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

Thrombotic disorders, characterized by abnormal blood clot formation, significantly contribute to global cardiovascular mortality. Targeting CFIXa offers a promising therapeutic strategy, balancing anticoagulation efficacy with reduced bleeding risk. This study investigates guava (Psidium guajava) leaf-derived bioactive compounds: 4-Androstene-3.α.,17.β.-diol (Compound 2), Methenolone acetate (Compound 3), and Amitriptyline-M-(CH₃)₂NOH AC (Compound 7) as potential CFIXa inhibitors through MDS, binding free energy calculations, and cluster network analysis. Compound 7 demonstrated superior stability, with the lowest RMSD (0.347 ± 0.110 nm) and strongest binding affinity (−15.475 ± 3.173 kcal/mol), driven by key interactions with residues H:ASN97 [264] (2.9–3.1 Å H-bond), H:TYR99 [266] (4.7–5.2 Å π-alkyl), and H:PHE174 [342] (4.8–5.3 Å π-alkyl) in the S2/S4 pockets. Cluster analysis revealed 40 conformational states, with Cluster 1 (15.8% occupancy) dominating due to these stable interactions. Compound 2 showed moderate stability (RMSD: 0.459 ± 0.307 nm; ΔG: −10.132 ± 4.414 kcal/mol), anchored by hydrophobic contacts with H:LYS239 [409] (4.40–5.17 Å) and L:PHE98 [98] (2.62 Å H-bond), but exhibited dynamic transitions between 49 clusters. Compound 3 was the least stable (RMSD: 0.965 ± 0.382 nm; ΔG: −9.541 ± 4.115 kcal/mol), with 74 clusters and frequent unbinding events (e.g., Cluster 24, 100% dissociation). Transition networks highlighted Compound 7’s kinetic robustness, with >97% of transitions occurring between stable, ligand-bound clusters (e.g., Cluster 1 ↔ Cluster 9). In contrast, Compound 3’s Markov model showed 96.3% self-transitions in unbound clusters, indicating poor residence time and kinetic trapping. Energy decomposition identified H:TYR99 [266] and H:PHE174 [342] as universal hotspots, contributing −0.51 ± 0.09 kcal/mol per residue in Compound 7 but only −0.20 ± 0.09 kcal/mol in Compound 2. These findings resolve the ambiguity surrounding guava leaves’ anticoagulant vs. procoagulant effects, confirming their CFIXa-inhibitory potential. Compound 7 emerges as a promising scaffold for rational drug design, leveraging conserved interactions with the S2/S4 subsites. This study provides a computational foundation for developing guava-derived antithrombotics, bridging structural insights with therapeutic optimization for safer anticoagulation strategies.