Li_2025_Bioorg.Chem_156_108183

Current immobilization approaches for ligand fishing often experience challenges such as limited protein loading capacity and difficulties in the recycling process. To overcome these challenges, we synthesized a magnetic metal-organic frameworks (MMOFs) composite, which can be rapidly separated and has a large specific surface area, and employed it to immobilize acetylcholinesterase (AChE). The synthesized MMOFs@AChE composite exhibited a high immobilization yield (129.7 mg/g) and excellent relative activity recovery (88.1 %). Furthermore, immobilized AChE can improve its resistance to alkaline environments and high temperatures. After being stored at 4 degreesC for a month, the immobilized enzyme maintained 91.4 % of its original activity, significantly higher than the free enzyme (77.6 %). Furthermore, it preserved more than 80 % of its initial activity after five cycles and 68.7 % after eight cycles. The composite MMOFs@AChE was then incubated with Qi-Fu-Yin extract to fish for ligands binding to AChE. Notably, Qi-Fu-Yin can alleviate Alzheimer's disease (AD) symptoms by modulating the AChE pathway, while active compounds remain unclear. Sixteen potential AChE inhibitors were identified through UHPLC-Q-Exactive-Orbitrap-MS/MS. The results of ligand fishing were validated through molecular docking studies, molecular dynamics simulation, surface plasmon resonance and AChE inhibitory activity assays. The screened compounds may exert inhibitory effects on AChE by altering the spatial configuration of the catalytic site or by influencing the binding of the substrate to the catalytic site, catalytic anionic site and peripheral anionic site regions. The MMOFs@AChE-based ligand fishing platform offers an efficient, effective, and convenient approach for enzymatic inhibitors discovery from natural products.