In vitro evaluation of heteroaryl-hydrazones as modulators of α-synuclein aggregation

Several neurodegenerative diseases are associated with the formation of toxic protein aggregates, such as Parkinson's disease (PD), which is characterized by the progressive loss of dopaminergic neurons and the presence of Lewy bodies—cytoplasmic inclusions containing aggregated alpha-synuclein (αSyn). αSyn is a 140-amino acid protein involved in various synucleinopathies and features three distinct structural domains: an amphipathic N-terminus, a hydrophobic NAC region, and an acidic C-terminus. In this study, we investigated the effect of heteroaryl hydrazones from the RPJ series - derived from 4-aminoquinolines originally developed as myeloperoxidase inhibitors - on αSyn aggregation. Given that these compounds possess a hydrazone fused to the 4-aminoquinoline core capable of interacting with the prion protein, and that αSyn shares a prion-like aggregation mechanism, the activity of these compounds against αSyn aggregation was evaluated. Initially, compounds RPJ6 and RPJ12 were studied, and based on preliminary results, derivatives RPJ20, RPJ21, RPJ22, RPJ23, and RPJ24 were designed and synthesized. Recombinant human αSyn was expressed in E. coli and purified by ammonium sulfate precipitation (salting out). Aggregation kinetics were monitored by thioflavin T fluorescence, while the final species were characterized by dynamic light scattering and transmission electron microscopy. The protein was obtained with a high degree of purity, predominantly in a random coil conformation, as expected. In the presence of the compounds, an increase in the population of particles with a smaller hydrodynamic radius was observed, except for RPJ12, alongside the formation of less electron-dense aggregates. Aggregation kinetics showed a reduction in thioflavin T fluorescence, indicating decreased amyloid aggregate formation. These results demonstrate that heteroaryl hydrazones modulate αSyn aggregation, with compounds RPJ20 to RPJ24 being the most promising. Further studies are underway to investigate the interaction of these compounds with αSyn fibrils and to elucidate their mechanisms of action.