Multidimensional assessment of natural ligands fisetin, morin and rutin against factor XIIa activity using in vitro and in silico studies

The discovery of selective inhibitors of factor XIIa (FXIIa) is an attractive approach for development of new antithrombotics that do not interfere with normal hemostasis. Here we report an in vitro chromogenic assay and in silico molecular modeling-based integrated protocol for predicting the inhibitory aptitude of natural flavonoids, Fisetin, Morin, Rutin and the synthetic derivative (FXIIa-IN-4) of the FXIIa enzyme. The frontier molecular orbitals (FMO) analysis, accompanied by the electrostatic potential (ESP) maps and a non-covalent interaction (NCI) map, provided the information about electronic reactivity of the title compounds. Chromogenic assays data of assessed flavonoids, Fisetin, Morin and Rutin have been shown a dose-dependent inhibition of FXIIa activity, in which at higher concentration (1000 µM) they exhibited about 52.6%, 57.1% and 71.9% inhibition, respectively. Molecular docking studies showed that Rutin has the lowest binding energy with FXIIa (8.6 kcal/mol) and Fisetin has with optimal balance between affinity, structural compactness and reactivity. In addition, molecular dynamics enables comparison of the stability and flexibility of the various ligand-protein complexes. Fisetin is the ligand that provides best structural stability, and Rutin causes a greater conformational variation, but with more hydrogen bond interactions. A detailed absorption, distribution, metabolism, and excretion (ADMET) analysis revealed that FXIIa-IN-4 has the best ADMET profile, while Fisetin is the second. Morin and Rutin, on the other hand, were found to have less clear toxic effects. Taken together, although the in vitro chromogenic assessment results demonstrated that Rutin has superiority in inhibition of FXIIa enzyme activity, the overall obtained data indicated the importance of Fisetin among all tested flavonoid compounds as an equilibrative natural inhibitor demanding in vivo experimental confirmation and focused molecular modification.