DOI: 10.5061/dryad.95x69p8r7
title: Modelling data for: Short-course combination treatment for experimental chronic Chagas disease

description: Chagas disease, caused by the protozoan parasite Trypanosoma cruzi,
 affects millions of people in the Americas and across the world leading to
 considerable morbidity and mortality. Current treatment options,
 benznidazole (BNZ) and nifurtimox, offer limited efficacy and often lead
 to adverse side effects due to long treatment durations. Better treatment
 options are therefore urgently required. Here we describe a
 pyrrolopyrimidine series, identified through phenotypic screening, that
 offers a clear opportunity to improve on current treatments. In vitro
 cell-based washout assays demonstrate that compounds in the series are
 incapable of killing all parasites, however, combining these
 pyrrolopyrimidines with a sub-efficacious dose of BNZ can clear all
 parasites in vitro after five days. Importantly, these findings were
 replicated in a clinically predictive in vivo model of chronic Chagas
 disease, where five days of treatment with the combination was sufficient
 to prevent parasite relapse. Comprehensive mechanism of action studies,
 supported by ligand-structure modelling, show that compounds from this
 pyrrolopyrimidine series inhibit the Qi active site of T. cruzi cytochrome
 b, part of the cytochrome bc1 complex of the electron transport chain.
 Knowledge of the molecular target enabled a cascade of assays to be
 assembled to evaluate selectivity over the human cytochrome b homologue.
 As a result, a highly selective and efficacious lead compound was
 identified. The combination of our lead compound with BNZ rapidly clears
 T. cruzi parasites, both in vitro and in vivo, and shows great potential
 to overcome key issues associated with currently available
 treatments. 
description: Protein and ligand
 preparation for molecular modelling
 Our molecular modelling
 studies were based on a previously generated homology model of
 <em>T. cruzi</em> cytochrome
 <em>b</em>.
 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
 <em>b</em> 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 <em>T. cruzi
 </em>cytochrome <em>b</em> 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 <em>b</em> 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<sup>+</sup> 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.
description: **Modelling data for Short-course combination treatment for experimental
 chronic Chagas disease.** https://doi.org/10.5061/dryad.95x69p8r7 The
 dataset contains PDB files and molecular dynamics files for the modelling
 section of our manuscript. Description of the data and file structure
 Datafiles are provided for Figure 5, and Supplemental Information Figures
 S10, S11, S12, S13, S14, S15, S16 and S17. Figure 5: Docking poses for
 compounds 1 – 4 into a homology model of T. cruzi cytochrome b. Files:
 Fig5A.pdb: PDB file for docking pose of compound 1
 (5-(2-methoxyphenyl)-4,6-dimethyl-7H-pyrrolo[2,3-d]pyrimidin-2-amine)
 Fig5B.pdb: PDB file for docking pose of compound 3
 (5-(4-Chloro-2-methoxyphenyl)-4,6-dimethyl-7H-pyrrolo[2,3-d]pyrimidin-2-amine) Fig5C.pdb: PDB file for docking pose of compound 2 (5-(4-Fluoro-2-methoxyphenyl)-4,6-dimethyl-7H-pyrrolo[2,3-d]pyrimidin-2-amine). Fig5D.pdb: PDB file for docking pose of compound 4 (5-(2,4-Dimethoxyphenyl)-4,6-dimethyl-7H-pyrrolo[2,3-d]pyrimidin-2-amine). --- FigS10: Comparison of binding mode of antimycin A and compound 3. Files: FigS10A.pdb: Crystal structure of antimycin A in the Qi site of the bc1 complex (PDB ID: 1PPJ) FigS10B.pdb: Predicted binding mode of compound 3 (pink) at the T. cruzi cytochrome b pocket (light grey) superimposed with the experimentally determined binding mode of antimycin A (blue; PDB ID: 1PPJ) --- FigS11: Molecular dynamics simulation of compound 3 in complex with T. cruzi cytochrome b. FigS11A PL-Contacts_HBond.dat: Datafile for