Mechanistic Insights into the Binding Stability and Covalent Inhibition of Lactone- Based Pancreatic Lipase Inhibitors Using Molecular Dynamics and Binding-Pose Metadynamics

Abstract Pancreatic lipase (PL) is a key digestive enzyme involved in dietary lipid hydrolysis and represents an important target for the development of lipid-lowering agents. In the present study, the binding dynamics, stability, and covalent inhibition characteristics of microbial and synthetic lactone-based PL inhibitors were investigated using molecular docking, molecular dynamics (MD) simulations, binding free-energy calculations, binding-pose metadynamics, and water-tunnel analysis. The inhibitors predominantly occupied the S1–S2 region of the catalytic site in the open conformation of PL, where their structural features, including the β-lactone ring, amide-containing substituents, and hydrophobic alkyl chains, contributed to ligand recognition and stabilization. Comparative binding free-energy calculations using the Amber ff14SB and OPLS3e force fields showed that Esterastin and Orlistat exhibited the most favourable computed binding energetics, driven predominantly by electrostatic and van der Waals interactions. Binding-pose metadynamics further demonstrated enhanced stability of the covalently bound inhibitor complexes and provided insight into differences in their conformational changes and residence time within the catalytic pocket. Further, the unbinding of inhibitor dependence through dynamic solvent pathways connecting the protein surface to the catalytic region and indicated that water molecules in the vicinity of the catalytic residue Ser153 may influence hydrolytic accessibility and thereby modulate the reversibility of lactone inhibition. Collectively, these findings provide a molecular-level understanding of how lactone architecture, active-site interactions, covalent-complex stability, and catalytic-site hydration govern binding affinity to pancreatic lipase inhibition.