Protein preparation wizard tool

All of the crystal structures were meticulously preprocessed with the assistance of the Schrödinger Maestro’s Protein Preparation Wizard tool. This preparation and minimization process was executed under a pH of 7.4, rectifying ionization states as necessary. Polar hydrogens were incorporated, and non-essential water molecules were excluded from the structures. The entire protein structure was subjected to a thorough minimization and optimization procedure utilizing the OPLS3 force field. This optimization aimed to enhance protein energies and eliminate any steric hindrance, with a default root mean square deviation (RMSD) value of 0.30 Å applied to non-hydrogen atoms [49 (link)].

The Schrödinger Suite was adopted for computing all theoretical investigations (Software: Schrödinger, LLC, New York, NY. 2017). The 3D structure of AC-18, AC-20 and AC-22 were built using the Maestro GUI (Software: Maestro Schrödinger, LLC, New York, NY. 2017) and further submitted to LigPrep version 3.9 tool (Software: LigPrep Schrödinger, LLC, New York, NY. 2017) to take into account the most stable protomeric forms at pH 7.4. The Protein Data Bank (https://www.rcsb.org/) crystallographic entry structure 2PRG (Nolte et al., 1998 (link)) was prepared by using the Protein Preparation Wizard tool (Schrödinger, LLC, New York, NY, 2017). The molecular recognition evaluation was carried out by means of Glide software ^[g] in combination with Schrödinger Induced Fit docking (IFD) (Glide Schrödinger, LLC, New York, NY, 2017) protocol. The binding site of the target model was defined by means of a regular grid box of about 64,000 Å centered on the co-crystallized ligand. Docking simulations were computed using Glide ligand flexible algorithm version 6.7 at standard precision (SP).

Protein-peptide docking analysis was performed using Glide module in Schrödinger. The protein structure of YAP WW1 domain (PDB ID: 2LTW) was downloaded from PDB [96 (link)] and was prepared with the Protein Preparation Wizard tool in Schrodinger. The peptides were built using Maestro and generated multiple conformers using the MacroModel sampling method. The receptor grid for peptide docking purposes was prepared with default settings using the peptide ligand to define the grid center. The peptide conformers were docked using Glide SP-PEP protocol [97 (link)].

Molecular docking was conducted in order to assess the binding interactions of the promising compounds. Initially, by using the LigPrep module synthesized compounds, their structures were transformed from 2D to low-energy 3D conformers with satisfactory bond lengths and angles. The 3D structures of S. aureus tyrosyl-tRNA synthetase, tyrosine kinases, and human pancreatic α-amylase were obtained from the PDB database under the accession codes 1JIJ, 2HCK and 2QV4, respectively. Before docking, all protein crystal structures were prepared using the Protein Preparation wizard tool by Schrodinger to address any structural problems. This process involves changing the bond order, adding hydrogens, looking for disulfide bonds and filling in side chains and loops that are lacking [64 (link),65 (link)]. Constrained minimization was additionally applied to the protein structure. In which, heavier atoms in the structure are constrained to reduce torsional stress throughout this reduction phase, while hydrogens are left unconstrained. Using the Schrodinger Receptor Grid Generation tool, a crystalized ligand structure was selected to generate a 3D grid with accurate dimensions to represent the active part of the receptor [66 ]. The binding free energy of protein–ligand complexes was calculated using Prime of Schrodinger.

The OR5K1
AF2 model was downloaded from the AlphaFold 2 database (https://alphafold.ebi.ac.uk/entry/Q8NHB7). OR5K1 AF2 and HM were superimposed through the Protein Structure
Alignment module available in Maestro (Schrödinger Release
2021-3, Maestro, Schrödinger, LLC, New York, NY, 2021). RMSD
values were calculated with visual molecular dynamics (VMD).^{118 (link)} Hydrogen atoms and side chains of both models
were optimized with the Protein Preparation Wizard tool at physiological
pH (Schrödinger Release 2021-3, Maestro, Schrödinger,
LLC, New York, NY, 2021). Histidine residues 56, 159, and 176 were
protonated on the epsilon nitrogen, while all others were protonated
on the delta nitrogen. Ramachandran plots were generated to verify
the reliability of the backbone dihedral angles of amino acid residues
in the models. The A100 tool was used to investigate the activation
state of the models.^{66 (link)}