3.1. Molecular Docking

Molecular docking was conducted using the Surflex-Dock module in the SYBYL-X 1.3 software (Tripos, Inc., St. Louis, MO, USA) [32,33,34,35]. All 320 molecules from our in-house natural compound database were downloaded from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/) in mol2 format. All hydrogen atoms were added, and the partial atomic charges of the atoms of each compound were assigned using the Gasteiger-Hückel method. Each structure was energy-minimized using the Tripos force field with a distance-dependent dielectric constant and the Powell conjugate gradient algorithm convergence criteria, which partially accounts for the shielding effects of the aqueous environment on electrostatic interactions [36]. These conformations were used as starting conformations to perform molecular docking. The crystal structure of Syk (PDB ID: 4PUZ), determined by X-ray diffraction at a 2.09 Å resolution, was chosen as a docking protein model [37]. All co-crystallized water molecules of the protein model were removed, and polar hydrogen atoms were added using SYBYL X-1.3. The protein model was assigned a force field using Gasteiger-Marsili charges and then energy-optimized for 1000 iterations using the default parameters in SYBYL X-1.3. The amino acid residues within 0.5 Å around the CG9 ligand were defined as a docking pocket using a ProtoMol-based method [38]. The ProtoMol in Surflex-Dock utilized various molecular fragments, such as CH₄, C=O, and N-H, to represent hydrophobic groups, hydrogen bond donors and hydrogen bond acceptors, respectively. The remaining parameters for docking were used on the default settings.

To validate the performance of the docking procedure, the co-crystallized CG9 ligand was extracted and re-docked into the active sites of the model. The root-mean-square deviation (RMSD) between the re-docking and co-crystallized conformation of CG9 was calculated according to the following formula [39,40]:

where d is the distance between N pairs of equivalent atoms excluding hydrogen. A lower RMSD indicates a greater overlap between the re-docked and co-crystallized conformation of theCG9 ligand. The compounds were docked into the active pocket of Syk, resulting in a hit list with a total score for each molecule. A higher total score indicates a higher binding force between the ligand and protein model. A cut-off value of six was used to select a reasonable number of virtual hits for further investigation [41].