Discovery Studio Software and Ligand Library
Discovery Studio 4.5 software (BIOVIA, San Diego, California, USA) is a suite of software for simulating small molecule and macromolecule systems. It aims to provide protein modeling, optimization, and drug design tools by applying protein structure and structural biologic computation. LibDock module of Discovery Studio was employed for virtual screening, CDOCKER module was used for docking study, and ADME module was analyzed for pharmacologic properties. The Natural Products database in the ZINC15 database was selected to screen STING agonists. The ZINC15 database is a free database of commercially available compounds provided by the Irwin and Shoichet Laboratories, Department of Pharmaceutical Chemistry, University of California, San Francisco (San Francisco, California, USA).
Structure-Based Virtual Screening Using LibDock
Ligand-binding pocket region of BCL2 was selected as the binding site to screen compounds that could potentially inhibit BCL2. Virtual screening was carried out using the LibDock module of Discovery Studio 4.5[21]. All ligand poses were ranked based on the ligands score. The 2.0-Å crystal structure of human BCL2(Protein Data Bank identifier: 4IEH) and the inhibitor Obatoclax (Protein Data Bank identifier: ZINC29052268) was downloaded from the Protein Data Bank and imported to the working circumstance of LibDock. The chemical structure of BCL2 is shown in Fig. 1. The protein was prepared by removing crystal water and other heteroatoms around it, followed by addition of hydrogen, protonation, ionization, and energy minimization[22]. The minimization performed 2000 steps with a root mean square gradient tolerance of 12.277, and the final root mean square gradient was 0.690. The prepared protein was employed to define the binding site. Using the ligands (Obatoclax) binding position, the active site for docking was generated. Based on the LibDock score, all the docked poses were ranked and grouped, and all compounds were ranked according to the LibDock score.
Absorption, Distribution, Metabolism, and Excretion and Toxicity Prediction
The ADME module of Discovery Studio 4.5 was employed to calculate absorption, distribution, metabolism, and excretion (ADME) of selected compounds, including their aqueous solubility, blood-brain barrier penetration, cytochrome P-450 2D6 (CYP2D6) inhibition, hepatotoxicity, human intestinal absorption and plasma protein binding level. TOPKAT module of Discovery Studio 4.5 was employed to calculate the toxicity and other properties of all the potential compounds. These pharmacologic properties were fully considered when selecting proper drug candidates for BCL2.
Molecule Docking
CDOCKER module of Discovery Studio 4.5 was used for molecular docking study. CDOCKER is a molecular docking method based on CHARMM force field, which can produce high-precision docking results. The CHARMM force field was used for both receptors and ligands. For each complex pose, the CHARMM energy and the interaction energy, which indicated ligand binding affinity, were calculated. Crystal structure of BCL2 was obtained from the protein data bank. The crystal water molecules were generally removed in a rigid and semi-flexible docking process, causing the fixed water molecules to possibly affect the conformation of the receptor-ligand complex[23, 24]. After the water molecules were removed, hydrogen atoms were added to the protein.
Molecular Dynamics Simulation
The best binding conformations of the ligand-BCL2 complexes among the poses predicted by the molecule docking program were selected and prepared for molecular dynamics simulation. To simulate the physiologic environment, sodium chloride was added to the system with the ionic strength of 0.145. Then the system was subjected to the CHARMM force field and relaxed by energy minimization (500 steps of steepest descent and 500 steps of conjugated gradient), with the final root mean square gradient of 0.227. The particle mesh Ewald algorithm was used to calculate long-range electrostatics, and the linear constraint solver algorithm was adapted to fix all bonds involving hydrogen. With initial complex setting as a reference, potential energy, and structural characteristics through the Discovery Studio 4.5 analysis trajectory protocol.