Protein preparation wizard workflow

The structure of AKT was prepared by the Protein Preparation Wizard Workflow provided in the Maestro module of Schrödinger software. The PDB ID of AKT is 6S9X. The protein was processed through the default pipeline. Subsequently, the Glidedock module of Schrödinger software was used to generate the protein grid of these complexes, and the grid boxes were defined as 12 × 12 × 12 Å, and the spatial region was centered on the original ligand of the complex structures. The 3D structure of the Mon compound was constructed by Chem3D Ultra 8.0; stereoisomers and tautomers were generated by LigPrep, and the protonation states of ligands at pH 7.0 ± 2.0 were predicted by Epik. Other parameters were set as default. Ligand docking module in Schrödinger was used to choose the receptor grid file and ligand file with extra-precision (XP) mode, which was generated from ligand preparation and protein preparation. The binding mode was visualized by Pymol [17 ].

Currently, there are 10 available X-ray crystal structures of IDO1 complexed with different inhibitors in the Protein Data Bank, and their PDB codes are 2D0T, 2D0U, 4PK5, 4PK6, 4U72, 4U74, 5EK2, 5EK3, 5EK4, and 5ETW, respectively. These structures were prepared with the Protein Preparation Wizard Workflow provided in the Maestro module of Schrödingersoftware (Schrödinger, LLC: New York, NY, 2015). The protein structure was firstly fixed by verifying proper assignment of bonds, adding hydrogens, creating zero-order bong to metals, creating disulfide bonds, deleting water molecules beyond 5 Å from the het group, removing waters with less than 3 H-bonds to non-waters, and a restrained minimization that allows only hydrogen atoms to be freely minimized. Subsequently, the receptor grids of these complexes were generated with Glide module of Schrödinger software, and the grid boxes were defined as a 20 × 20 × 20 Å space region centered at the original ligand of the complex structures. In addition, one metal-coordination was introduced into heme iron during grid box generation. For the other parameters, the default values were assigned.

Docking study was conducted by Maestro 10.1. Crystal structure of JAK2 (4Z32) was downloaded from RCSB (Protein Date Bank). Protein Preparation Wizard Workflow (Schrödinger program suite) was used to prepare the protein and Ligand Preparation was used to prepare the compound. PN was docked into the defined binding site without constraint. Results were generated by Pymol based on the Prime-score.