TY - JOUR T1 - Elimination of a hydroxyl group in FTY720 dramatically improves the phosphorylation rate JF - Molecular Pharmacology JO - Mol Pharmacol DO - 10.1124/mol.110.064873 SP - mol.110.064873 AU - Eve Jary AU - Thomas Bee AU - Scott R Walker AU - Sung-Kee Chung AU - Kyung-Chang Seo AU - Jonathan C Morris AU - Anthony S Don Y1 - 2010/01/01 UR - http://molpharm.aspetjournals.org/content/early/2010/07/07/mol.110.064873.abstract N2 - The new immunosuppressant FTY720 (Fingolimod™), an analogue of the endogenous lipid sphingosine, induces transient lymphopenia through the sequestration of lymphocytes in secondary lymphoid organs. Phosphorylation of FTY720 by sphingosine kinase 2 (SphK2) yields the active metabolite, FTY720-phosphate (FTY-P), which induces lymphopenia through agonism of the sphingosine 1-phosphate receptor S1P1 on endothelial cells and lymphocytes. Dephosphorylation of circulating FTY-P creates an equilibrium between FTY720 and its phosphate, and results with human patients indicate that phosphorylation of FTY720 could be rate limiting for efficacy. We report that the FTY720 derivative 2-amino-4-(4-heptyloxyphenyl)-2-methylbutanol [AAL(R)] is phosphorylated much more rapidly than FTY720 in cultured human cells and whole blood. The Kcat for AAL(R) with recombinant SphK2 is 8-fold higher than for FTY720, whilst the Km for the two substrates is very similar, indicating that the increased rate of phosphorylation results from faster turnover by SphK2, rather than a higher binding affinity. Consequently, treating cells with AAL(R), but not FTY720, triggers an apoptotic pathway that is dependent on excessive intracellular accumulation of long chain base phosphates. In agreement with the in vitro results, phosphorylation of AAL(R) is more complete than that of FTY720 in vivo (mice), and AAL(R) is a more potent inducer of lymphopenia. These differences may be magnified in humans, as phosphorylation of FTY720 is much less efficient in humans compared to rodents. Our results suggest that AAL(R) is a better tool than FTY720 for in vivo studies with S1P analogues, and would probably be a more effective immunosuppressant than FTY720. ER -