In silico and experimental characterization of fipronil-membrane interactions: Implications for pesticide selectivity.

Fipronil is a widely used phenylpyrazole insecticide that acts by blocking γ-aminobutyric acid type A receptors (GABAA-R) in insects. Fipronil exhibits significant toxicity in vertebrates, despite having higher affinity for invertebrate receptors. This suggests that other mechanisms beyond the receptor-binding affinity contribute to its biological effects. This study investigates how membrane composition influences fipronil solubility and bioavailability using molecular dynamics simulations and experimental approaches. We compared fipronil interactions with vertebrate and invertebrate model membranes' compositions. The former are composed of varying concentrations of phosphatidylcholine-cholesterol while the latter present different concentrations of phosphatidylcholine-phosphatidylethanolamine. Our results demonstrate that fipronil maintains high membrane solubility across all lipid systems, in contrast to the structurally related insecticide fluralaner, which aggregates in cholesterol-rich membranes. Free energy calculations reveal that fipronil partitioning is entropy-driven, with the molecule adopting a stable orientation at the membrane interface through strong van der Waals interactions with lipid tails and hydrogen bonding with polar headgroups. Unlike fluralaner, fipronil preserves its interaction network and solubility even at high cholesterol concentrations, remaining dispersed rather than aggregating. Langmuir monolayer experiments confirm fipronil integration into lipid films without disrupting membrane architecture. These findings provide a mechanistic framework to explain FIP's unexpectedly high toxicity in vertebrates, whose membranes often contain elevated cholesterol. Our results highlight a membrane-mediated mechanism that complements receptor-level interactions and highlights the importance of membrane composition in understanding insecticide selectivity.