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Probing the activity of Sphingomyelinases by Fluorescence Resonance Energy Transfer (FRET)

Zainelabdeen Ahmed and Christoph Arenz

Institut für Chemie, Humboldt-Universität zu Berlin, Brook-Taylor-Str. 2, 12489, Berlin, GER

Sphingomyelinases (SMases) hydrolyse sphingomyelin, releasing ceramide, and creating a cascade of bioactive lipids. These lipids include sphingosine and sphingosine-1-phosphate, all of which have a specific signalling capacity. Thus SMases are involved in lipid signaling processes that render it a key regulator of the cell’s fate. The molecular details of the most studied acid Sphingomyelinase (ASM) like its localization during signaling and its interplay with other proteins; however appear to be very complex. Different probes were designed to monitor and quantify the activity of the ASM enzyme either in vitro or within living cells by means of Förster resonance energy transfer (FRET) techniques with real time resolution. The target of the design was to combine both higher substrate recognition, better 2P excitabillity and Synthetic potential. Concerning the substrate recognition we manged to synthesize a probe with a quaternary nitrogen center highly mimicking the natural sphingomyelin which we expect that it will not only Speed up the enzymatic cleavage but may also allow the recognition of the probe by the neutral sphingomyelinase enzyme or other species comparable enzymes. For better 2P excitabillity, a bromo coumarin dye derivative with high 2PE cross sections 2 was selected to be used as a FRET doner with NBD as acceptor. With a perfectly 2P excitable probe we plan to reduce phototoxicity and increase imaging depth through our future live cell/tissue experiments. With those probes in hand we are looking forward to start abroad range of applications which do not include only the searching and optimization of SMases inhibitors but also studying the complex nature of SMases production and localization in addition to the related physiological disorders.

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The probes are forming a FRET system as long as they are intact. Upon hydrolysis with the sphingomyelinases, the FRET system is broken and this can be monitored quantitatively.