C-18

Biocide disinfectants are essential chemicals in infection control, but their use may cause adverse effects by inhibiting neurosteroid biosynthesis. In the current study we systematically examine the inhibitory effect of the biocide benzalkonium chloride (BAC), which is widely used for surface disinfection and as preservative in many consumer products, on human and rat 5α-reductase 1 (5α-R1) activity. The effects of BAC component (C1-C18) on brain 5α-R1 were investigated using HPLC-MS/MS measurement of steroid substrate testosterone and product dihydrotestosterone for enzyme inhibition screening assay and kinetics analysis, surface plasmon resonance (SPR) binding, intact cells, in silico docking and molecular dynamic simulations as well as structure-activity relationship (SAR)/3D-QSAR modeling. BAC component inhibited both human and rat 5α-R1, depending on alkyl chain length, with C10⁺ components exhibiting significant activity. C18 component emerged as the most potent inhibitor (IC₅₀ = 9.54 μM for human 5α-R1; 34.36 μM for rat 5α-R1). SPR assay showed that C18 had a high affinity binding with KD of 6.42 μM with a relatively slow dissociation rate, indicating stable target engagement once bound. Mechanistic studies identified mixed/noncompetitive inhibition of 5α-R1 by BAC components by competing with NADPH (SPR assay). In intact SF126 cells, C12-C18 components suppressed 5α-dihydrotestosterone formation at 10-100 μM. Pearson correlation analysis linked inhibitory strength to structural parameters (LogP, molecular weight, alkyl carbon count, heavy atoms, and rotatable bonds). A 3D-QSAR pharmacophore model highlighted hydrophobic interactions as critical for inhibition. Molecular docking further elucidated binding mechanisms, showing that BAC components occupy the NADPH-binding site via van der Waals forces, hydrogen bonds, hydrophobic contacts, and charge interactions. Our results suggest that BAC components have the potential to inhibit human and rat 5α-R1 activity and cause the neural disorders. These findings underscore BAC as potential endocrine disruptor by targeting neurosteroid biosynthesis, warranting further investigation into their neurotoxicological implications. This work was funded by the Zhejiang Nature and Science Foundation. The funders had no role in study design, interpretation or decision to publish.

Benzylalkyldimethylammonium (BAC) quaternary disinfectants are extensively used as antimicrobial agents. However, the specific BACs responsible for inhibition and their structure-activity relationship (SAR) for human 3β-hydroxysteroid dehydrogenase 2 (3β-HSD2) and its homolog rat 3β-HSD1, the critical enzyme in gonadal steroidogenesis, remain poorly understood. This study evaluated nine BACs for their inhibitory effects on human 3β-HSD2 and rat 3β-HSD1, determining potency (IC₅₀), inhibition kinetics, cellular impact, and binding interactions via molecular docking and 3D-QSAR. Inhibitory potency (IC₅₀) revealed C18 (11.84 μM) > C16 (15.51 μM) > C14 (25.25 μM) > C12 (30.63 μM) > C10 (56.86 μM) > C1-C8 BACs (no inhibition at 100 μM) against human 3β-HSD2 and C18 (8.23 μM) > C16 (10.74 μM) > C14 (12.21 μM) > C12 (14.38 μM) > C10 (26.62 μM) > C1-C8 BACs (no inhibition at 100 μM) against rat 3β-HSD1. Both enzymes exhibited mixed-type inhibition. Cellular suppression showed that C14, C16, and C18 BACs inhibited progesterone synthesis in human KGN granulosa tumor cells at ≥ 5 μM. Docking and SAR revealed binding at the NAD+ /steroid site via hydrogen bonding, hydrophobic, and van der Waals interactions. Lipophilicity (LogP), steric bulk, Fsp3, and flexibility correlated positively with potency, indicating that longer alkyl chains (≤C18) enhance inhibition. 3D-QSAR confirmed the importance of hydrophobic regions in binding affinity due to long alkyl chain. In conclusion, BACs inhibit gonadal 3β-HSD activity in a chain-length-dependent manner (C10-C18), with lipophilicity as a key determinant. These findings suggest that BAC disinfectants may act as potential endocrine disruptors by interfering with steroidogenesis.