Protein preparation wizard

Preparation of the compounds for docking included the generation of tautomeric and ionisation states at pH 6–8 and the creation of 3D structures using LigPrep (Schrödinger Release 2021–3: LigPrep, Schrödinger, LLC, New York, NY, 2021). Proteins were prepared by adding H-atoms, protonating side chains at pH = 7, and refining H-bond network with the default settings of Protein Preparation Wizard (Schrödinger Release 2021–3: Protein Preparation Wizard, Schrödinger, LLC, New York, NY, 2021). Ser460 was mutated to Gly for noncovalent docking performed by Glide (Schrödinger Release 2021–3: Glide, Schrödinger, LLC, New York, NY, 2021). Covalent docking was performed by CovDock⁴⁶ (link).

The 3-dimensional (3D) structure of Plasmodium falciparum Hexose Transporter 1 protein (PfHT1) was retrieved from Protein Data Bank (PDB) (https://www.rcsb.org). After considering residual factor (R)-value free, R-value work, R-value observed and overall resolution of the PfHT1 structures on PDB 6M20, 6ML2 and 6RW3, structure 6M20 was found to have the lowest values in all the parameters which indicated it could make a good target [19 ]. The PfHT1 protein with PDB ID 6M20 was refined by using Protein Preparation Wizard of Schrödinger-Maestro Release 2021–4 [19 ]. We assigned charges, bond orders and deleted water molecules to avoid inaccurately high binding scores [20 (link)]. Subsequently, hydrogens were added to the heavy atoms. The heavy atom root-mean-square deviation (RMSD) was fixed to 0.30Å using the optimized potentials for liquid simulations (OPLS) 2005 force field [21 ]. Lastly, we optimized amino acids using neutral pH. To determine the selectivity of our compounds as PfHT1 inhibitors versus, we downloaded the human GLUT1 (6THA) protein from the protein data bank https://www.rcsb.org, prepared the protein using the Protein Preparation Wizard of Schrodinger suite. Subsequently, a receptor glide grid was generated and molecular docking of the receptor with our hit compounds was performed [22 (link)].

The protein structure for docking studies, crystal complexes of HA with siallylactose (LTSc) and complex of EGFR with erlotinib were selected from the PDB (PDB ID: 1RVT, resolution 2.50 Å and PDB ID: 1M17, resolution 2.60 Å) and prepared in Protein Preparation Wizard [Protein Preparation Wizard; Epik, Schrödinger, LLC, New York, NY, 2016; Impact, Schrödinger, LLC, New York, NY, 2016; Prime, Schrödinger, LLC, New York, NY, 2016], under the default settings. Three dimensional structure, conformation and protonation states of SM were generated by LigPrep [LigPrep, Schrödinger, LLC, New York, NY, 2016] (at pH 7.4). Finally, Glide [Glide, Schrödinger, LLC, New York, NY, 2016] was used for docking at the SP level under default settings (sampling nitrogen inversion, sampling ring conformations with energy window equal to 2.5 kcal/mol, penalizing nonplanar conformation of amides up to 100 steps during energy minimization and performing postdocking optimization) of each SM conformer to protein model. Each pose was ranked according to affinity score, and the highest scored pose was chosen for further analysis.

The crystal structures of human FcγRIIIa in complex with Fc (PDB ID: 3SGJ) and human FcγRIIb in complex with Fc (PDB ID: 3WJJ) were used in the modeling. The structures were prepared with the “Protein Preparation Wizard” in Schrödinger (2020-2) (Protein Preparation Wizard; Epik, Schrödinger, LLC, New York, NY, 2016; Impact, Schrödinger, LLC, New York, NY, 2016; Prime, Schrödinger, LLC, New York, NY, 2020.). The residues Q38, Q74, V158, Q169 on FcγRIIIa in 3SGJ and D238 on Fc in 3WJJ were mutated back to their wild-type residues respectively. Then they were refined using “Predict Side Chains” (Schrödinger Release 2020-2: Prime, Schrödinger, LLC, New York, NY, 2020) with backbone sampling. The virtual mutation calculation was performed with “Residue Scanning Calculations” in Schrödinger (Schrödinger Release 2020-2: BioLuminate, Schrödinger, LLC, New York, NY, 2020). The mutated residue and nearby residues were refined with “side-chain prediction with backbone sampling”. The residues within 6 Å were refined to handle mutations in flexible hinge region.

The cocrystal structure of wild-type ALK complexed with ceritinib (PDB: 4MKC) and HDAC6 complexed with trichostatin A (PDB: 5EDU) were taken from the Protein Data Bank (http://www.rcsb. org). Molecular modelling was performed using the Schrödinger small molecule drug discovery suite 2021–4. The receptors were prepared by Protein Preparation Wizard (Schrödinger Release 2021–4: Protein Preparation Wizard; Epik, Schrödinger, LLC, New York, NY, 2021; Impact, Schrödinger, LLC, New York, NY; Prime, Schrödinger, LLC, New York, NY, 2021.). The centroid of the original ligands was selected to be the site of the grid box to cover the surrounding residues of the binding pockets. The 2D ligand structures were prepared using Chemdraw 8, and the 3D structures were generated by LigPrep (Schrödinger Release 2021–4: LigPrep, Schrödinger, LLC, New York, NY, 2021.). The molecular docking simulations were performed using Glide (Schrödinger Release 2021– 4: Glide, Schrödinger, LLC, New York, NY, 2021.) and the results indicated that the original ligands (ceritinib and trichostatin A) can be reproduced. The binding pose with the lowest score in each case is selected to represent the predicted binding mode. The 3D and 2D protein − ligand interaction plots were presented using Maestro (Schrödinger Release 2021–4: Maestro, Schrödinger, LLC, New York, NY, 2021.).