Ribone_2026_Methods.Mol.Biol_3021_369

The two more relevant isoforms of human carboxylesterases (CES), CES1 and CES2, are responsible for catalyzing the hydrolysis of numerous approved drugs and prodrugs. Elucidating the substrate specificity of each CES isoform is a highly significant topic in medicinal chemistry. The general rule suggests that selectivity toward CES1 or CES2 depends on the relative sizes of the acyl and alkyl moieties present in the substrate structure; however, numerous exceptions to this trend have been reported. In this chapter, stepwise methodologies combining classical molecular mechanics (MM) and hybrid quantum mechanics/molecular mechanics (QM/MM) simulations are applied to support experimental enzymatic assays aimed at elucidating CES1 and CES2 substrate selectivity. The classical MM strategies are employed to investigate substrate binding affinity to the catalytic site ( KM ) through molecular docking, molecular dynamic simulations (MD), and free-energy interaction analyses. The hybrid QM/MM-MD simulations are used to study the hydrolytic reaction coordinates, thereby modeling the enzymatic turnover rate ( kcat ).