4.4. Molecular Docking

To calculate the binding affinity of the screened hits to JNK1, we performed a semi-flexible docking. The crystallographic structure of JNK1 was downloaded from Protein Data Bank (https://www.rcsb.org/, accessed on 18 September 2022) and preprocessed as follows: the missing atoms were inserted, the water and the co-crystallized ligand (PubChem CID: 25113171) were removed, the polar hydrogen atoms were added, and the Gasteiger charges were recalculated. The 3D structures of the small-molecule ligands were downloaded from PubChem (https://pubchem.ncbi.nlm.nih.gov/, accessed on 18 September 2022) and minimized with the MM2 force field. The grid box was generated with the co-crystallized ligand as the center, and the parameters were set as follows: center_x = 22.78, center_y = 38.34, center_z = 31.17, size_x = size_y = size_z = 12.5, and exhaustiveness = 30. To verify the feasibility of the docking process, the extracted co-crystallized ligand was re-docked into the target protein and the root-mean-square deviation (RMSD) was calculated. All candidate molecules were subjected to molecular docking studies by AutoDock Vina software [29]. After docking was completed, the protein–ligand complexes were analyzed for interactions using LigPlot⁺ software [30].