BChE Inhibitors(University of Ljubljana)

Selective butyrylcholinesterase (BChE) inhibition is gaining renewed attention as a potential therapeutic strategy for Alzheimer's disease (AD), particularly in advanced stages marked by a shift from acetylcholinesterase (AChE) to BChE dominance. Beyond cholinergic regulation, BChE participates in metabolic, inflammatory, and affective pathways, including the enzymatic control of acyl ghrelin that influences appetite, energy balance, and mood. Preclinical and experimental evidence suggests that selective BChE inhibition may modulate cholinergic tone, enhance cognition, and exert antidepressant- and anti-anhedonic-like effects, although clinical evidence remains limited and inconclusive. Genetic polymorphisms in BChE further shape disease progression and responses to cholinesterase therapy. This review integrates advances in BChE inhibitor development with evolving insights into BChE-dependent metabolic and neuropsychiatric mechanisms, highlighting selective BChE inhibition as a multifaceted therapeutic approach in AD.

The development of selective butyrylcholinesterase (BChE) inhibitors from natural sources represents a strategic frontier in discovering novel therapeutics for Alzheimer's disease (AD). Employing a rigorous hierarchical virtual screening protocol encompassing high-throughput screening, ensemble molecular docking, and MM/GBSA binding affinity refinement, we systematically interrogated a natural product library containing 60,580 phytochemicals. This computational cascade identified three alkaloid candidates (hirsutine, picrasidine I, and picrasidine T) exhibiting potent BChE inhibition (IC₅₀ < 10 μM) validated through enzymatic assays. Detailed kinetic characterization further established hirsutine and picrasidine I as reversible, mixed-type inhibitors, targeting both the catalytic active site and the peripheral aromatic site (PAS) of BChE. To elucidate the mechanism of interaction, picrasidine I was selected for comprehensive spectroscopic analysis, including molecular fluorescence, time-resolved fluorescence, three-dimensional fluorescence, circular dichroism (CD), and isothermal titration calorimetry (ITC). ITC analysis confirmed a strong affinity between picrasidine I and BChE, yielding a dissociation constant (Kd = 2.97 μM), while fluorescence studies indicated a static quenching mechanism. Molecular dynamics (MD) simulations (50 ns) substantiated the stability of the ligand-protein complex, demonstrated by a backbone RMSD <0.3 nm. Furthermore, ADME predictions suggested favorable blood-brain barrier (BBB) permeability and minimal cytotoxicity in PC-12 neurons. This synergistic computational-experimental paradigm not only successfully identifies new chemotypes as potent BChE inhibitors and unveils their precise molecular mechanism of action, but also provides a practical and highly effective strategy for the rational discovery and development of natural anti-AD agents.