In Vitro, In Silico and Structure-Activity Relationship Evaluation of 3-Amino-thiophene-2-carbohydrazides as...

In this study, 18 arylidenehydrazide derivatives (1-18) were systematically evaluated for their inhibitory activity against lactoperoxidase (LPO, EC 1.11.1.7). The inhibitory potential of the derivatives was investigated using in vitro spectrophotometric assays, yielding nanomolar-level K_i values. Among the tested compounds, compound 18 exhibited the most potent activity (IC₅₀ = 0.33 nM, K_i = 0.05 nM) through a competitive inhibition mechanism, whereas compound 10 demonstrated strong non-competitive inhibition (IC₅₀ = 0.52 nM, K_i = 0.20 nM). Complementary in silico analyses, including molecular docking, MM-GBSA calculations, and 250 ns molecular dynamics (MD) simulations, confirmed stable ligand-protein interactions at the active site for compound 18 and at a putative allosteric site for compound 10. The computational findings supported the experimental results by revealing favorable binding affinities driven by hydrogen bonding, π-π stacking, and halogen interactions, maintaining stable interaction profiles throughout the equilibrated simulation period. Furthermore, a structure-activity relationship (SAR) analysis revealed that specific molecular modifications markedly affected the inhibitory potency. In particular, the presence of a hydroxyl (-OH) group at the C1 position and a bromide (-Br) group at the C4 position significantly enhanced the inhibitory activity against LPO. Overall, this integrated experimental and computational study provides deep mechanistic insights into LPO inhibition and establishes a robust foundation for the rational design of novel arylidenehydrazide-based LPO inhibitors.