Thymoquinone mediated inhibition of α-Synuclein fibrillation: Insights from biophysical and thermodynamic studies

Abstract

The fibrillation of presynaptic α-Synuclein (α-Syn) protein and its subsequent accumulation in the nerve cells is a major causative factor responsible for neurodegenerative Parkinson’s disease (PD). Understanding the biophysical and thermodynamic aspects of the mechanism of inhibition of α-Syn fibrillation by inhibitor molecules is pivotal for designing the therapeutic interventions targeted at PD. The current study explores the biophysical and thermodynamic aspects of the binding, inhibition, modulation and disintegration of α-Syn fibrils by thymoquinone (THQ). The fluorescence spectroscopy shows that THQ interacts with α-Syn with affinity of (2.1 ± 0.2) × 10⁴ mol⁻¹⋅kg. Molecular docking and isothermal titration calorimetry studies reveal that thymoquinone (THQ) primarily binds to α-Syn through hydrophobic interactions, with docking pinpointing the NAC region as the key binding site. This region, crucial for aggregation, aligns with ITC findings that highlight the dominance of hydrophobic forces in THQ’s interaction. Kinetic studies using ThT fluorescence and light scattering studies demonstrate that THQ inhibits α-Syn fibrillation, further confirmed by TEM morphological analysis. Seeding experiments reveal that THQ forms seeding-incompetent oligomers incapable of inducing fibrillation in monomeric α-Syn. Additionally, THQ not only halts fibrillation after it begins but also disintegrates preformed amyloid fibrils. These findings will offer insightful understandings into the therapeutic effects of THQ on α-Syn fibrillation and contribute towards the ongoing efforts for therapeutic interventions targeted at PD.