Sphingosine-1-phosphate is a bioactive signaling molecule that mediates important cellular functions such as cell proliferation, cytoskeletal rearrangement, angiogenesis, mobilization of intracellular calcium and immune cell trafficking. FTY720, a synthetic analog of sphingosine that has immunosuppressive properties, is the first oral drug approved by the U.S. FDA for treatment of multiple sclerosis (under the trade name GilenyaTM). This thesis project is focused on the cellular uptake, partitioning, and diffusion studies on a fluorescent analog of this drug (namely, Bodipy-FTY720) in cultured C3H10T1/2 cells, derived from mouse embryos. Our working hypothesis is that Bodipy-FTY720 will be phosphorylated by sphingosine kinase 2 (SphK2) prior to binding to sphingosine-1-phosphate receptor 1 (S1PR1) followed by internalization and degradation of the receptor. Our results indicate that Bodipy-FTY720 resides in the endoplasmic reticulum (ER) of C3H10T1/2 cells and is excluded from the nucleus. Our single-molecule diffusion measurements also indicate that Bodipy-FTY720 binds with the cellular ER membrane prior to its phosphorylation, secretion via the ABC transporter, and then binding to S1PR1 receptors. These studies represent a step forward towards elucidating the action mechanism of FTY720 at the single-cell level.
University of Minnesota M.S. thesis. December 2011. Major: Chemistry. Advisor: Ahmed A. Heikal. 1 computer file (PDF); xi, 48 pages, appendices p. 47-48.
Wickramasinghe, W.P.M. Dhanushka.
Partitioning and diffusion studies of bodipy-fingolimod analogs, immune suppressors, in mouse embryonic fibroblast cells.
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