Computational Drug Discovery and Design

1. Cap protein termini with ACE and NME residues if the protein
   is not fully resolved at the extremities (seeNote 1). Then, save
   the protein with the ligand for the unbinding simulation using
   the US and without ligand for conformational sampling
   with aMD.


2. Predict and assign the protonation states of protein residues at
   pH¼7 using the PROPKA program (http://propka.org/).


3. Calculate automatic charges and generate mol2 file for the
   ligand using Antechamber program in AmberTools package.
   Next, construct additional frcmod file for the ligand with
   parmchk2.


4. With tleap program in AmberTools, we protonate, solvate, and
   create the topology and coordinate files using ff14SB and Gaff
   force fields to describe respectively the protein and the ligand.
   The TIP3P model of water is used to represent explicit water
   molecules as solvent. Na+and Clare chosen as counterions to
   neutralize system.

At the end of these steps, we obtain the system topology file

(.prmtop) describing the parameters and the topology of the mole-

cules in the system, and the initial molecular coordinates of the

system (.rst7).

3.2 System
Minimization and
Equilibration

Now that the system parameters have been set, a preliminary pro-

tocol has to be carried out before running MD simulations. This

protocol, constituting of both minimization and equilibration

steps, allows the relaxation of the 3D structure of the protein and

the equilibration of water molecules around the system. Most of

the time, the equilibration begins with a thermalization step, in

which the system reaches the thermal equilibrium at the desired

temperature. Our equilibration protocol was carried out in five

steps (seeNote 2):

1. Minimization of experimentally structure to find a stable state
   (a minimum) on the potential energy surface from which to
   begin the simulation. The minimization was performed in two
   steps: first we minimize the solvent box and second we mini-
   mize all the system (protein/ligand/solvent box).


2. Thermal equilibration: the system is incrementally thermalized
   from 0 to 300 K for 10 ps.


3. Density equilibration: equilibrate the density of the system
   (NPT) with weak harmonic restraints on the complex during
   200 ps.


4. Relaxation of the system in the NVT ensemble by gradually
   decreasing the harmonic restraints during 600 ps.


5. cMD production with the complete relaxation of the system
   (no harmonic restraint) during 50 ns.

408 Sonia Ziada et al.