Rational Design of α-Conotoxin RegIIA Analogues Specifically Inhibiting the Human α3β2 Nicotinic Acetylcholine Receptor through Computational Scanning.

Engineering the selectivity of α-conotoxins for nicotinic acetylcholine receptors (nAChRs) presents considerable complexity and challenges, as it involves the optimization of their binding affinities to multiple highly conserved nAChR subtypes. Here we applied computational based scanning approach for rational design of α-conotoxin RegIIA analogues specifically targeting the human (h) α3β2 versus hα3β4 nAChRs. Binding mode analysis suggested that several residues in loop II of RegIIA (position 9, 10 and 11) formed non-conserved interactions with residues of the β2 and β4 subunits. The molecular mechanics Generalized Born surface area (MMGB-SA) method was applied for in silico sequence scanning of RegIIA position 9, 10 and 11 on frames extracted from single molecular dynamics simulation trajectory. RegIIA analogues with favourable predicted binding affinity solely to the hα3β2 nAChR were synthesized and tested electrophysiologically. We report three RegIIA analogues, with position 9 aromatic residue substitutions, exhibiting 10- to 37-fold subtype selectivity improvement for hα3β2 compared to hα3β4 nAChR. The in silico scanning method proposed from this study has considerable potential for efficient design of nAChR subtype selective antagonists in the future.