PARKINSON'S DISEASE: VALIDATION OF A NOVEL TARGET THROUGH THE DEVELOPMENT OF METHYLPHENIDATE ANALOGS AS POTENTIAL MODULATOR AND FLUORESCENT PROBES

Parkinson’s Disease (PD) is a neurological disorder characterized by the progressive degeneration of dopaminergic striatal neurons, which in turn leads to the onset of a typical set of motor and non-motor symptoms. α-synuclein is the most studied protein involved in the pathogenesis of PD and its accumulation, misfolding and aggregation are strongly correlated with dopaminergic neuronal derangement. Synapsin-III is a neuronal protein involved in trafficking, fusion and binding of dopaminergic presynaptic vesicles. A growing body of evidence implicates the cooperation between α-synuclein and Synapsin-III in the pathogenesis of the disease and opens up to the possibility of pharmacological modulation of their interaction as a novel therapeutic approach for the treatment of PD. In Chapter 1 of this work, the design and synthesis of methylphenidate analogs as possible modulators of the α-synuclein/Synapsin-III interaction is presented. The parent molecule has been systematically modified in order to evaluate the effect of structural changes on biological activity. The obtained compounds have been tested for their biological activity and toxicity profile with the use of specific assays. The ability of these compounds to promote the interaction between the two target proteins, as well as their ability to slow down or prevent the aggregation of α-synuclein has been evaluated. Interestingly, one of the analogs showed a promising biological activity and, possibly, cytoprotective effects in cell models of PD. The activity data for the analogs will support the characterization of the binding modes to Synapsin-III and, hopefully, the identification of the preferential binding pocket. Moreover, very low or absent cytotoxicity corroborate our interest in these analogs as possible novel antiparkinsonian agents. In Chapter 2, the design, synthesis and radioligand binding evaluation of a novel group of fluorescent small molecule ligand-based probes for imaging the norepinephrine transporter (NET) is presented. NET is a member of the monoamine transporters family and is implicated in PD as well as in depression, ADHD and substance abuse. The selective imaging of NET in live cells, in particular with the aid of super resolution microscopy techniques, would contribute in a better understanding of its function, localization and trafficking at a molecular level. Herein, we identified a functional design for a NET-selective high affinity probe and verified its binding capabilities at NET. The most promising candidate is at the moment under evaluation in live-cell microscopy experiments. The work presented in Chapter 3 applies the fluorescent probes chemistry and concepts shown in Chapter 2 to the design and synthesis of novel fluorescent probes based on a methylphenidate scaffold and represents the ideal connection between the two projects previously described. The synthesized probes are now under evaluation in FRET experiments, in order to characterize the binding of methylphenidate on Synapsin-III, which would make a crucial contribution to our understanding of the interaction of the compounds presented in Chapter 1 with their target.