Functional and structural interactions of novel acrylamide-based compounds with the α7 nicotinic acetylcholine receptor

The primary objective of this study was to structurally and functionally characterize a series of novel acrylamide-based compounds at the rat α7 nicotinic acetylcholine receptor (nAChR). Electrophysiological analyses revealed that DM497 [(E)-3-(thiophen-2-yl)- N -(p-tolyl)acrylamide] exhibited dual activity: at low acetylcholine (ACh) concentrations, it potentiated the α7 nAChR by delaying desensitization, whereas at high concentrations, it inhibited receptor activity. In contrast, ERR91 [(E)-3-(benzofuran-2-yl)- N -(p-tolyl)acrylamide] displayed only potentiating effects. Several compounds exhibited purely inhibitory effects, with DM490 [(E)-3-(furan-2-yl)- N -methyl- N -(p-tolyl)acrylamide] and ERR6 [(E)-3-(furan-2-yl)-1-(isoindolin-2-yl)prop-2-en-1-one] identified as the most potent negative allosteric modulators (NAMs). The activity of both NAMs (e.g. DM490) and positive allosteric modulators (PAMs) (e.g. DM497) was voltage-independent, suggesting that these compounds do not interact directly with the ion channel pore. Notably, at higher agonist concentrations, DM497′s PAM activity transitioned to NAM activity, while DM490′s NAM activity was further enhanced, an effect inconsistent with steric interactions at the orthosteric site. Molecular docking simulations excluded luminal interactions but identified two distinct intersubunit binding sites for DM497 and PAM-4 [(E)-3-furan-2-yl- N -phenylacrylamide], corresponding to PAM activity. In contrast, two non-overlapping intrasubunit sites were identified for DM490, likely contributing to its NAM activity. Additionally, a unique intersubunit site for DM497 was observed, which may explain its NAM activity at high ACh concentrations. The distinct binding locations of DM497 and DM490 align with functional competition assays, indicating that these compounds bind to different sites. Together, these findings suggest that the determination of PAM versus NAM activity may depend on selective interactions with intersubunit versus intrasubunit binding sites.