Modelling data for: Short-course combination treatment for experimental chronic Chagas disease

Description

Notes

Methods

Protein and ligand preparation for molecular modelling

Our molecular modelling studies were based on a previously generated homology model of T. cruzi cytochrome b. The protein structure with a conserved water molecule interacting with F33 was prepared using the Protein Preparation module in the Schrödinger suite (Schrödinger Release 2021-2). Protonation states were assigned by PROPKA at pH 7.0, and the hydrogen bonding network was consequently optimized. A restrained energy minimization step was then executed using the OPLS4 force field with default settings. Models of cytochrome b bearing either L197I or F222L mutations were prepared in Maestro by mutating one residue at a time. Subsequently, the mutated versions of cytochrome b were processed with the Protein Preparation tool in an identical manner to the wild-type enzyme.

Ligand structures (compounds 1-4) were prepared with Schrödinger's LigPrep (Schrödinger Release 2021-2). All possible tautomeric forms and stereoisomers were generated at pH 7.0 ± 0.4 using Epik. 

Docking studies

Molecular docking studies were carried out using Glide Standard Precision (SP) mode (Schrödinger Release 2021-2). First, the docking grid boxes were generated using the Receptor Grid Generation tool; bound ubiquinone (UQ2) was selected as the center of the grid, and a cube of 10 Å was defined as the inner box. During docking, a total of 20 poses per ligand were subjected to post-docking minimization, and a maximum of ten docking poses for each ligand were output. This setup was able to successfully reproduce the experimentally determined binding mode of antimycin A.

Molecular dynamic simulation

Molecular Dynamic (MD) simulation of the binding mode generated by molecular docking was carried out to analyze the stability of the pose and the interactions at the binding site. The docking pose of compound 3 in complex with T. cruzi cytochrome b was used as the initial coordinate for the generation of the MD system. Desmond software (Schrödinger Release 2021-2) was utilized to set up the system and run the MD simulation. As cytochrome b is embedded in the inner mitochondrial membrane, MD was performed in membrane; thus, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membranes were added to the model system by placing the two layers automatically. The system was then solvated using the SPC (simple point-charge) water model in a Periodic Boundary Conditions orthorhombic box and neutralized with Na⁺ ions at a salt concentration of 0.15 M. The option "exclude ions and salt placement" was turned on to allow a 10 Å buffer zone from the ligand. The default Desmond protocol for energy minimization and model relaxation was applied before performing the production simulation. The OPLS4 force field and NPAT (constant particle number (N), pressure (P), lateral surface area (A), and temperature (T)) ensemble were used. The temperature was kept constant at 300 K, while the pressure was kept at 1.01325 bar. Lastly, a 100 ns MD simulation with a trajectory interval of 5 ps was carried out with the same conditions. Simulation Interactions Diagram (SID) was used for the analysis of the MD simulation. The RMSF and RMSD values were then plotted using R packages.