Each protein-ligand complex was placed in a cubic TIP3P water box with a padding distance of 1 nm to the surface of the protein. Sodium and chloride ions were added to neutralize the system and obtain an ionic concentration of 150 mM. Simulations for heating and pressure adjustment were performed with Amber24 53 , with each structure undergoing five separate simulations, resulting in five replicas. The complex was energy-minimized in four stages using the steepest descent method, a maximum of 1500 steps and gradual reduction of the positional restraints applied on the protein's heavy atoms (500 kcal/mol/Å², 100 kcal/mol/Å², 5 kcal/mol/Å², 0). In the next step, the energy-minimized system underwent an NVT ensemble heating procedure, with the temperature being increased from 10 K to 300 K. Positional restraints of 50 kcal/mol/Å² were applied to the heavy atoms during this step. The simulations were performed for 25,000 steps with a time step of 0.002 ps using the Nose-Hoover thermostat and Langevin dynamics with a friction coefficient of 1.0. This was followed by an equilibration protocol consisting of four steps performed in an NPT ensemble with a constant temperature of 300 K using the Nose-Hoover thermostat , a constant pressure of 1 bar using the Berendsen barostat and gradual reduction of the positional restraints over the heavy atoms of the protein (50 kcal/mol/Å², 10 kcal/mol/Å², 2 kcal/mol/Å², 0 kcal/mol/Å²). Each step of the equilibration consisted of 10000 steps, with a time step of 0.002 ps. Van der Waals and electrostatic forces were computed using a cutoff of 1.0 nm.