3.4. Molecular Docking

Molecular docking served as a helpful tool to obtain the reasonable binding conformations of bioactive molecules and to identify core residues in the active site of target protein. The crystal structure of bovine XO protein (PDB ID: 1N5X), a very close homologue of human XO enzyme, was used for molecular docking by the surflex-docking package of SYBYL-X 2.1 with default parameters [1]. The sequence alignment of bovine (Bos taurus) and human (Homo sapiens) XO with approximately 90% sequence identity was shown in Figure S6, and particularly in the febuxostat binding site, the key amino acids were the same, which was consistent with the reported literatures [1,20]. Before docking, an online web service (http://www.mrc-lmb.cam.ac.uk/pca/ (accessed on September 2020)) was used to explore the non-covalent contacts between ligand and protein [25]. After the pretreatment steps of the original protein, including hydrogenating, adding electric charges, extracting the crystallographic ligands, and removing water and other unnecessary atoms, the applicable docking pocket was generated with a threshold of 5 Å by a “ligand” mode. The crystallographic ligand was first redocked into the pocket to examine the dependability of the docking method. The conformation differences between the redocked and original ligands were evaluated by the RMSD values [17]. The RMSD < 2.0 Å is considered as a reference criterion, indicating that the docking method is reasonable. The same docking method was then applied on 46 ODC XOIs (Table 1), and the appropriate docking conformations with different docking scores were then obtained [42]. Finally, the conformations of febuxostat, the most active compound 44, and the least active compound 26 were used for further analyses. The docking visualization was completed by PyMOL software (DeLano Scientific LLC, San Carlos, California, USA).