*3.4. MD Simulations*

Simulations were conducted using processor Intel(R) Xeon(R) CPU E5-2680 v4 @ 2.40GHz, 64 bit. MD simulation was performed using the optimal receptor–ligand complex position and an evaluation of the conformational space and inhibitory potential. MD simulation analysis with the GROMOS96 43a1 force field was performed using the Gronningen machine for chemical simulations (GROMACS, version 2019.2 package).

Both ligands0 parameter files and topologies were created using the latest CGenFF via CHARMM-GUI [25,26]. Online server CHARMM-gui was used to insert the dipalmitoylphosphatidylcholine (DPPC) membrane. Seventy two DPPC molecules were added to upperleaflet and lowerleaflet. SPC water models that extended 10 Å from the receptor were used to examine the receptor–ligand configurations in a rectangular box [27]. 37 K<sup>+</sup> and 46 Cl− ions (0.15 M salt) were administered to neutralize the systems and reproduce physiological salt concentrations (Figure 15).

**Figure 15.** Lateral view of CTtD complex incorporated in dipalmitoylphosphatidylcholine (DPPC) membrane in a rectangular box solvated with water molecules and neutralized with 37 K<sup>+</sup> and 46 Cl<sup>−</sup> ions (0.15 M salt).

A constant temperature (300 K) and constant pressure (1.0 bar) over 100 ns were used for simulations using the leap-frog MD complement in the NPT/NVT equilibration run [28]. The steepest descent technique with 5000 steps was also used to reduce improper contact within the system [29]. Hydrogen bonds checked with the gmx hbond tool. Rg and SASA were calculated using the gmx gyrate and gmx sasa programs, respectively. The RMSD of protein was calculated using the gmx rms tools. Trajectory examination was accomplished using GROMACS program [30]. The plots were made with Grace Software, and the visualization was done with PyMol/VMD [8,31,32].
