a3c8k-FreeEnergyProtocol.txt

Name

alchemical free energy protocol with average network analysis

Software

FESetup release 1.3dev, SUI version: 0.8.1, somd-freenrg 2016.1.1, pymbar version 3.0.0.dev0.dev-Unknown, Networkx v.1.11

Parameter

forcefield amber, ff14sb, tip3p, hfe
gaff2
mdengine, amber pmemd
AFE.type sire
AFE.separate_vdw_elec false
box.type = rectangular
box.length = 12.0
neutralize = False
temperature = 300 K
pressure = 1 atm
nmoves = 20000
ncycles = 100
buffered coordinates frequency = 5000
save coordinates = True
timestep = 2 * femtosecond
constraint = hbonds-notperturbed
hydrogen mass repartitioning factor = 1.0
cutoff type = cutoffperiodic
cutoff distance = 10*angstrom
barostat = True
andersen = True
energy frequency = 250
precision = mixed
minimise = True
equilibrate = False
equilibration iterations = 5000
center solute = True
reaction field dielectric = 82.0
minimal coordinate saving = True
lambda array varying between 9-27 evenly spaced lambda windows

Method

The ligands were prepared for relative free energy calculations from the output of the docking protocols. Each ligand was parameterized using GAFF inside FESetup. Ligands were then solvated in TIP3P water and minimized and equilibrated for 100 ps. For the relative free energy perturbations, a series of alchemical transformation was proposed and the necessary perturbation protocol was computed with FESetup, resulting in Sire compatible input for the solvated ligands as well as vacuum ligands. FESetup, calling AMBERtoolswas used to solvated the protein and ligand complexes without adding any counter ions resulting in a charged simulation box. The solvated complexes were minimized and equilibrated for 100 ps with all but solvent restraint. Alchemical morphs are obtained with FESetup with explicit mapping of atoms for different binding poses of the same ligand. Production simulations were carried out using Sire somd-frenrg. Prior to the production run, Sire was also used for a further minimization and 2 ps equilibration to each of the intermediate lambda values using an annealing protocol. All simulations were run with a 2 fs timestep. Reduced gradients and energies were saved every 400 fs with a total simulation time of 4 ns. The simulation temperature of 298.15 K was kept constant using an anderson thermostat and the pressure of 1 atm was maintained using a MC barostat as implemented in OpenMM. The barostat frequency is set to 25 and the collision frequency of the anderson thermostat is 1 /ps. Furthermore, multi state Bennet's acceptance ratio (MBAR) was also as a second free energy analysis method. All submitted results come from the MBAR estimator including the error estimates. 5% of the initial data was discarded to equilibration in the MBAR analysis. The individually estimated free energy differences were then read into a networkx digraph. Backward and forward simulations were averaged, as well as repeated runs. All possible paths within the graph were estimated and weighted averages of all possible paths between two compounds for the relative free energy computation were computed. Standard errors were computed based on forward and backward averages as well as repeated runs and then propagated along the paths using standard error propagations. Each path was then weighted based on the standard error along the path, the same was done for averaged path errors.

a3c8k-PosePredictionProtocol.txt

Name

Maestro/Marvisketch/rDock Visual

Software

Maestro 11 (Beta Version)/ fconv/ Open Source Pymol 1.7 /MarvinSketch 15.3.30/ rDock

System Preparation Parameters

Assumed pH 7.4
Tautomers considered

System Preparation Method

Maestro's prepwizard was used to add hydrogens using default parameters.
The FXR structure provided by the organizers was used as initial template.
The structure was converted to mol2 files using fconv for the docking calculations, after initial cleaning with Maestro.
For the alchemical free energy calculations it was necessary to model a missing fragment comprised of residues A459-K464.
To this end the protein residues between N448 and Q476 were replaced by the same fragment as crystallized in the 3OKH structure. Subsequently, ACE capping groups were added to residues M247 of the main chain and D743 of the co-activator fragment.
Similarly an NME capping group was attached to D755 of the co activator fragment.
Ligand 3D structures were generated from 2D sdf files provided using MarvinTools scripts.
No water molecules were retained for docking calculations.
For the alchemical free energy calculations coordinates for water molecules accompanying the X-ray structure provided by
the organisers were superimposed with the coordinates of the poses. It was found that 1 water molecule
was susceptible of interfering with the simulations of the largest compounds (such as fxr_102). Consequently it was manually displaced to a nearby position.

Pose Prediction Parameters

RECEPTOR_FLEX 3.0 # Receptor flexibility weight
SITE_MAPPER RbtSphereSiteMapper # Cavity definition function
SMALL_SPHERE 1.5 # Small sphere radius for the cavity mapper
LARGE_SPHERE 4.0 # Large sphere radius for the cavity mapper
SCORING_FUNCTION RbtCavityGridSF #Cavity scoring function
WEIGHT 2 # Pharmacophoric restraint penalty weight

Pose Prediction Method

Docking calculations were performed for compounds fxr_91, fxr_101 and fxr_102. Docking was performed with rDock, generating the cavity using the two sphere method available in the program, centering a 15 A cavity within residues M294, I356, S336 and Y373 using 1.5 and 4.0 A for the radius of small and large spheres respectively. As pharmacophoric restraint an aromatic ring was forced within 4 A of the the center of the cavity.
Coincident binding modes were obtained for the 3 compounds. However, to minimize the differences between binding modes of the different compounds, poses for the 17 compounds in the set were derived from a common scaffold.
To this end, the BM of the largest compound FXR_102 was selected as a template and subsequently modified in Maestro to obtain poses that were minimized using the internal forcefield to avoid steric clashes.