2.7. Covalent docking using AutoDockFR

To use AutoDockFR for covalent docking, the receptor and covalent ligand were prepared according to the method described previously [³⁷]. The ligand must be in the product form (Fig. S2), and the receptor and covalent ligand need to share 3 atoms. In this case, the atoms (all sidechain atoms and backbone carbon atoms) of cysteine should be included as part of the ligand structure to allow the conformational sampling of the sidechain of C145 during docking. The 2D structure of the product was converted to 3D structure by RDkit (version: 2020.3) [³⁸] and PDBQT format by OpenBabel (version 3.0) [³⁹], respectively. To meet the requirement of AutoDockFR, the atoms in the root section in PDBQT format must correspond to those atoms of backbone carbon, alpha carbon and beta carbon of cysteine, and this operation was achieved by using the MGLTools script, prepare_ligand4.py, with the “-R” option.

Crystal structure 6YNQ [⁴⁰] was processed using PyMOL and Autodock/Vina plugin [⁴¹] and was saved in PDBQT format. The center of docking grid was defined as the center of mass of the ligand in 6YNQ, and a cube with length of 22.5 Å was defined as the grid box. Residues of N142 and M165 were defined as flexible residues, and C145 was defined as the covalent residue. The detected flexible residues were chosen to be modeled explicitly during docking. The atom index of backbone carbon, alpha carbon and beta carbon of C145 was specifically defined as overlapping atoms for the ligand. Program “agfr” was used to generate the grid file.

Covalent docking was carried with default options using program “adfr”, and the result was visualized with PyMOL and Autodock/Vina plugin. We firstly conducted re-docking. The re-docking was done using the SARS-CoV-2 3CL^pro structure model (PDB: 6YNQ) with the same protocol (allowing M165, C145 and N142 to be flexible) as that of Vitamin K3 docking. As shown in Fig. S3, the PDB ligand 2-Methyl-1-tetralone docking pose (cyan stick model) was almost identical to that (gray stick model) in the crystal structure.