Figure 2.

Alchemical cycle to compute free energy of the transfer of the PL-indomethacin prodrug molecules to the PLA₂ transition state geometry. (ΔG₁ + ΔG₅), the energies for bond cutting/forming cancel out. Free energies of pulling the drug molecule to the end of the linker are computed using the umbrella sampling technique. (ΔG₂ + ΔG₄), the differences in free energies of changing the linker length in the complex with PLA₂ and in the initial state (lipid or water) are computed using the free energy perturbation (FEP) method.