Possible Thrombus-clearing Mechanism and Modification Suggestion of β-sitosterol.

INTRODUCTION

Thrombin (THR) is a key therapeutic target for anticoagulant therapy, yet the mechanism of β-sitosterol, a natural compound with antithrombotic potential, remains unclear.

METHODS

This study integrated AI-driven structural alignment, molecular docking, Molecular Dynamics (MD) simulations, binding free energy calculation, and Density Functional Theory (DFT) calculations to elucidate the recognition mechanism between THR and β-sitosterol.

RESULTS

Simulations revealed that β-sitosterol binding is stabilized primarily by hydrophobic and van der Waals interactions, leading to the closure of the active site and conformational changes in the EF_loop (i.e., γ-loop). The large conformational changes within EF_Loop may be dominated by weak interactions between W168/ P184/ Q183/ S185 and the ligand β-sitosterol. Based on these insights, a series of novel sterol derivatives was designed with improved binding affinity and predicted antithrombotic activity, as indicated by the lowest binding free energy.

DISCUSSION: This study not only reveals molecular recognition and inhibitory mechanism of β- sitosterol at the atomic level, but also provides suggestions for structural optimization of novel inhibitors against human thrombin. Future work should include in vitro binding assays and in vivo functional studies to confirm the inhibitory activity.

CONCLUSION

The conformational change of EF_loop with the recognition of β-sitosterol effectively occludes the catalytic site, thereby impairing thrombin's proteolytic activity. Among 13 designed sterol derivatives, the compound d3 was identified as a promising inhibitor with excellent ADMET properties. This work provides an anticoagulant mechanism for the dynamic identification of β- sitosterol and supports the rational design of allosteric THR inhibitors.