Drug Development

Proteostasis, ensuring proteome stability and functionality, relies on components such as the ubiquitin-proteasome system (UPS) and autophagy. Dysregulation of these pathways, particularly in aging, leads to protein aggregation in neurons-a hallmark of neurodegenerative diseases. Activating proteasome has received most attention, however recent evidence suggests that UPS can clear aggregate proteins and a potential therapeutic target for Alzheimer disease and other protein misfolding diseases.

Phenothiazine- and imidazoline-based small molecules were synthesized and screened in SH-SY5Y neuroblastoma cells for proteasome activation and cell viability improvement. Docking and molecular dynamics simulations (PDB: 4R3O) showed selective binding to the α1/α2 intersubunit pocket of the proteasome, involving stabilizing interactions such as hydrogen bonding and π-stacking. Protein activity assays confirmed enhanced 20S proteasome activity. The compounds were highly biocompatible across various cell lines (e.g., HeLa, HEK293T, SH-SY5Y), with no toxicity even at 200 µM. Pretreatment protected cells from H₂O₂-induced apoptosis and significantly reduced reactive oxygen species (ROS). In vivo studies in APP/PS1 Alzheimer's mice evaluated effects on memory, amyloid plaque clearance, and proteasome activity.

Phenothiazine- and imidazoline-based molecules, designed with diverse functional groups and halogen substituents, selectively bound and activated the proteasome via gate-opening mechanisms. Among tested compounds, one exhibited superior efficacy, enhancing proteasome activity at nanomolar concentrations without toxicity. In APP/PS1 mice, administering compound one (50 mg/kg) for 14 days improved contextual memory, reduced amyloid plaques, and restored proteasome function. In SH-SY5Y cells, the compound provided neuroprotection against oxidative stress induced by H₂O₂.

This study introduces direct proteasome activation via gate-opening as a novel therapeutic approach for neurodegenerative diseases. Validated in SH-SY5Y cells and Alzheimer's mouse models, the findings demonstrate reduced amyloid plaques, improved memory, and enhanced neuroprotection. These results establish a robust foundation for advancing proteasome activators as therapeutic agents.