494
S. Hanessian et al. / Bioorg. Med. Chem. Lett. 17 (2007) 491–494
vention in disease as well.2 While potent S1P1 selective
compounds have recently been described,19,20 selective
agents in particular for S1P4 and S1P5 receptors are
not known. Therefore, compounds 12 and 18 could be
interesting probes to explore the effects of pharmacolog-
ical intervention with S1P4 and S1P5, using compounds
devoid of activity on S1P1. In addition they also could
be used to assist molecular modeling of the binding site
of these receptors.
volumes of 100 ll with D-erythro-sphingosine, FTY-720
or derivatives 9 or 17 (20 lM; added from stock solutions
in DMSO), 1 mM ATP, and 2 lCi [c-32P]ATP in 50 mM
Hepes buffer (pH 7.4) containing 15 mM MgCl2, 0.005%
Triton X-100, 10 mM KCl, 10 mM NaF, and 1.5 mM
semicarbazide. Following incubations for different time
points up to 2 h, lipids were extracted and separated by
thin-layer chromotography (TLC) plates (Merck) using
butanol/acetic acid/water 3:1:1 as mobile phase Radiola-
beled derivatives were visualized and quantified using a
Molecular Dynamics Storm PhosphorImager (Sunnyvale,
CA). The calculated phosphorylation efficiency is reported
as value relative to sphingosine (=100%) for which the rate
was 41 and 25 nmol/min/mg with SPHK1 and SPHK2,
respectively.
References and notes
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13. The phosphorylation reactions were performed essentially
as described in Ref. 12. Briefly, the cytoplasmic fraction of
recombinant HEK-293 cells overexpressing human
SPHK1 or SPHK2 was incubated at 30 °C in total
18. The calcium flux assay using FLIPR (fluorometric imaging
plate reader) was performed essentially as described in
Ref. 12. Briefly, CHO cells expressing the recombinant
human receptors were plated in black Costar plate (384
well, 12,500 cells) in aMEM containing 10% FCS and
cultured for 20–24 h at 37 °C in a CO2 incubator. After the
removal of the culture medium cells were incubated in
HBSS medium containing 2 lM Fluo4AM (molecular
probes) and 5 mM probenicid for 1 h at 37 °C, rinsed with
HBSS buffer/2.5 mM probenicid, and overlaid with the
same medium (50 ll). The plates were transferred to the
FLIPR. After measuring the baseline for 40 s, 25 ll of the
agonist solution in HBSS was added and the fluorescence
was measured at intervals of 2 s for 3–5 min. Calculations
of the EC50 were performed using a nonlinear regression
fit program provided in the Origin 7 RS2 software package
(Origin LabCorporation).
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