Design and synthesis of fluorescent-methylphenidate analogues for FRET-based assay of Synapsin III binding

We previously described Synapsin III (Syn III) as a synaptic phosphoprotein that controls dopamine release in cooperation with alpha-synuclein (aSyn). Moreover, we found that in Parkinson's disease (PD), Syn III also participates in alpha-synuclein (aSyn) aggregation and toxicity. Our recent observations point to threo -methylphenidate (MPH), a monoamine reuptake inhibitor that efficiently counteracts the freezing gait characteristic of advanced PD, as a ligand for Syn III. Here, we designed and synthesized two different fluorescently-labelled MPH derivatives, one with Rhodamine Red (RHOD) and one with 5-Carboxytetramethylrhodamine (TAMRA), to be used for assessing MPH binding to Syn III by fluorescence resonance energy transfer (FRET). TAMRA-MPH exhibited the ideal characteristics to be used as a FRET acceptor, as it was able to enter into the SK-N-SH cells and could interact specifically with human green fluorescent protein (GFP)-tagged Syn III but not with GFP alone. Moreover, uptake of TAMRA-MPH and co-localization with Syn III was also observed in primary mesencephalic neurons. These findings support that MPH is a Syn III ligand and that TAMRA-conjugated drug molecules may represent valuable tools to study drug-ligand interactions by FRET or to detect Syn III in cytological and histological samples.