Histidine Focused Covalent Inhibitors Targeting Acetylcholinesterase: A Computational Pipeline for Multisite...

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 R_g 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.