Next, we estimated dU dλ at five different lambda values: 0, 0.25, 0.5, 0.75, and 1.0 for the same time interval. Simulations were conducted for both the ligand in water and the proteinligand complex in water. Additionally, dU dλ was assessed under four different conditions: (i) SHAKE constraints for hydrogen atoms with Hydrogen Mass Repartitioning (HMR) applied to both the ligand and the protein-ligand complex, (ii) SHAKE constraints for hydrogen atoms without HMR for both the ligand and protein-ligand complex, (iii) no SHAKE con-straints for hydrogen atoms with HMR applicable to both the ligand and protein-ligand complex, and (iv) no SHAKE constraints and no HMR applied to either the ligand or the protein-ligand complex. In total, 120 simulations were performed for each run. We will evaluate dU dλ with SHAKE and HMR using different time steps. The dU dλ values were collected using in-house Python code.