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

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 (IC 50 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 (IC 50 of 30 μM). The α-ketoacid-hydroxylamine (KAHA) method generated CTD linked to the NTD (VxXXB-NC; α7 IC 50 of 27 nM) and full-length synthetic VxXXB variant (α7 IC 50 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.