. No targets could be attributed to the peptides, pointing to novel functionalities. Moreover, in the quest of identifying novel pharmacological targets, the peptides were tested for antagonistic activity against a broad panel of Gram-negative and Gram-positive bacteria, as well as two yeast strains.

Sea anemone venoms comprise multifarious peptides modulating biological targets such as ion channels or receptors. The sequence of a new Kunitz-type peptide, HCRG21, belonging to the Heteractis crispa RG (HCRG) peptide subfamily was deduced on the basis of the gene sequence obtained from the Heteractis crispa cDNA. HCRG21 shares high structural homology with Kunitz-type peptides APHC1–APHC3 from H. crispa, and clusters with the peptides from so named “analgesic cluster” of the HCGS peptide subfamily but forms a separate branch on the NJ-phylogenetic tree. Three unique point substitutions at the N-terminus of the molecule, Arg1, Gly2, and Ser5, distinguish HCRG21 from other peptides of this cluster. The trypsin inhibitory activity of recombinant HCRG21 (rHCRG21) was comparable with the activity of peptides from the same cluster. Inhibition constants for trypsin and α-chymotrypsin were 1.0 × 10−7 and 7.0 × 10−7 M, respectively. Electrophysiological experiments revealed that rHCRG21 inhibits 95% of the capsaicin-induced current through transient receptor potential family member vanilloid 1 (TRPV1) and has a half-maximal inhibitory concentration of 6.9 ± 0.4 μM. Moreover, rHCRG21 is the first full peptide TRPV1 inhibitor, although displaying lower affinity for its receptor in comparison with other known ligands. Macromolecular docking and full atom Molecular Dynamics (MD) simulations of the rHCRG21–TRPV1 complex allow hypothesizing the existence of two feasible, intra- and extracellular, molecular mechanisms of blocking. These data provide valuable insights in the structural and functional relationships and pharmacological potential of bifunctional Kunitz-type peptides.

Sponges of the genus Agelas produce compounds that modulate the activity of voltage-gated sodium ion channels and contribute novel scaffolds for the development of compounds with activity against a plethora of biological targets. In particular, clathrodin and dibromosceptrin were reported to decrease the average maximum amplitude of inward sodium currents in isolated chick embryo sympathetic ganglia cells; we envisaged these compounds as a starting point to design novel Na-v channel modulators. This endeavor was part of our long-term goal of designing a comprehensive library of Agelas alkaloid analogs that would cover a broader chemical space and allow us to examine the activity of such compounds on Na-v channels. Our series of compounds was designed by maintaining the terminal structural features found in clathrodin while rigidizing the central part of the molecule and replacing the 3-aminopropene linker with a 4-methylenepiperazine moiety. Synthesised compounds were screened for inhibitory action against the human voltage-gated sodium channel isoforms Na-v 1.3, Na-v 1.4, cardiac Na-v 1.5, and Na-v 1.7 using an automated patch clamp electrophysiology technique. The results demonstrate that we have obtained a series of compounds with a modest but selective inhibitory activity against the Na-v 1.3 channel isoform. The most potent compound showed selective activity against the Na-v 1.3 channel isoform with an IC50 of 19 mu M and is a suitable starting point for further development of selective Na-v 1.3 channel modulators. Such compounds could prove to be beneficial as a pharmacological tool towards the development of novel therapeutically useful compounds in the treatment of pain.

Cone snails (Conus sp.) are poisonous animals that can be found in all oceans where they developed a venomous strategy to prey or to defend. The venom of these species contains an undeniable source of unique and potent pharmacologically active compounds. Their peptide compounds, called conotoxins, are not only interesting for the development of new pharmaceutical ligands, but they are also useful for studying their broad spectrum of targets. One conotoxin family in particular, the a-conotoxins, acts on nicotinic acetylcholine receptors (nAChRs) which dysfunctions play important roles in pathologies such as epilepsy, myasthenic syndromes, schizophrenia, Parkinson's disease and Alzheimer's disease. Here we define a new subclass of the a-conotoxin family. We purified the venom of a yet unexplored cone snail species, i.e. Conus australis, and we isolated a 16-amino acid peptide named a-conotoxin AusIA. The peptide has the typical a-conotoxin CC-Xm-C-Xn-C framework, but both loops (m/n) contain 5 amino acids, which has never been described before. Using conventional electrophysiology we investigated the response of synthetically made globular (I–III, II–IV) and ribbon (I–IV, II–III) AusIA to different nicotinic acetylcholine receptors. The a7 nAChR was the only receptor found to be blocked with a similar potency by both peptide-configurations. This suggests that both a5/5 conotoxin isomers might be present in the venom gland of C. australis. NMR spectroscopy showed that no secondary structures define the peptides' three-dimensional topology. Moreover, the ribbon configuration, which is generally considered to be non-native, is more stable than the globular isomer. Accordingly, our findings show relevancy concerning the a-conotoxin classification which might be helpful in the design of novel therapeutic compounds.

