Ca2+ is one of the most versatile and important intracellular messenger since it controls numerous cellular process. Nicotinic acid adenine dinucleotide phosphate (NAADP) functions as an intracellular 2nd messenger causing Ca2+ release from acidic and vacuolar Ca2+ stores. Signaling through NAADP is widespread and important, but the process is not yet been completely understood. To determine cellular localization and potential changes in cell distribution of NAADP binding proteins we propose to use NAADP-dye conjugates. NAADP-dye conjugates can be produced from 5-(3-aminopropyl)-NAADP, linking it through an amide linkage to a dye which in turn contains a carboxylic acid. An oligo-ethylene glycol could also be introduced between the NAADP analog and the dye as a hydrophilic spacer. To start the synthesis, BODIPY dyes are particularly attractive, since they are intense, do not photobleach, are uncharged, and can be produced with long wavelength fluorescent emission if desired. Once the first generation of NAADP dye conjugates has been produced its biological activity will be characterized using sea urchin NAADP binding proteins. We can further apply NAADP-dye conjugates for the development of fluorescence depolarization assays of NAADP binding in future work. A successful depolarization assay would enable us to develop automated assays of NAADP binding to sea urchin egg homogenates and subsequently to screen libraries of drug like molecules to find stable and bioavailable NAADP agonists and antagonists.

NAADP-BODIPY dye conjugates for characterizing NAADP binding proteins

256th National Meeting (2018)

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