Chain-length-selective inhibition of human and rat steroid 5α-reductase type 1 by alkyltrimethylammonium chloride disinfectants: kinetic, surface plasmon resonance, computational, and network toxicology analyses with implications for neurosteroidogenesis.

Alkyltrimethylammonium chlorides (ATMAs) are a major subclass of cationic quaternary ammonium compound (QAC) surfactants whose human exposure has risen sharply since 2020, but their effect on the brain neurosteroidogenic enzyme steroid 5α-reductase type 1 (SRD5A1) is unknown. Eight ATMAs from C1 to C22 were tested on human and rat SRD5A1 microsomes using HPLC-MS/MS, complemented by surface plasmon resonance (SPR), molecular docking, three-dimensional quantitative structure-activity relationship (3D-QSAR) modelling, and a network toxicology workflow. Screening at 100 μM identified ATMA-C14 and ATMA-C16 as the only active inhibitors against either species. The C14 and C16 compounds inhibited human SRD5A1 with IC50 of 50.94 μM and 46.98 μM respectively (Ki 55.51 and 52.95 μM); against rat SRD5A1, ATMA-C16 retained equal potency (IC50 46.75 μM) but ATMA-C14 lost approximately half its activity (IC50 108.62 μM), revealing a species shift around the C14 homologue. An equimolar C14/C16 mixture was synergistic against the human enzyme but antagonistic against the rat enzyme, mirroring the species difference in single-agent potency. Both compounds behaved as mixed/non-competitive inhibitors with respect to testosterone, and cofactor-titration kinetics together with SPR competition data placed the binding site at the NADPH pocket. SPR returned KD of 52.8 and 59.3 μM with rapid dissociation, consistent with reversible non-covalent binding. In intact SF126 cells the order was reversed, ATMA-C14 suppressing DHT production more than ATMA-C16, in keeping with differential membrane permeability. Docking located both ligands in the NADPH pocket (ΔG -5.78 to -6.65 kcal/mol), and a 3D-QSAR pharmacophore emphasised one hydrogen-bond acceptor and four hydrophobic features. Network toxicology pointed, in silico, to Alzheimer's disease as the most enriched disease intersection. ATMA-C14 and ATMA-C16 thus emerge as moderate, reversible SRD5A1 inhibitors with chain-length and species-dependent potency, raising potential endocrine-disruption concerns that warrant exposure-relevant assessment.