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F. Caijo et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4421–4426
Binding assays were performed in 96-well plate format,
using a classical filtration assay with a human full-length
PPARc construct (GST-PPAR LBD (25 lg/ml))
expressed in bacteria with some modifications to the
experimental conditions. The membrane-associated
PPARc was used as a biological source, as previously
described. The binding buffer consisted of 10 mM Tris/
HCl, pH 8.2, containing 50 mM KCl and 1 mM dithio-
threitol. Membrane preparations (5 lg/mL) were
incubated for 180 min at 4 ꢁC in the presence of
[3H]Rosiglitazone [BRL49653, Amersham] (4 nM) and
the tested compounds. Nonspecific binding was defined
using an excess of unlabelled Rosiglitazone (10 lM).
Incubation was terminated by the addition of ice-cold
50 mM Tris/HCl buffer, pH 7.4, followed by rapid filtra-
tion under reduced pressure through Whatman GF/C
filter plates presoaked with ice-cold buffer, followed by
three successive washes with the same buffer. Radioac-
tivity was measured in a TopCount apparatus (Pack-
ard). The receptor preparation used during these
experiments presented a Bmax of 49 pmol/mg proteins
and a Kd of 5.58 nM for [3H]Rosiglitazone. The com-
pounds were solubilized in pure DMSO and diluted to
the appropriate working concentrations (100 lM to
0.1 nM). For each compound tested, plots of ligand con-
centration versus DPM of radioligand binding were con-
structed and apparent Ki values were estimated from
nonlinear least-squares fit of the data assuming simple
competitive binding. The details of this assay have been
reported elsewhere.14
ised and seeded in 96-well plates at a density of 2 · 104
cells/well and incubated for 6 h in 10% FCS containing
DMEM. Cells were then incubated for 16 h in DMEM
containing 0.2% FCS and increasing concentrations of
the compound tested (10 lM to 10 nM) or vehicle
(DMSO). At the end of the experiment, cells were
washed once with ice-cold PBS and the luciferase activ-
ity was measured and normalized to internal control
b-galactosidase activity, as described previously.15 Com-
pounds, which elicited on an average at least 80% activa-
tion of PPAR(s) versus Rosiglitazone (PPARc) or
WY14.643 (PPARa) (positive controls), were considered
full agonists. EC50 were estimated using Prism software
(GraphPad). All transactivation and binding experi-
ments were performed once. For each concentration
tested, the measurements were made in triplicate.
Acknowledgment
We thank Mrs. M. Liutkus for stimulating discussions.
References and notes
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Compounds were screened for functional potency in a
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