Computational design and evaluation of Factor Xa inhibitory peptides derived from the Tamarind Kunitz inhibitor reactive loop.

Blood coagulation is a crucial process, and its dysregulation can lead to life-threatening conditions. Factor Xa (FXA) is a well-established therapeutic target for the management of vascular diseases, as it acts as a converging point of both intrinsic and extrinsic coagulation pathways. Currently available FXA inhibitors display major side effects and toxicity, necessitating novel inhibitors. We previously reported that the inhibition of FXA from the natural source by the Tamarind Kunitz Inhibitor (TKI), a Kunitz-type protease inhibitor from Tamarindus indica seeds, inhibits FXA through its seven-residue reactive loop. In this study, in-silico mutagenesis of the TKI loop (SRARISH) was performed to design improved peptide inhibitors. A peptide library was constructed and screened using molecular docking, and the top mutants were selected and subjected to MD simulations. The binding free energies calculations, principal component analysis and free energy landscape analysis were also performed to elucidate the conformational dynamics. Selected peptides showed significantly improved binding affinities over the wild-type, with total binding free energies ranging from -16.75 to -10.59 kcal/mol compared to -6.83 kcal/mol for the wild-type. The Mutant 2 (SAWWISH) emerged as the best predicted high-affinity binder with -16.75 kcal/mol binding free. The improvement results from the elimination of the electrostatic desolvation penalty and the formation of an extended aromatic scaffold that effectively interacts with the hydrophobic sub-pockets of the FXA active site. These findings provide a strong computational foundation for the utilization of TKI-derived peptide inhibitors as natural anticoagulants against vascular diseases.