Inhibition and Molecular Recognition of Lactoperoxidase by Acetohydrazide-Based Sulfonamide Derivatives

Abstract

Lactoperoxidase (LPO) is a heme-containing enzyme widely distributed in mammalian secretions, contributing to host defense through the generation of antimicrobial oxidants. Unintended inhibition of LPO by therapeutic agents may impair mucosal immunity, making the investigation of potential inhibitors clinically relevant. In this study, the inhibitory effects of 11 acetohydrazide-based sulfonamide derivatives on bovine milk LPO were investigated using combined biochemical and computational approaches. The compounds exhibited diverse inhibition profiles, with IC₅₀ values ranging from 2.32 ± 0.14 to 13.4 ± 2.05 μM. The corresponding Kᵢ values were determined in the low micromolar range, varying from 1.25 ± 0.05 to 7.53 ± 0.03 μM. Among them, 4b-2 (3-pyridyl) and 4a-5 (2-naphthyl) derivatives were identified as the most potent inhibitors, acting through a competitive inhibition mechanism with low micromolar Kᵢ values. The binding mechanisms of relatively active compounds in the enzymatic assay were explored through molecular modeling. The docking study disclosed that compounds 4a-5 and 4b-2 would show higher binding potential to the LPO structure. The two compounds formed stable complexes with the enzyme structure according to the molecular dynamics (MD) simulation study. These interactions were mainly governed by hydrogen bonding and π–π stacking interactions involving key residues in the heme-containing active site, supporting a competitive binding mode consistent with the kinetic data. Overall, the combined biochemical and computational findings suggest that acetohydrazide-based sulfonamides represent promising scaffolds for the development of potent and selective LPO inhibitors and provide new insights into the molecular recognition patterns governing their interaction with the LPO active site.