AI-Driven Discovery of an Allosteric Thrombin-Targeting Anticoagulant Peptide from Traditional Chinese Medicine Dilong (Pheretima aspergillum)

Ethnopharmacological relevanceDilong, the dried body of Pheretima aspergillum, is a classic traditional Chinese medicine that has been clinically used for millennia to promote blood circulation and resolve blood stasis. Modern pharmacological studies have validated its marked oral antithrombotic activity, yet its bioactive component has not been identified.Aim of the studyThis study aimed to establish an artificial intelligence (AI)-driven screening strategy to efficiently pinpoint the primary bioactive antithrombotic component from Dilong, and to elucidate the molecular mechanism of its anticoagulant action.Materials and methodsA machine learning model was trained using a dataset of well-characterized anticoagulant peptides, and was subsequently applied to screen the peptide library derived from the active fraction of Dilong, for rapid identification of potential bioactive candidates. The in vitro and in vivo anticoagulant and antithrombotic activities of the screened candidate were systematically evaluated. Furthermore, circular dichroism (CD), fluorescence spectroscopy, and microscale thermophoresis (MST) were employed to explore its underlying pharmacological mechanism.ResultsThe anticoagulant peptide LMWFSDRNK (LK9) from Dilong exhibited significant anticoagulant and antithrombotic activities both in vitro and in vivo. Further mechanistic investigations revealed that LK9 allosterically regulates thrombin which enhanced its binding to antithrombin, with an EC50 value of 66.88 μM, thereby exerting its anticoagulant effect.ConclusionsThis study provides robust scientific evidence to support the clinical application of Dilong in the management of thrombotic diseases. Meanwhile, the identified LK9 as a promising lead compound for the development of novel anticoagulant drugs, also offering a reference paradigm for efficient discovery of bioactive components from traditional Chinese medicines.