PT  - JOURNAL ARTICLE
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
TI  - Elimination of a Hydroxyl Group in FTY720 Dramatically Improves the Phosphorylation Rate
AID  - 10.1124/mol.110.064873
DP  - 2010 Oct 01
TA  - Molecular Pharmacology
PG  - 685--692
VI  - 78
IP  - 4
4099  - http://molpharm.aspetjournals.org/content/78/4/685.short
4100  - http://molpharm.aspetjournals.org/content/78/4/685.full
SO  - Mol Pharmacol2010 Oct 01; 78
AB  - The new immunosuppressant FTY720 (fingolimod), an analog 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, whereas 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, because phosphorylation of FTY720 is much less efficient in humans compared with rodents. Our results suggest that AAL(R) is a better tool than FTY720 for in vivo studies with S1P analogs and would probably be a more effective immunosuppressant than FTY720.