Sea anemone (Cnidaria, Anthozoa) venom is an important source of bioactive compounds used as tools to study the pharmacology and structure-function of voltage-gated K+ channels (KV). These neurotoxins can be divided into four different types, according to their structure and mode of action. In this work, for the first time, two toxins were purified from the venom of Bunodosoma caissarum population from Saint Peter and Saint Paul Archipelago, Brazil. Sequence alignment and phylogenetic analysis reveals that BcsTx1 and BcsTx2 are the newest members of the sea anemone type 1 potassium channel toxins. Their functional characterization was performed by means of a wide electrophysiological screening on 12 different subtypes of KV channels (KV1.1–KV1.6; KV2.1; KV3.1; KV4.2; KV4.3; hERG and Shaker IR). BcsTx1 shows a high affinity for rKv1.2 over rKv1.6, hKv1.3, Shaker IR and rKv1.1, while Bcstx2 potently blocked rKv1.6 over hKv1.3, rKv1.1, Shaker IR and rKv1.2. Furthermore, we also report for the first time a venom composition and biological activity comparison between two geographically distant populations of sea anemones.

Cone snail venoms provide a largely untapped source of novel peptide drug leads. To enhance the discovery phase, a detailed comparative proteomic analysis was undertaken on milked venom from the mollusk-hunting cone snail, Conus textile, from three different geographic locations (Hawai’i, American Samoa and Australia's Great Barrier Reef). A novel milked venom conopeptide rich in post-translational modifications was discovered, characterized and named a-conotoxin TxIC. We assign this conopeptide to the 4/7 a-conotoxin family based on the peptide's sequence homology and cDNA pre-propeptide alignment. Pharmacologically, a-conotoxin TxIC demonstrates minimal activity on human acetylcholine receptor models (100 µM, < 5% inhibition), compared to its high paralytic potency in invertebrates, PD50 = 34.2 nMol kg-1. The non-post-translationally modified form, [Pro]2,8[Glu]16a-conotoxin TxIC, demonstrates differential selectivity for the a3ß2 isoform of the nicotinic acetylcholine receptor with maximal inhibition of 96% and an observed IC50 of 5.4 ± 0.5 µM. Interestingly its comparative PD50 (3.6 µMol kg-1) in invertebrates was ~100 fold more than that of the native peptide. Differentiating a-conotoxin TxIC from other a-conotoxins is the high degree of post-translational modification (44% of residues). This includes the incorporation of ?-carboxyglutamic acid, two moieties of 4-trans hydroxyproline, two disulfide bond linkages, and C-terminal amidation. These findings expand upon the known chemical diversity of a-conotoxins and illustrate a potential driver of toxin phyla-selectivity within Conus.

To date, cone snail toxins (“conotoxins”) are of great interest in the pursuit of novel subtype-selective modulators of voltage-gated sodium channels (Navs). Navs participate in a wide range of electrophysiological processes. Consequently, their malfunctioning has been associated with numerous diseases. The development of subtype-selective modulators of Navs remains highly important in the treatment of such disorders. In current research, a series of novel, synthetic, and bioactive compounds were designed based on two naturally occurring µ-conotoxins that target Navs. The initial designed peptide contains solely 13 amino acids and was therefore named “Mini peptide.” It was derived from the µ-conotoxins KIIIA and BuIIIC. Based on this Mini peptide, 10 analogues were subsequently developed, comprising 12–16 amino acids with two disulfide bridges. Following appropriate folding and mass verification, blocking effects on Navs were investigated. The most promising compound established an IC50 of 34.1 ± 0.01 nm (R2-Midi on Nav1.2). An NMR structure of one of our most promising compounds was determined. Surprisingly, this structure does not reveal an a-helix. We prove that it is possible to design small peptides based on known pharmacophores of µ-conotoxins without losing their potency and selectivity. These data can provide crucial material for further development of conotoxin-based therapeutics.

Sea anemone venom is a known source of interesting bioactive compounds, including peptide toxins which are invaluable tools for studying structure and function of voltage-gated potassium channels. APETx3 is a novel peptide isolated from the sea anemone Anthopleura elegantissima, containing 42 amino acids cross-linked by 3 disulfide bridges. Sequence alignment reveals that APETx3 is a natural occurring mutant from APETx1, only differing in 1 amino acid at position 3. APETx1 is believed to be a selective modulator of human ether-á-go-go related (hERG) potassium channels. In this study, APETx1, 2 and 3 have been subjected to an electrophysiological screening on a wide range of 21 ion channels expressed in Xenopus leavis oocytes: 10 cloned voltage-gated sodium channels (NaV1.2-NaV1.8, the insect channels DmNaV1, BgNaV1-1a and the arachnid channel VdNaV1) and 11 cloned voltage-gated potassium channels (KV2.1, KV3.1, KV4.2, KV4.3, KV7.1, KV7.2, KV7.3, KV7.4, KV7.5, hERG, the insect channel Shaker IR). Surprisingly, the Thr3Pro substitution results in a complete abolishment of APETx3 modulation on hERG channels. However, the same substitution provides this toxin the ability to become a potent modulator of voltage-gated sodium channels (NaVs). APETx3 slows down the inactivation of mammalian and insect channels similar to site 3 toxins such as a-scorpion toxins and sea anemone NaVs toxins. Our screening reveals that the homologous toxins APETx1 and APETx2 display promiscuous properties as they are also capable of recognizing NaV channels, causing an inhibition of the sodium conductance.All together, these data provide new insights in key residues which allow these toxins to recognize distinct ion channels with similar potency but with different modulatory effects. Furthermore, we describe for the first time the target promiscuity of a family of sea anemone toxins believed to be highly selective.