The immobilization of neuraminidase onto MOF-808 by in-situ precipitation for targeted screening of antiviral components from traditional Chinese medicine

Influenza poses a severe threat to global public health, with viral neuraminidase (NA) serving as a key target for antiviral drug development. However, the poor stability of free NA and low targeting efficiency of conventional screening approaches render the screening of NA inhibitors from complex traditional Chinese medicine (TCM) matrices a major unresolved challenge. Herein, a cross-linker-free in situ precipitation strategy was developed to immobilize NA on MOF-808 via ammonium sulfate induction, leveraging the synergistic effect of electrostatic and hydrophilic interactions to achieve an enzyme capacity of 19.1 mg·g −1 . Notably, the moderate agglomeration of MOF-808 particles forms a unique “protective cage” structure, which can effectively preserve the native conformation of NA and facilitate the local enrichment of substrates at the enzyme active sites. MOF-808@NA was further employed as an affinity sorbent for ligand fishing from the extract of Mahuang-Xixin-Fuzi Decoction (MXF), a clinically used TCM formula, and 12 potential NA inhibitors were successfully captured and identified via HPLC-Q-TOF-MS/MS. The activity evaluation results demonstrated that, compared with the crude MXF extract, the MOF-808@NA-enriched fraction exhibited a 26-fold enhancement in NA inhibitory potency with an IC₅₀ of 0.18 mg·mL −1 , as well as a 2.4-fold improvement in cellular antiviral efficacy against influenza A/H1N1 with an EC₅₀ of 0.70 mg·mL −1 . This work verifies that the developed cross-linker-free in situ precipitation strategy enables the efficient and targeted screening of bioactive components from complex TCM matrices, thereby providing a highly promising technical tool for the targeted discovery of novel antiviral drugs derived from natural products.