Free energy perturbation calculations
SchrödingerRelease2016v2/Maestro10.6/Desmondv4.6/Glidev7.1/FEP+
OPLS3 force field
Windows per transformation: 12
Assumed pH 7.4
Typical simulation time per window: 5 ns
Water Model: SPC
Ensemble for production run: NPT
Free Energy Perturbation Protocol
The free energy calculations were repeated for this set of congeneric ligands, using as reference the native binding mode. The calculations were based to initial poses of the ligands into the receptor which were created according to the crystallographic poses of compounds FXR_10 and FXR_12. The ID of these crystal structures are 1sjp and 1kjyp.
Crystal structures 1sjp and 1kjyp was prepared with Protein Preparation Wizard of Schrodinger.
The protein preparation took place using the following procedure from within the Protein Preparation Wizard, Schrödinger Release 2016-2, Maestro 10.6:
1) Crystal structures 1sjp and 1kjyp imported
2) Automatically added missing hydrogen atoms
3) Enumerate bond orders to HET groups
4) Removed or kept co-crystallized water molecules as described below (see following section)
5) Capped protein termini with ACE and NMA residues
6) Highlighted residues with missing atoms or multiple occupancies
7) Pre-processed structures for Prime, Schrödinger's program for protein structure prediction
8) Determined the most likely ligand protonation state as well as the energy penalties associated with alternate protonation states with Epik
9) Determined optimal protonation states for histidine residues
10) Corrected potentially transposed heavy atoms in arginine, glutamine, and histidine side chains
11) Optimize the protein's hydrogen bond network by means of a systematic, cluster-based approach, which greatly decreases preparation times
12) Perform a restrained minimization that allows hydrogen atoms to be freely minimized, while allowing for sufficient heavy-atom movement to relax strained bonds, angles, and clashes using Impact
Schrödinger Release 2016-3: Schrödinger Suite 2016-3 Protein Preparation Wizard; Epik, Schrödinger, LLC, New York, NY, 2016; Impact, Schrödinger, LLC, New York, NY, 2016; Prime, Schrödinger, LLC, New York, NY, 2016.
Compounds FXR_38, FXR_73, FXR_75 and FXR_79, which all possess a thiophene ring, were aligned to FXR_10 crystal structure with the Flexible ligand alignment tool of the Schrodinger Suite.
Accordingly,the rest of the compounds, which have a benzene ring, were aligned to FXR_12 crystal structure.
Since the ortho substituted benzene ring of FXR_12 displays double occupancy in the crystallographic data, all ortho substituted spiros compounds were considered as having double occupancy as long as they have a unique ortho substitution. So for this subset of compounds, two poses for each ligand were considered for free energy calculations. These poses were constructed with the assumption that the common binding mode of FXR_10 and FXR_12 is conserved in all ligands. Because during Free Energy perturbation calculations the charge cannot change, ligands that have a carboxylic acid in their structure were calculated in their protonated state.
Subsequently, the free energy calculations were conducted with the aid of FEP+ module of Schrodinger.
FEP+ results gave us the final relative free energy differences ranking. We submitted the final snapshot of the MD trajectory for each ligand. All water molecules were removed. Since we can upload only one pose for each ligand in the main submission directory, the poses of AB occupancy for each compound are included in the SuppInfo directory.
The relative binding free energies for the ligands with the two binding modes (AA and AB according to double occupancy) were calculated according to equation 8 in publication:
Lingle Wang et al, How to deal with multiple binding poses in alchemical relative protein-ligand binding free energy calculations, J. Chem. Theory Comput., 2015, 11.
This publication describes a technique to calculate relative binding free energies of ligands that have two or more different binding modes. The corrected binding free energy combines the free energy results of each binding mode.
Contributors: Christina Athanasiou, Sofia Vasilakaki, Dimitris Ntellis, Zoe Cournia
Biomedical Research Foundation, Academy of Athens, Greece
Pose prediction for free energy perurbation calculations
Schrödinger Release 2016-2: /SchrödingerRelease2016v2/Maestro 10.6/ProteinPreparationWizard/Primev4.4/Impactv71014/Desmondv4.6/Phasev4.7/Epikv3.6
Using Schrödinger's Protein Preparation Wizard, we converted the raw PDB structure into an all-atom, fully prepared protein model.
The protein preparation took place using the following procedure from within the Protein Preparation Wizard, Schrödinger Release 2016-2, Maestro 10.6:
1) Imported 1sjpr and 1kjyp crystal structures
2) Automatically added missing hydrogen atoms
3) Enumerate bond orders to HET groups
4) Removed or kept co-crystallized water molecules
5) Capped protein termini with ACE and NMA residues
6) Highlighted residues with missing atoms or multiple occupancies
7) Pre-processed structures for Prime, Schrödinger's program for protein structure prediction
8) Determined the most likely ligand protonation state as well as the energy penalties associated with alternate protonation states with Epik
9) Determined optimal protonation states for histidine residues
10) Corrected potentially transposed heavy atoms in arginine, glutamine, and histidine side chains
11) Optimize the protein's hydrogen bond network by means of a systematic, cluster-based approach, which greatly decreases preparation times
12) Perform a restrained minimization that allows hydrogen atoms to be freely minimized, while allowing for sufficient heavy-atom movement to relax strained bonds, angles, and clashes using Impact
Schrödinger Release 2016-3: Schrödinger Suite 2016-3 Protein Preparation Wizard; Epik, Schrödinger, LLC, New York, NY, 2016; Impact, Schrödinger, LLC, New York, NY, 2016; Prime, Schrödinger, LLC, New York, NY, 2016.
Sastry, G.M.; Adzhigirey, M.; Day, T.; Annabhimoju, R.; Sherman, W., "Protein and ligand preparation: Parameters, protocols, and influence on virtual screening enrichments," J. Comput. Aid. Mol. Des., 2013, 27(3), 221-234
Flexible ligand alignment: align maximum common substructure
The pose prediction methodology is described below.
1) Protein Preparation of 1sjpr and 1kjyp crystal structures
The default parameters from Maestro Protein Preparation Module were used as described above.
2) Flexible ligand alignment
Compounds FXR_38, FXR_73, FXR_75 and FXR_79, which all possess a thiophene ring, were aligned to FXR_10 crystal structure with the Flexible ligand alignment tool of the Schrodinger Suite.
Accordingly,the rest of the compounds, which have a benzene ring, were aligned to FXR_12 crystal structure.Since the ortho substituted benzene ring of FXR_12 displays double occupancy in the crystallographic data, all ortho substituted spiros compounds were considered as having double occupancy as long as they have a unique ortho substitution. So for this subset of compounds, two poses for each ligand were considered for free energy calculations. These poses were constructed with the assumption that the common binding mode of FXR_10 and FXR_12 is conserved in all ligands. Because during Free Energy perturbation calculations the charge cannot change, ligands that have a carboxylic acid in their structure were calculated in their protonated state. We made the assumption that all ligands have the same binding mode.
Contributors: Christina Athanasiou, Sofia Vasilakaki, Dimitris Ntellis, Zoe Cournia
Biomedical Research Foundation, Academy of Athens, Greece