Affinity ultrafiltration and UPLC-HR-Orbitrap-MS based screening of thrombin-targeted small molecules with...

This study aims to screen potential anticoagulant components from leeches, a representative animal-sourced traditional Chinese medicine using thrombin (THR)-targeted ultrafiltration combined with ultrahigh performance liquid chromatography and high-resolution Orbitrap mass spectrometry (UPLC-HR-Orbitrap-MS). As a result, five small molecules in leech extract were discovered to interact with THR for the first time. Among them, two new compounds were isolated and their structures were identified by IR, HR-MS and NMR data. Furthermore, their THR inhibitory activity was confirmed with IC₅₀ values of 4.74 and 8.31 μM, respectively. In addition, molecular docking analysis showed that the active (catalytic) site of THR might be the possible binding site of the two hits. Finally, reverse screening analysis indicated that LTA4-H, ACE and ALOX5AP were potential anticoagulant targets of the two new compounds. This study will broaden our understanding of the medicinal substance basis in leeches and further contribute to the discovery and development of clinical anticoagulant drugs from leeches.