DDOX expands the repertoire of tetracyclines for Parkinson's disease by preventing the cellular uptake and intracellular impact of α-synuclein preformed fibrils.

The increasing prevalence of Parkinson's disease (PD) requires innovative multi-targeted disease-modifying therapies to counteract the toxicity associated with the amplification, propagation, and accumulation of alpha-synuclein (α-Syn) aggregates in the brain. Tetracyclines, particularly doxycycline, have demonstrated multimodal neuroprotective effects, both in vitro and in vivo. The non-antibiotic derivative of doxycycline 4-dedimethylamino-12a-deoxydoxycycline (DDOX), has been recently shown to rescue neurons from oxidative injury. Here, we demonstrate that DDOX showcases a diverse range of mechanisms targeting α-Syn aggregates. Notably, DDOX inhibited the aggregation of α-Syn and the seeding ability of α-Syn pre-formed fibrils (PFF) in biophysical and cellular assays. In addition, the compound ameliorated the relocalization of total and phospho-α-Syn, triggered by exogenous α-Syn PFF. Surprisingly, DDOX drastically mitigated lysosomal stress induced by these aggregates. Moreover, we determined that DDOX effectively impeded the internalization of fluorescently labeled α-Syn PFF. Biophysical techniques and molecular docking simulations suggest that DDOX binds to hydrophobic patches on α-Syn fibrils. Our findings reveal novel neuroprotective attributes of tetracyclines, wherein a direct extracellular interaction between DDOX and α-Syn aggregated species mitigates their intracellular impact. These results provide a promising foundation for DDOX, a drug that aims to interfere with the intracellular seeding, propagation and uptake of α-Syn fibrils in neurodegenerative conditions.