Hierarchical enrichment driven ultrafiltration for rapid identification of 3-chymotrypsin-like protease inhibitors from Forsythia suspensa (Thunb.) Vahl

The discovery of high-quality natural product-based enzyme inhibitors remains a major challenge due to the chemical complexity of herbal extracts. Although affinity-based ligand fishing and ultrafiltration are rapid methods for screening active compounds, non-specific interactions often interfere with the binding of high-quality active compounds to target enzymes, resulting in the fished components with weak or no biological activity. To overcome this limitation, we developed a novel hierarchical enrichment-driven ultrafiltration strategy by integrating affinity ultrafiltration (AUF) with solid-phase extraction (SPE). A case study was conducted on Forsythia suspensa (Thunb.) Vahl to screen for 3-chymotrypsin-like protease (3CL^pro) inhibitors. Initially, the crude extract was fractionated into three polarity-based portions using SPE to reduce chemical complexity and enrich bioactive constituents. Enzyme activity assays guided the selection of the most active fraction. Subsequently, an optimized AUF approach (pH 6.0, 30 °C, 20 min) coupled with liquid chromatography-mass spectrometry was employed to screen potential bioactive compounds from the selected 50 % methanol polar fraction. Three 3CL^pro inhibitors were successfully identified. Notably, IFT was reported for the first time as a 3CL^pro inhibitor, exhibiting significant bioactivity, with an IC₅₀ of 10.86 ± 2.39 μM against 3CL^pro and an EC₅₀ of 11.12 ± 1.05 μM (SI = 6.91) against SARS-CoV-2. These findings highlight its potential as a promising lead compound for antiviral drug development. In summary, this study provides a simple and effective strategy for the rapid discovery of high-quality active natural products.