2.10. In Silico Assay

The metabolites identified in EHGV were schematically designed in 3D models on GaussView 5.0.8 [31] and had their geometric, electronic and vibrational properties optimized with Gaussian 09 [32] using the density functional theory (DFT) method, combining the hybrid functional B3LYP and the basis set 6-31 ++ G (d, p).

All MD protocol utilized Autodock Vina [33]. The structure of the human cyclo-oxygenase 2 (COX-2) (PDB ID 5F19) and nuclear factor kappa-B (NF-κB) NEMO/IKKβ association (PDB ID 3BRV) and ligands were prepared for MD with AutoDock Tools, version 1.5.7 [34]. Docking methodology described in literature were used [35] with some modifications [36,37]. Gasteiger partial charges were calculated after addition of all hydrogens both in ligands, COX-2 and NEMO/IKKβ association structures. Non-polar hydrogens from COX-2, NEMO/IKK IKKβ and EHGV metabolites were subsequently merged. The dimensions of cubic box in the x-, y- and z-axes were 30 × 30 × 30. Grid box was centered on oxygen atom from residues Arg120 from COX-2 and Glu89 of NEMO domain. In addition to visual inspection, the initial coordinates of interaction complexes were chosen based on the criterion of better docking conformation of the lowest energy score.