A Dual Approach To Combat Alzheimer's Disease through Concomitant hBChE Inhibition and S1R Activation.

Alzheimer's disease (AD) remains an incurable neurodegenerative disorder, requiring novel therapeutic strategies. We developed multitarget-directed ligands designed to inhibit human butyrylcholinesterase (hBChE) and activate the sigma-1 receptor (S1R), addressing both cholinergic dysfunction and neuroinflammation, the latter being reduced through action on both targets. The (pseudo-)irreversible carbamate inhibitor 18c emerged as the most promising compound, exhibiting potent and selective hBChE inhibition (IC50 = 3.3 nM, 45-fold selectivity over human acetylcholinesterase) and strong S1R agonistic activity (IC50 = 25 nM, EC50 = 99 nM) determined in a radioligand binding assay and by S1R-BiP dissociation, respectively. Its cleavage product 14c (after carbamate hydrolysis by hBChE) retained dual activity (IC50(hBChE) = 269 nM, IC50(S1R) = 20 nM, and EC50(S1R) = 279 nM). Both compounds reduced the lipopolysaccharide-induced pro-inflammatory activation profile in microglial N9 cells while preserving anti-inflammatory marker expression, thereby indicating an overall immunomodulatory effect. In vivo, inhibitor 18c improved cognitive deficits in a mouse model with Aβ25-35-induced neurotoxicity, enhancing short- and long-term memory in Y-maze and passive avoidance tests at dosages as low as 0.1-1 mg/kg. These findings highlight the potential of dual-targeting hBChE/S1R strategies for AD therapy.