Magnetic beads-based neuraminidase enzyme microreactor as a drug discovery tool for screening inhibitors from compound libraries and fishing ligands from natural products

•Neuraminidase functionalized-magnetic beads (NA-MBs) were prepared for the first time.•A screening assay for pure compound libraries was established using NA-MBs coupled with LC–MS/MS.•NA-MBs were successfully applied for the NA ligand fishing from Flos Lonicerae.•The NA-MBs based assay offers a novel anti-flu drug discovery tool. Neuraminidase (NA) is a glycoside hydrolase that has been proposed as a potential therapeutic target for influenza. Thus, the identification of compounds that modulate NA activity could be of great therapeutic importance. The aim of this study is to develop a drug discovery tool for the identification of novel modulators of NA from both compound libraries and natural plant extracts. NA was immobilized onto the surface of magnetic beads and the inherent catalytic activity of NA-functionalized magnetic beads was characterized. Based on the enzymatic activity (hydrolysis ratio), the inhibitory activities of 12 compounds from plant secondary metabolites were screened, and the desired anti-NA activities of flavonoids were certified. Ligand fishing with the immobilized enzyme was optimized using an artificial test mixture consisting of oseltamivir, lycorine and matrine prior to carrying out the proof-of-concept experiment with the crude extract of Flos Lonicerae. The combination of ligand fishing and HPLC-MS/MS identified luteolin-7-O-β-D-glucoside, luteolin, 3,5-di-O-caffeoylquinic acid and 3,4-di-O-caffeoylquinic acid as neuraminidase inhibitory ligands in Flos Lonicerae. This is the first report on the use of neuraminidase functionalized magnetic beads for the identification of active ligands from a botanical matrix, and it sets the basis for the de novo identification of NA modulators from complex biological mixtures.