Ligand-Receptor Complex Molecular Dynamics Simulation

Docking consideres the receptor to be rigid, which may affect the precision of the result. To simulate realistic conditions, Molecular Dynamics (MD) simulations were conducted Using GROMACS 2024.4 to evaluate system dynamics, stability, and predicted interactions over time [49].

To investigate the dynamic interaction of synthesized compounds compared to Morphine, molecular dynamic (MD) simulations were performed using 3d, as a promising compound from the docking studies, and Morphine, as a well-known agonist. For docking simulations, the DOCK 6.11 software was employed as previously described [48]. For the protein, the structure was obtained from the Protein Data Bank (PDB code: 5C1M). Chimera software was used to clean the protein structure by removing chain B, water molecules, and all ligands in preparation. Hydrogen atoms and atomic charges were added, and the resulting structure was saved in mol2 format. A duplicate of the structure was created without hydrogen atoms for molecular surface generation. Using the sphere generation algorithm, spheres were created on the protein surface to represent binding pockets, which were clustered. Ligand binding site spheres within a 10 Å radius of the crystallized ligand were selected for search space determination.

The CHARMM-GUI web server was used to construct the simulation system. Protein-ligand complexes were uploaded via the Membrane Builder module, and PPM2 was employed to determine receptor orientation in the membrane [50]. The system included a Lipid bilayer of 128 POPC molecules in each layer, Water, and ions for neutralizing the system. For the forcefield, CHARMM36 was applied for topology and parameter files.

Afterwards, the system underwent energy minimization using steepest descent (SD) method to eliminate spatial clashes and achieve stability. Minimization continued until the maximum force was less than 1000 kJ/mol/nm. Equilibration was performed in two steps of NVT ensemble, stabilized system at a temperature of 310 K, and NPT ensemble, Stabilizing system pressure and density using the Parrinello-Rahman barostat. Following equilibration, MD simulations were executed for 100 ns using the GROMACS 2024 software with the full-atom CHARMM force field.