Synthesis of full-length homodimer αD-VxXXB that targets human α7 nicotinic acetylcholine receptors

Abstract

αD-Conotoxin VxXXB is a pseudo-homodimer that allosterically inhibits nicotinic acetylcholine receptors (nAChRs) with high potency and selectivity. However, challenges in synthesizing αD-conotoxins have hindered further structure–function studies on this novel class of peptides. To address this gap, we synthesized and characterized its C-terminal domain (CTD) and N-terminal domain (NTD). The CTD inhibited α7 nAChRs (IC₅₀ of 23 nM, measured via FLIPR assays) and bound at the acetylcholine binding protein (Ls-AChBP) through an allosteric binding mode determined from radioligand binding assays. The anti-parallel dimeric NTD synthesised via a regioselective strategy also inhibited α7 nAChRs but with reduced potency (IC₅₀ of 30 μM). The α-ketoacid-hydroxylamine (KAHA) method generated CTD linked to the NTD (VxXXB-NC; α7 IC₅₀ of 27 nM) and full-length synthetic VxXXB variant (α7 IC₅₀ of 11 nM), while the three other native chemical ligation approaches proved unsuccessful. This work underpins further characterisation of the structural components contributing to αD-conotoxin affinity, selectivity and allosteric inhibition of nAChR function that may prove useful in the development of new treatments for nAChR-related disorders.