Structurally Minimized μ‐Conotoxin Analogues as Sodium Channel Blockers: Implications for Designing Conopeptide‐Based Therapeutics

Abstract

Disulfide bridges, which stabilize the native conformation of conotoxins impose a challenge in the synthesis of smaller analogs. In this work, we describe the synthesis of a minimized analog of the analgesic μ-conotoxin KIIIA that blocks two sodium channel subtypes, the neuronal NaV1.2 and skeletal muscle NaV1.4. Three disulfide-deficient analogs of KIIIA were initially synthesized in which the native disulfide bridge formed between either C1-C9, C2-C15 or C4-C16 was removed. Deletion of the first bridge only slightly affected the peptide’s bioactivity. To further minimize this analog, the N-terminal residue was removed and two non-essential Ser residues were replaced by a single 5-amino-3-oxapentanoic acid residue. The resulting “polytide” analog retained the ability to block sodium channels and to produce analgesia. Until now, the peptidomimetic approach applied to conotoxins has progressed only modestly at best; thus, the disulfide-deficient analogs containing backbone spacers provide an alternative advance toward the development of conopeptide-based therapeutics.

Interactions of Key Charged Residues Contributing to Selective Block of Neuronal Sodium Channels by μ-Conotoxin KIIIA

“…However, removal of only one of the three disulfide linkages yielded a lower k on the C2-C15 linkage (Han et al, 2009;Khoo et al, 2009). This decrease in k on fits with the KIIIA-wt pH results, the concomitant 10-fold increase in k off , together with the rapid reversibility of our observed pH effects, makes disulfide bond disruption an unlikely rationale for our observations.…”

Design of Bioactive Peptides from Naturally Occurring μ-Conotoxin Structures

“…They illustrated that the first disulfide bridge between Cys 1 and Cys 9 in KIIIA is removable, almost without affecting the original activity of the peptide on Na v 1.2 and Na v 1.4 (34). Ultimately, the first disulfide bridge was excluded in our chimeric peptide.…”

“…We integrated recent results by Han et al (34) into our design strategy. They illustrated that the first disulfide bridge between Cys 1 and Cys 9 in KIIIA is removable, almost without affecting the original activity of the peptide on Na v 1.2 and Na v 1.4 (34).…”

“…We integrated this in our design strategy, resulting in the two analogues Mini-R5A and Mini-R5E. A third peptide was based on results of Han et al (34). They established that the adjacent Ser 5 and Ser 6 in KIIIA could be replaced by a PEG backbone without severely affecting the block on Na v 1.2 (Table 2).…”

Early Engineering Approaches to Improve Peptide Developability and Manufacturability

“…From a manufacturing perspective, exploring the removal of disulfide bonds to reduce the complexity of the peptide can be initiated early in a molecule's lifetime using standard biophysical techniques, such as nuclear magnetic resonance in combination with a functional assay, to evaluate the impact of these changes. The utility of disulfide bond deletion was demonstrated with μ-conotoxin KIIIA, a disulfide-rich venom peptide isolated from Conus kinoshitai that blocks mammalian neuronal voltage-gated sodium channels and is a potent analgesic (43). The authors systematically removed cysteine pairs, by making simple cysteine to alanine mutations, and screened for functional activity by testing the disulfide-deficient analogues against mammalian Na v 1.2 and Na v 1.4.…”