A novel strategy for screening angiotensin-converting enzyme inhibitors from natural products based on enzyme-immobilized ligand fishing combined with active-site blocking and directional enrichment.

Some natural products are important sources of treatments for hypertension based on their potential inhibitory effects on angiotensin-converting enzyme (ACE); however, it is difficult to identify natural ACE inhibitors (ACEIs) due to the complex secondary metabolite environment of natural products. Enzyme immobilization is an important method for screening active constituents in natural products, but this method can sometimes return false-positive and false-negative results. To improve the accuracy and reliability of ligand-fishing methods, we established a novel strategy based on enzyme-immobilized ligand fishing combined with active-site blocking and directional enrichment technologies. We first synthesized ACE-immobilized mesoporous magnetic beads and then verified the screened compounds by molecular docking and in vitro activity detection. We then used active-site blocking to exclude non-specific binding constituents and applied directional enrichment to enrich the low-content constituents for ligand fishing. The screening identified six potential ACEIs from Scutellariae Radix and eight potential ACEIs from Lonicerae japonicae flos, and their inhibitory activity was confirmed by molecular docking simulations and in vitro activity detection. This process screened six additional compounds and excluded two false-positive results as compared with results exclusively using enzyme immobilization. This strategy provides a feasible method for screening active compounds in natural products.