Lipophilicity and alkyl chain length govern pyridine quaternary ammonium disinfectants as gonadal 3β-hydroxysteroid dehydrogenase inhibitors: Implications for endocrine disruption.

Pyridine quaternary amino disinfectants (PYRs) are extensively used as antimicrobial agents. However, the specific PYRs responsible for inhibition and their structure-activity relationship (SAR) for human 3β-hydroxysteroid dehydrogenase 2 (h3β-HSD2) and its homolog rat r3β-HSD1, the critical enzyme in gonadal steroidogenesis, remain poorly understood. This study evaluated eleven PYRs for their inhibitory effects on h3β-HSD2 and r3β-HSD1, determining potency (IC50), inhibition kinetics, cellular impact, binding interactions, and toxicology prediction via molecular docking, 3D-QSAR, and network toxicology analysis. Inhibitory potency (IC50) revealed C18 (8.68 μM) > C16 (15.45 μM) > C14 (21.70 μM) > C20 (36.10 μM) > C12 (57.83 μM) > C1-C8 PYRs (no inhibition at 100 μM) against h3β-HSD2 and C18 (1.61 μM) > C16 (2.54 μM) > C20 (5.56 μM) > C14 (7.91 μM) > C12 (38.06 μM) > C1-C8 PYRs (no inhibition at 100 μM) against r3β-HSD1. Mechanism of action revealed mixed/noncompetitive inhibition for both enzymes. Cellular suppression showed that C12-C18 PYRs effectively inhibited progesterone synthesis in human KGN granulosa tumor cells at 1 and 10 μM. Molecular docking and SAR showed that binding occurs at the NAD+/steroid interface via hydrogen bonds, hydrophobic interactions, and van der Waals forces, and lipophilicity (LogP), steric bulk (molecular weight, alkyl chain length and volume), Fsp3, and flexibility positively correlated with potency (pIC50), indicating that longer alkyl chains (up to C18) enhance inhibition, and 3D-QSAR confirmed the importance of hydrophobic regions in binding affinity due to long alkyl chain. Network toxicology analysis revealed common pathway (nuclear receptor signaling/steroidogenesis) in male hypospadias. In conclusion, PYRs inhibit gonadal 3β-HSD activity in a chain-length-dependent manner (C12-C18), with lipophilicity as a key determinant. These findings suggest that PYR disinfectants may act as potential endocrine disruptors by interfering with steroidogenesis.