Histidine Focused Covalent Inhibitors Targeting Acetylcholinesterase: A Computational Pipeline for Multisite Therapeutic Discovery in Alzheimer’s Disease

Alzheimer’s disease affects over 10% of individuals above the age of 65, yet current treatments offer only limited and temporary relief. Acetylcholinesterase, a key enzyme in neurotransmitter breakdown, also contributes to disease progression by promoting β-amyloid aggregation. While previous studies have focused on the catalytic serine, a key proton transfer residue, His447 remains unexplored as a potential covalent binding site. In this study, we aim to interrupt the activation of Ser203 by covalently modifying His447, thereby shutting down the entire catalytic process. Here, we reported a computational pipeline to identify epoxide-based small molecules that covalently engage His447 and modulate AChE activity. From a curated library of >7,000 epoxides, three ligands (L5, L6, L7) were selected via covalent docking, molecular dynamics simulations, and drug-likeness profiling. Microsecond-scale simulations revealed stable binding across multiple subsites, with L5 exhibiting the most consistent RMSD and compact Rg values. Covalent engagement of L5 and L6 induced modest shifts in His447 (2.48 and 1.43 Å), whereas L7 maintained apo-like geometry. Furthermore, ADMET predictions indicated favorable profiles, with no cardiotoxicity risk. Our findings highlight His447 as a novel covalent target in AChE and support further in vivo investigation of the specificity and inhibitory mechanisms of these ligands.