Molecular Dynamics and Free Energy Perturbation Analysis of Guava (Psidium guajava) Leaf-Derived Inhibitors Against Coagulation Factor IXa

There is an ambiguity whether guava (Psidium guajava) leaves support or inhibits coagulation as it was commonly used as wound dressing. Our previous study extracted, identified, and screened using docking, bioactive compounds from guava leaves that has the potential to be coagulation factor IXa inhibitor. Docking has static limitations that require further evaluation [1]. To investigate the potential of bioactive compounds as inhibitors of coagulation factor IXa (CFIXa) using molecular dynamics (MD) simulations and binding free energy perturbation analyses.Top three docking-scored CFIXa-ligand complexes from our previous study were evaluated using MD simulations with GROMACS 2024 and CHARMM force fields. Simulations included minimization, NVT, NPT with and without restraints, followed by a 10 ns production run. Structural analysis was performed using RMSD, RMSF, SASA, and secondary structure analysis in UCSF ChimeraX. Binding free energies (ΔGbind) were calculated using gmx_mmpbsa with PB and GB models, and decomposition analysis identified key residues contributing to binding. Compound 7 showed the strongest binding with CFIXa, with a binding free energy ranging from -10 to -20 kcal/mol and stable interactions, particularly at H:THR245, while compound 2 demonstrated moderate binding with a binding free energy of -5 to -15 kcal/mol. Compound 3 exhibited instability, with RMSD fluctuations from 0.06 to 7.71 nm, and binding free energy approaching 0 kcal/mol, confirming its unbinding during the simulation. Compound 7 demonstrated the strongest binding interactions and stability, making it the most promising candidate for further development, while compound 2 showed moderate binding and potential for optimization through structural refinements. In contrast, compound 3 exhibited instability and weak binding, suggesting that modifications could improve its binding efficiency and specificity.