Design and Synthesis of Fluorescent Methylphenidate Analogues for a 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 α-synuclein (aSyn). Moreover, we found that in Parkinson's disease (PD), Syn III also participates in aSyn aggregation and toxicity. Our recent observations point to threo-methylphenidate (MPH), a monoamine re-uptake inhibitor that efficiently counteracts the freezing-gait characteristic of advanced PD, as a ligand for Syn III. We have designed and synthesised 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 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, the 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 might be valuable tools to study drug-ligand interactions by FRET or to detect Syn III in cytological and histological samples. © 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Citation

Andrea Casiraghi, Francesca Longhena, Valentina Straniero, Gaia Faustini, Amy H Newman, Arianna Bellucci, Ermanno Valoti. Design and Synthesis of Fluorescent Methylphenidate Analogues for a FRET-Based Assay of Synapsin III Binding. ChemMedChem. 2020 Jul 20;15(14):1330-1337

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PMID: 32452650

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