Hinokitiol as a Potential GPX4 Allosteric Modulator to Prevent Ferroptosis-mediated Neurodegeneration in Parkinson's Disease.

Ferroptosis is a form of cell death driven by iron-dependent lipid peroxidation. Recently, it was identified as a key factor in Parkinson's disease (PD). Through gene network analysis, we identified glutathione peroxidase 4 (GPX4) as a bottleneck node in the ferroptosis pathway. Allosteric activation of GPX4, or increased gene/protein expression of GPX4, was shown to prevent neurodegeneration. Accordingly, we sought to identify novel phytochemicals that could act as allosteric modulators of GPX4. In our study, hinokitiol showed the best ligand efficiency for GPX4's allosteric pocket. We further validated the binding stability using molecular dynamics (MD) simulations. Hinokitiol showed stable binding throughout the 500ns MD run. Notably, hinokitiol induced conformational changes in residues near the catalytic binding site, which might influence substrate binding to GPX4. We examined the neuroprotective effects of hinokitiol in retinoic acid-differentiated SH-SY5Y cells exposed to rotenone (10 µM). Malondialdehyde (MDA), intracellular iron content, and GPX4 gene expression were quantified in SH-SY5Y cells. Pretreatment with hinokitiol (25 and 50 µM) significantly protected SH-SY5Y cells from rotenone-induced toxicity. Hinokitiol markedly reduced rotenone-induced MDA and iron levels and increased GPX4 gene expression. In Drosophila melanogaster, rotenone reduced mean survival to approximately 19 days, whereas co-treatment with hinokitiol extended lifespan to ~ 23 days (500 ppm) and ~ 27 days (1000 ppm). Similarly, in rotenone-induced PD mice, hinokitiol reduced catalepsy and improved locomotion and motor coordination. Collectively, these findings suggest that hinokitiol could mitigate ferroptosis-mediated neurodegeneration, possibly by modulating GPX4 activity.