Using Constellation Pharmacology to Characterize a Novel α-Conotoxin from Conus ateralbus.

The venom of cone snails has been proven to be a rich source of bioactive peptides that target a variety of ion channels and receptors. α-Conotoxins (αCtx) interact with nicotinic acetylcholine receptors (nAChRs) and are powerful tools for investigating the structure and function of the various nAChR subtypes. By studying how conotoxins interact with nAChRs, we can improve our understanding of these receptors, leading to new insights into neurological diseases associated with nAChRs. Here, we describe the discovery and characterization of a novel conotoxin from Conus ateralbus, αCtx-AtIA, which has an amino acid sequence homologous to the well-described αCtx-PeIA, but with a different selectivity profile towards nAChRs. We tested the synthetic αCtx-AtIA using the calcium imaging-based Constellation Pharmacology assay on mouse DRG neurons and found that αCtx-AtIA significantly inhibited ACh-induced calcium influx in the presence of an α7 positive allosteric modulator, PNU-120596 (PNU). However, αCtx-AtIA did not display any activity in the absence of PNU. These findings were further validated using two-electrode voltage clamp electrophysiology performed on oocytes overexpressing mouse α3ß4, α6/α3ß4 and α7 nAChRs subtypes. We observed that αCtx-AtIA displayed no or low potency in blocking α3ß4 and α6/α3ß4 receptors, respectively, but improved potency and selectivity to block α7 nAChRs when compared with αCtx-PeIA. Through the synthesis of two additional analogs of αCtx-AtIA and subsequent characterization using Constellation Pharmacology, we were able to identify residue Trp18 as a major contributor to the activity of the peptide.

Non-Peptidic Small Molecule Components from Cone Snail Venoms.

Venomous molluscs (Superfamily Conoidea) comprise a substantial fraction of tropical marine biodiversity (>15,000 species). Prior characterization of cone snail venoms established that bioactive venom components used to capture prey, defend against predators and for competitive interactions were relatively small, structured peptides (10-35 amino acids), most with multiple disulfide crosslinks. These venom components ("conotoxins, conopeptides") have been widely studied in many laboratories, leading to pharmaceutical agents and probes. In this review, we describe how it has recently become clear that to varying degrees, cone snail venoms also contain bioactive non-peptidic small molecule components. Since the initial discovery of genuanine as the first bioactive venom small molecule with an unprecedented structure, a broad set of cone snail venoms have been examined for non-peptidic bioactive components. In particular, a basal clade of cone snails (Stephanoconus) that prey on polychaetes produce genuanine and many other small molecules in their venoms, suggesting that this lineage may be a rich source of non-peptidic cone snail venom natural products. In contrast to standing dogma in the field that peptide and proteins are predominantly used for prey capture in cone snails, these small molecules also contribute to prey capture and push the molecular diversity of cone snails beyond peptides. The compounds so far characterized are active on neurons and thus may potentially serve as leads for neuronal diseases. Thus, in analogy to the incredible pharmacopeia resulting from studying venom peptides, these small molecules may provide a new resource of pharmacological agents.

Characterization of the First Conotoxin from Conus ateralbus, a Vermivorous Cone Snail from the Cabo Verde Archipelago.

Conus ateralbus is a cone snail endemic to the west side of the island of Sal, in the Cabo Verde Archipelago off West Africa. We describe the isolation and characterization of the first bioactive peptide from the venom of this species. This 30AA venom peptide is named conotoxin AtVIA (δ-conotoxin-like). An excitatory activity was manifested by the peptide on a majority of mouse lumbar dorsal root ganglion neurons. An analog of AtVIA with conservative changes on three amino acid residues at the C-terminal region was synthesized and this analog produced an identical effect on the mouse neurons. AtVIA has homology with δ-conotoxins from other worm-hunters, which include conserved sequence elements that are shared with δ-conotoxins from fish-hunting Conus. In contrast, there is no comparable sequence similarity with δ-conotoxins from the venoms of molluscivorous Conus species. A rationale for the potential presence of δ-conotoxins, that are potent in vertebrate systems in two different lineages of worm-hunting cone snails, is discussed.

Small Molecules in the Cone Snail Arsenal.

Cone snails are renowned for producing peptide-based venom, containing conopeptides and conotoxins, to capture their prey. A novel small-molecule guanine derivative with unprecedented features, genuanine, was isolated from the venom of two cone snail species. Genuanine causes paralysis in mice, indicating that small molecules and not just polypeptides may contribute to the activity of cone snail venom.