Sulfonamide Derivatives as Competitive Lactoperoxidase Inhibitors Through Integrated Experimental and Multi‐Level Computational Analysis

ABSTRACT Lactoperoxidase (LPO; EC 1.11.1.7) is a milk‐derived heme oxidoreductase that plays a central role in the innate antimicrobial defense of mammals by catalyzing the H 2 O 2 ‐dependent oxidation of substrates such as thiocyanate. Beyond this physiological role, the selective inhibition of LPO has gained increasing attention in both mechanistic enzymology and therapeutic development, particularly in oxidative stress‐related contexts. In this study, three sulfonamide derivatives, naphthalene‐2‐sulfonamide (Naphthalene‐2‐SA), 2,5‐dichlorothiophene‐3‐sulfonamide (2,5‐DCTSA), and 6‐aminopyridine‐3‐sulfonamide (6‐APSA), were evaluated as bovine LPO inhibitors using a combined experimental and multi‐level computational approach. Bovine LPO was purified approximately 509‐fold from skimmed milk by affinity chromatography, with a final specific activity of 168 EU/mg. Kinetic assays revealed that all three compounds behaved as competitive inhibitors, with IC 50 values of 0.69, 1.19, and 11.95 µM and Kᵢ values of 0.50, 2.50, and 4.71 µM, respectively. Naphthalene‐2‐SA emerged as the most potent inhibitor and consistently ranked highest across all computational analyses, including molecular docking (−8.54 kcal/mol), 100 ns molecular dynamics simulations, and MM‐GBSA binding free energy calculations (−20.24 kcal/mol). DFT‐based reactivity descriptors (HOMO–LUMO gap, chemical softness) followed the experimental potency trend, while ADMET profiling further confirmed favorable drug‐like properties for all three compounds. The convergence of experimental kinetics with multiple independent computational observables, namely docking, MD stability, MM‐GBSA energetics, and DFT reactivity, provides new mechanistic insights into sulfonamide–LPO interactions and identifies Naphthalene‐2‐SA as a promising lead scaffold for the rational design of next‐generation LPO inhibitors.