Benzalkonium chloride disrupts human placental progesterone synthesis via locking NAD+ pocket in 3β-Hydroxysteroid dehydrogenase 1 and rat homolog: docking evidence and 3D-QSAR, and experiment risk assessment.

Benzalkonium chloride (BAC) disinfectants are widely used, but their potential to disrupt reproductive endocrine systems remains known. Human placental 3β-hydroxysteroid dehydrogenase 1 (h3β-HSD1) plays a key role in progesterone biosynthesis and is the potential target. This study investigates the inhibitory effects of 9 BACs on h3β-HSD1 and rat placental (r3β-HSD4) homolog. Kinetic analyses revealed h3β-HSD1 exhibited a Michaelis constant Km of 0.261 μM, while r3β-HSD4 showed similar Km (0.254 μM). Screening identified C12-C18 BACs as potent inhibitors (IC50: 1.15-20.43 μM for h3β-HSD1; 1.90-20.75 μM for r3β-HSD4), with mixed/noncompetitive inhibition confirmed by Lineweaver-Burk plots. A BAC mixture demonstrated antagonistic effects. Functional cell assays in JAr cells showed that C12-C18 suppressed progesterone biosynthesis, with potency influenced by membrane permeability and inhibitory strength. Structure-activity relationship (SAR) and 3D-QSAR models highlighted hydrophobicity, alkyl chain length as well as steric features as key determinants, validated by pharmacophore mapping. Molecular docking revealed conserved NAD+-binding interactions, with C18 exhibiting the highest affinity via hydrogen bonds and hydrophobic contacts as well as van der Waals. Molecular dynamics simulations confirmed stable enzyme-ligand complexes. Pharmacokinetics prediction profiling indicated variable solubility (C12 > C14 > C16 = C18). These findings elucidate BAC-mediated endocrine-disrupting mechanisms and support structure-based optimization of inhibitors.