X1INH, an improved next-generation affinity-optimized hydrazonic ligand, attenuates abnormal copper(I)/copper(II)-α-Syn interactions and affects protein aggregation in a cellular model of synucleinopathy

Although normal aging presents an accumulation of copper and iron in the brain, this becomes more relevant in neurodegeneration. alpha-Synuclein (alpha-Syn) misfolding has long been linked with the development of Parkinson's disease (PD). Copper binding promotes aggregation of alpha-Syn, as well as generalized oxidative stress. In this sense, the use of therapies that target metal dyshomeostasis has been in focus in the past years. Metal-Protein Attenuating Compounds (MPACs) are moderate chelators that aim at disrupting specific, abnormal metal-protein interactions. Our research group has now established that N-acylhydrazones compose a set of truly encouraging MPACs for the bioinorganic management of metal-enhanced aggregopathies. In the present work, a novel ligand, namely 1-methyl-1H-imidazole-2-carboxaldehyde isonicotinoyl hydrazone (X1INH), is reported. We describe solution studies on the interaction and affinity of this compound for copper(ii) ions showing that a fine tuning of metal-affinity was achieved. A series of in vitro biophysical NMR experiments were performed in order to assess the X1INH ability to compete with alpha-Syn monomers for the binding of both copper(i) and copper(ii) ions, which are central in PD pathology. A preference for copper(i) has been observed. X1INH is less toxic to human neuroglioma (H4) cells in comparison to structure-related compounds. Finally, we show that treatment with X1INH results in a higher number of smaller, less compact inclusions in a well-established model of alpha-Syn aggregation. Thus, X1INH constitutes a promising MPAC for the treatment of Parkinson's disease.