Using Constellation Pharmacology to Characterize a Novel -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. alpha-Conotoxins (alpha Ctx) 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, alpha Ctx-AtIA, which has an amino acid sequence homologous to the well-described alpha Ctx-PeIA, but with a different selectivity profile towards nAChRs. We tested the synthetic alpha Ctx-AtIA using the calcium imaging-based Constellation Pharmacology assay on mouse DRG neurons and found that alpha Ctx-AtIA significantly inhibited ACh-induced calcium influx in the presence of an alpha 7 positive allosteric modulator, PNU-120596 (PNU). However, alpha Ctx-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 alpha 3 beta 4, alpha 6/alpha 3 beta 4 and alpha 7 nAChRs subtypes. We observed that alpha Ctx-AtIA displayed no or low potency in blocking alpha 3 beta 4 and alpha 6/alpha 3 beta 4 receptors, respectively, but improved potency and selectivity to block alpha 7 nAChRs when compared with alpha Ctx-PeIA. Through the synthesis of two additional analogs of alpha Ctx-AtIA and subsequent characterization using Constellation Pharmacology, we were able to identify residue Trp18 as a major contributor to the activity of the peptide.