hydrogen bonding interactions reported in 2D ligand-protein interaction diagram (FigS11A). Columns: \# Frame#: MD frame number Residue#: Protein Residue Number Chain: Protein Chain ResName: Protein Residue Name AtomName: Atom Name of the protein residue LigandFragment: Ligand Fragment part LigandAtomName: Atom Name of the ligand FigS11A PL-Contacts_Pi-Pi.dat: Datafile for π-π stacking interactions reported in 2D ligand-protein interaction diagram (FigS11A). Columns: \# Frame#: MD frame number Residue#: Protein Residue Number Chain: Protein Chain ResName: Protein Residue Name LigandFragment: Ligand Fragment part Distance: Interaction distance (Å) Type: Type of π-π stacking interactions, Edge-to-face (e2f), Face-to-face (f2f) S11-B.CSV: Ligand root mean square fluctuation (RMSF) values for each atom (as indicated in FigS11C in the manuscript). --- FigS12: 100 ns MD simulation of compound 3 in complex with T. cruzi cytochrome b. Files: FigS12A.csv: Root mean square deviation (RMSD) values of the protein (backbone atoms) and ligand (heavy atoms) during 100ns MD simulation. Columns: Timestep: Timepoint in picoseconds. RMSD-Lig: Root mean square deviation for ligand heavy atoms RMSD-Protein_Backbone: Root mean square deviation for protein backbone atoms FigS12B.pdb: PDB for binding mode of compound 3 observed during the MD simulation at 0 ns FigS12C.pdb: PDB for binding mode of compound 3 observed during the MD simulation at 25 ns FigS12D.pdb: PDB for binding mode of compound 3 observed during the MD simulation at 50 ns FigS12E.pdb: PDB for binding mode of compound 3 observed during the MD simulation at 75 ns FigS12F.pdb: PDB for binding mode of compound 3 observed during the MD simulation at 100 ns --- FigS13: Summary of 100 ns MD simulation of compound 3 in complex with T. cruzi cytochrome b. Files report the observed interactions and contacts for each frame of the molecular dynamics stimulation. Files: FigS13 PL-Contacts_HBond.dat: data for hydrogen bonds FigS13 PL-Contacts_Hydrophobic.dat : data for hydrophobic interactions FigS13 PL-Contacts_Pi-Cation : data for π-cation interactions FigS13 PL-Contacts_Pi-Pi : data for π-π interactions FigS13 PL-Contacts_WaterBridge: data for waterbridges Columns (combined for all files): \# Frame#: MD frame number Residue#: Protein Residue Number Chain: Protein Chain ResName: Protein Residue Name AtomName: Atom Name of the protein residue LigandFragment: Ligand Fragment part LigandAtomName: Atom Name of the ligand Distance: Interaction distance (Å) Type: Type of π-π stacking interactions, Edge-to-face (e2f), Face-to-face (f2f) --- Fig S14: Analysis of resistance mutations in Qi site of T. cruzi cytochrome b. Files: FigS14A.pdb: Homology model for T. cruzi cytochrome b. FigS14B.pdb: Homology model for T. cruzi cytochrome b with L197I mutation. FigS14C.pdb: Homology model for T. cruzi cytochrome b with F222L mutation. FigS14D.pdb: PDB file with predicted binding mode of compound 3 (pink) at the T. cruzi cytochrome b pocket (light grey) superimposed with I197 (blue) and L222 (magenta) mutations. --- Figs S15-S16: Predicted binding modes for compounds 1 - 4 in the T. cruzi cytochrome b pocket bearing L197I mutation. FigS15-16-A.pdb: Docking pose of compound 1 in the T. cruzi cytochrome b pocket bearing L197I mutation. FigS15-16-B.pdb: Docking pose of compound 3 in the T. cruzi cytochrome b pocket bearing L197I mutation. FigS15-16-C.pdb: Docking pose of compound 2 in the T. cruzi cytochrome b pocket bearing L197I mutation. FigS15-16-D.pdb: Docking pose of compound 4 in the T. cruzi cytochrome b pocket bearing L197I mutation. --- FigS17: Predicted binding modes of compound 3 in the T. cruzi cytochrome b pocket bearing F222L mutation. FigS17_lig-1.pdb: Docking pose 1 for compound 3. FigS17_lig-2.pdb: Docking pose 2 for compound 3. FigS17_lig-3.pdb: Docking pose 3 for compound 3. FigS17_pro: Homology model for T. cruzi cytochrome b with F222L mutation.