Salvianolic acid A

The traditional method for natural products discovering could be time-consuming and labor-intensive, thus ligand fishing has attracted a lot of attention as a fast-screening method. Nevertheless, ligand fishing often relied on immobilized enzyme with solid carriers, which can be expensive and complicated to prepare. In this study, a carrier-free enzyme immobilization technology, cross-linked enzyme aggregates (CLEAs), was introduced by preparing carbonic anhydrase cross-linked enzyme aggregates (CA-CLEAs), aiming to develop a method for ligand fishing that is simpler, faster and more cost-effective. CA-CLEAs were synthesized with optimal conditions, and were proven to have the ability to capture CA inhibitors and recycle. The crude extract of Salvia miltiorrhiza (SM) was determined to have inhibitory activity against CA, and two major components of SM, i.e., phenolic acids and diterpenoids, were prepared and incubated with CA-CLEAs, respectively. Seven potential inhibitors were fished out and identified using liquid chromatography-mass spectrometry (LC-MS), and all of them were verified to have inhibitory activity, with two novel inhibitors, salvianolic acid C and tanshinone IIA, being discovered. Molecular docking simulations revealed the different inhibitory mechanisms of phenolic acids and diterpenoids. Furthermore, combination effect analysis was carried out, and the synergistic effects were discovered between two components, as well as between their representative compounds, i.e., salvianolic acid B and tanshinone IIA. In this study, a ligand fishing method based on carrier-free immobilized enzyme was established and applied to discover CA inhibitors. A variety of inhibitors with different inhibitory mechanisms were identified successfully, which could accelerate the development of CA inhibitors, demonstrating the efficiency and potential of the method in new bioactive compounds discovery.