Protein crystal structure preparation
Three-dimensional (3D) crystal structures of (PDB ID:2J4Z) Aurora Kinase A protein was retrieved from RCSB Protein Data Bank. The protein retrieved (PDB ID:2J4Z) has resolutions of 2.0Å. Before performing the docking calculations, the crystal structures of Aurora kinase A were prepared at pH 7 ± 1 by the PROPKA program and used protein preparation wizard of Schrodinger (Protein Preparation Wizard: Schrodinger Release 2019-2). The OPLS3e force field was used to optimize the crystal structure and the side chains.
Fluoroflavone derivative of flavones (CID: 261400) and its compounds similar (90% similar) are taken as inhibitors against Aurora Kinase A. 3882 compounds were obtained from PubChem database (https://pubchem.ncbi.nlm.nih.gov/). These compounds are further filtered using different methods. Firstly, these compounds were pushed to Entrez and a filter of Lipinski’s rule of 5 was added and 3882 compounds were filtered in we have 3430 compounds. There are different filters of CANVAS of Schrodinger 2019-software, which filter compounds based on different parameters. REOS is one of the filters which sets the criteria to identify lead-like small compounds and the compounds are filtered. Filtering 3430 compounds to 2449 compounds. The next filter of CANVAS is PAINS. PAIN filter compounds with the substructure. PAINS1, PAINS2, and PAINS3 filtered 2449 compounds to 2448 compounds.
Ligand structure retrieval and its preparation
Derivatives of Fluoroflavone were filtered and are used as an inhibitor to inhibit the overexpression of Aurora Kinase A. The 2 dimensional (2D) chemical structures of Fluoroflavone (PubChem CID: 261400) and its derivatives were retrieved from the PubChem database and was filtered. LigPrep (Schrodinger Release 2019-2) is used for the 2D structures of the ligands and was subjected to energy minimization and appropriate bond order assignment with Optimized Potentials for Liquid Simulations (OPLS3e) force field (module of Schrodinger to get a stable conformation of the analogs of the Fluoroflavone).
The binding cavities of targeted receptors were defined considering all residues which come within the cut off distance of 2 Å from the crystal ligands which are present in the retrieved crystal structure of Aurora kinase A receptor and also, the binding cavity information for this receptor was taken from PDBsum (www.ebi.ac.uk/pdbsum/). The prepared 2448 ligands (filtered analogs of Fluoroflavone) were subjected to docking calculations within the defined binding cavity using the Extra Precision (XP) and Standard Precision (SP) different modes of Glide program (Glide: Schrodinger Release 2019-2) with the OPLS3e force field. Aurora Kinase A receptor, and binding cavity of Aurora Kinase A protein forms a complex structure of the ligands and the protein by docking into the binding cavity using SP and XP mode of Glide . We further extended the data to understand the interactions of residues with ligands.
“Enrichment” is the measure of known ligands ranks to a set of decoys. Enrichments of ligands is required to improve the binding affinity with specific residues in the cavity of targeted Kinase  using DUD.E database decoys was designed for decoy bias on enrichment. For Aurora Kinase A protein, a decoy set for Aurora Kinase is generated using the DUD.E database . Decoys are computed on the basis of similar physical properties but different chemical structures. So active set of decoys are generated, but they have not been tested. So, the random 1000 drug-like decoys for Aurora Kinase and 10 best XP docked ligands and Fluoroflavone molecules were docked with Aurora Kinase A (PDB ID: 2J4Z). A module for calculating Enrichment Factors and the effectiveness of ligands and decoys with target protein was calculated using the Enrichment Schrodinger analysis enrichment calculator module.
Molecular dynamic (MD) studies
Molecular dynamic studies are used to evaluate the strength to bind and activity of Fluoroflavone, and the best 2 analogs of Fluoroflavone were obtained after docking studies. The full-scale molecular dynamic simulation was performed for 100ns with a complex of Fluoroflavone and its 2 best analogs of Fluoroflavone with Aurora Kinase A. The process of Molecular dynamic simulations using Desmond helps to measure the movements of the atoms and calculate the forces. However, Desmond considers all detailed requirements including temperature, pressure, volume system, and all the functionality used to study the complex interactions. System builder of Desmond in the Maestro program is used, the system for the protein-ligand complex is immersed in a water-filled cubic box containing 1Å spacing water molecules using an extended TIP3 (three-point water model) with periodic boundary conditions. The solvate complex system is charged and is neutralized by adding counter ions randomly. The steepest descent method is used for energy minimization which is an important step for MD simulations. To apply boundary conditions to the protein-ligand complex, a cube box (box size 8.0) is taken for reducing the edge effects in the finite system. The total system is surrounded by translated copies of itself and the atoms are in the space-filling box. The OPLS3e force field (parameters used to describe the potential energy of a system) is chosen, which has an improved force field for MD simulation of proteins . Certain parameters as input such as constraints set as all-bonds, integrator as MD noise is considered in the studies of molecular dynamics. However, the chain thermostat method and it uses the Martyna-Tobias-Klein barostat method at a temperature of 300k. After the system gains the state of equilibrium, the stable conformation trajectories are taken into account and analyzed to inspect interactions and stability. The C-alpha backbone’s, conformational changes of Aurora Kinase A crystal structure have been compared with previous conformations. Besides, we have performed MD trajectory clustering analysis to find out the binding mode of the selected analogs of the Fluoroflavone within the active sites of the protein using trajectories generated during 100ns Molecular Dynamic simulations.
MD trajectory analysis and prime MM/GBSA calculations
The binding free energy of the complex system was calculated using the Prime module. To calculate the ligand binding free energies and ligand strain energies for the complexes of Aurora Kinase A with the ligands, the MM/GBSA (Molecular Mechanics, The Generalized Born Model, and Solvent Accessibility) was used. The non-polar solvation energies, polar solvation energies, and potential energy are comprised of binding free energy. Prime MM-GBSA works with the combination of advanced OPLS-3E force field, SGB solvation model for polar solvation (GSGB), non-polar solvation (GNP), and Molecular Mechanics Energies (EMM) that compiled different nonpolar solvent accessible surface area and van der Waals interactions. The free energy changes upon ligand binding were calculated using the following equations.
ΔGbind = Gcomplex– (Gprotein + Gligand)G = EMM + GSGB + GNP.
The Gcomplex represents complex energy, Gprotein is the receptor energy and Gligand is the unbound ligand energy. EMM represents molecular mechanics energies, GSGB is an SGB solvation model for polar solvation and GNP is a nonpolar solvation term .