Fig. 2. Molecular docking, enzyme inhibition, and cell-based studies of PREPi. (A) Lineweaver–Burk plot of PREPi inhibition at various PREPi concentrations. (B) Detailed Lineweaver–Burk plot of PREPi inhibition focusing on the concentration range, highlighting the shift in slope at different inhibitor concentrations. (C) Secondary plot showing the slope of the Lineweaver–Burk plot versus the PREPi concentration. (D) Predicted binding interactions of (±)-2-pentacosylcyclohexanol (PREPi) within the active site of prolyl endopeptidase (PREP), showing key hydrogen bonds and hydrophobic interactions. (E) Radius of Gyration (RoG) plot illustrating the compactness of the PREPi–PREP complex. (2F) RMSF (Root Mean Square Fluctuation) analysis showing minimal flexibility at the active site of PREP upon PREPi binding. (G) RMSD (Root Mean Square Deviation) analysis indicating the stability of the PREPi-PREP complex during molecular dynamics simulations (H) Dynamic Cross-Correlation Matrix (DCCM) analysis between active site residues upon PREPi binding. (I) HEPG2 cell viability at various concentrations of PREPi and Kyp-2047. (J) Effect of varying doses of PREPi on PA-induced lipotoxicity model in HepG2 cells. (K) Triglyceride (TG) quantification in PA-induced HepG2 cells. All data are presented as means ± SD. Statistical significance is indicated as follows: p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), p < 0.0001 (****), “ns” denotes non-significant difference.