Cone snails represent a large taxon of carnivorous gastropods that use specialized venom to hunt fish, worms, or fellow molluscs. The venom of these snails is prolific with small, disulfide-rich peptides that bind to various physiological targets. Because the disulfide bonding leads to highly stable and constrained 3-dimensional conformations [
1], conotoxins, as they are known, have repeatedly shown high specificity for their targets, making them an excellent resource for the discovery of novel, medically significant compounds [
1,
2,
3].
Due to recent transcriptomic sequencing of
Conus venom glands, the discovery rate of novel conotoxins has increased dramatically [
4,
5,
6]. Additionally, it has been revealed that, despite the large number of conotoxins found in a single snail’s venom, they originate from a relatively small number of genes [
4]. In fact, it has been found that in a single species of cone snail, approximately only 100 genes are responsible for producing thousands of peptides [
4]. This molecular diversity is possible via variable peptide processing (VPP), in which the use of alternative cleavage sites, post-translational modifications (PTMs), and variable N- and C-terminal truncations create a plethora of peptides from a single gene precursor, resulting in biological “messiness” at the proteomic level. Of particular interest was the gene coding for the χ-conotoxin MrIA (sequence NGVCCGYKLCHOC-NH2) because of its proven pharmacological relevance and its high expression in the venom. MrIA specifically inhibits human norepinephrine transporters (hNET) at an allosteric site, leading to an attenuation of neuropathic pain [
7]. Because of this, an optimized version of MrIA, known as Xen2174 (sequence: ZGVCCGYKLCHOC-NH
2), was progressed into phase II clinical trials to treat pain in post-surgical and cancer patients [
8]. The high hNET selectivity of MrIA’s targeting is modulated by its pharmacophore, which is well understood [
9]. The pharmacophore includes the stabilizing scaffold of two disulfide bonds joined in a 1-4, 2-3 ribbon connectivity. The scaffold stabilizes the pharmacophore residues, Tyr7, Lys8, and Leu9, and creates an inverse gamma turn that presents the pharmacophore residues and allows for selective binding on the hNET target [
9] (
Figure 1). It was found that modifications to any of the pharmacophore residues as well as slight structural changes could have large impacts on the hNET inhibition exhibited by the peptide [
9].
In the study by Dutertre et al. [
4] on
C. marmoreus venom, 72 unique peptide masses related to MrIA were identified via proteomic methods that corresponded to various peptides originating from the MrIA parent peptide. A variety of different truncations contributed to this remarkable diversity, as well as PTMs, including C-terminal amidation and the inclusion of non-typical amino acids, such as pyroglutamic acid. MrIA and its deamidated form were much more dominant in the venom, with the next most intense mass precursor ion having an intensity of only approximately 4% the intensity of the deamidated form and 90% of the peptides with intensities of less than 1% of MrIA [
4]. Currently, it is unknown how, if at all, peptides expressed at such low levels affect venom lethality.
A number of the MrIA analogs identified contained either the entire MrIA pharmacophore [
4], or portions of it. However, the purpose of the analogs as venom components is unclear. Therefore, the aim of the present study was to investigate the activity of these MrIA analogs on hNET and ion channels that act as common conotoxin targets to gain a better understanding of their biological significance.