Integrating high-resolution bioassay profiling with affinity-based ligand fishing for unveiling galloylated derivatives as novel catechol-O-methyltransferase inhibitors in Paeonia lactiflora Pall.

Catechol-O-methyltransferase (COMT) inhibition is a critical therapeutic strategy for Parkinson's disease (PD), yet clinical inhibitors face limitations in bioavailability and hepatotoxicity, driving demand for novel natural scaffolds. In this study, we developed an integrated analytical platform by coupling high-resolution bioassay profiling (HRBP) and affinity-based ligand fishing system to effectively characterize bioactive compounds targeting COMT in Paeonia lactiflora Pall. (the most frequently used core herb in tradition Chinese medicine prescriptions for PD treatment). Parallel high-performance liquid chromatography with diode-array detection and tandem mass spectrometry (HPLC-DAD-MS/MS) coupled with nanofractionation enabled real-time bioactivity mapping via 384-well COMT inhibition assays, while semi-preparative liquid chromatography (LC) was employed to further identify co-eluted components. HRBP and immobilized COMT ligand fishing identified 16 and 21 candidates, respectively, with 5 overlapping bioactive markers. Notably, the potent inhibitors galloylpaeoniflorin (half maximal inhibitory concentration (IC50) = 16.2 ± 3.4 μM) and 1,2,3,4,6-O-pentagalloylglucose (IC50 = 3.1 ± 0.5 μM) exhibited comparable potency to the positive control morin (IC50 = 10.1 ± 0.7 μM). Molecular docking results further revealed the critical interactions and binding sites between the active compounds and COMT. The validated platform demonstrates significant potential for rapid discovery of plant-derived enzyme inhibitors, bridging advanced separation, bioactivity screening, and mechanistic validation in neurodegenerative therapeutic development.