Abstract
Objective:
This study was devoted to identifying natural thrombin inhibitors from traditional Chinese medicine (TCM) and evaluating its biological activity in vitro and binding characteristics.
Methods:
A combination strategy containing molecular docking, thrombin inhibition assay, surface plasmon resonance (SPR) and molecular dynamics simulation were applied to verify the study result.
Results:
Gallic acid was confirmed as a direct thrombin inhibitor with IC50 of 9.07 μmol/L and showed a significant inhibitory effect on thrombin induced platelet aggregation. SPR-based binding studies demonstrated that gallic acid interacted with thrombin with a KD value of 8.29 μmol/L. Molecular dynamics and binding free energy analysis revealed that thrombin-gallic acid system attained equilibrium rapidly with very low fluctuations, the calculated binding free energies was -14.61 kcal/mol. Ala230, Glu232, Ser235, Gly258 and Gly260 were the main amino acid residues responsible for thrombin inhibition by gallic acid, providing a mechanistic basis for further optimization.
Conclusion:
This study proved that gallic acid is a direct thrombin inhibitor with platelet aggregation inhibitory effect, which could provide a basis for the follow-up research and development for novel thrombin inhibitors.
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