Modulation of amyloid formation in the hSOD1 R115G mutant by an ionic liquid ([BMIM][SCN]).

Protein aggregation is crucial to the molecular pathogenesis of amyotrophic lateral sclerosis (ALS), particularly in cases involving superoxide dismutase 1 (hSOD1) mutants. There is increasing focus on the development of small-molecule modulators that can disrupt aggregation pathways. Recently, ionic liquids (ILs) have been recognized as effective modulators of protein aggregation due to their tunable physicochemical properties. This study employed a combined computational and experimental approach to assess the inhibitory efficacy of 1-butyl-3-methylimidazolium thiocyanate ([BMIM][SCN]) on amyloid formation induced by the ALS-associated R115G mutation in hSOD1. Molecular dynamics (MD) simulations were conducted to obtain atomic-level insight into the inhibitory mechanism, demonstrating that [BMIM][SCN] primarily interacts with aggregation-prone loop regions in the R115G mutant, diminishing local flexibility and stabilizing partially folded intermediates. These interactions likely disrupt early nucleation processes essential for fibril propagation. The anti-amyloidogenic effects of [BMIM][SCN] were further confirmed under aggregating conditions using Thioflavin T (ThT) fluorescence kinetics, which exhibited a significant, concentration-dependent decrease in fibril formation. This trend was confirmed by transmission electron microscopy (TEM), which demonstrated a distinct suppression of fibrillar structures. Furthermore, ANS binding assays indicated reduced exposure of hydrophobic regions, implying a shift toward more compact, less aggregation-prone conformations. Fourier-transform infrared (FTIR) spectroscopy supported these findings by demonstrating a decrease in β-sheet-rich secondary structures commonly linked to mature amyloids. These findings indicate that [BMIM][SCN] modulates aggregation of the R115G mutant, providing mechanistic insights into how [BMIM][SCN] influences amyloid formation. These results may guide the rational design of biocompatible ionic-liquid-based analogs with potential therapeutic applications for ALS.