168
A. Moulin et al. / Bioorg. Med. Chem. Lett. 18 (2008) 164–168
5. Guerlavais, V.; Boeglin, D.; Mousseaux, D.; Oiry, C.;
into the temperature-regulated FlexStation machine. The
machine records the fluorescence output over a period of
60 s, with the compounds being automatically distributed
into the wells containing the cells after 15 s. The Fluo-
4AM exhibits a large fluorescence intensity increase on
binding of calcium and therefore the fluorescence output is
used directly as a measure of intracellular calcium mobi-
lization. The excitation and emission wavelengths were
485 and 525 nm, respectively. The basal fluorescence
intensity of dye-loaded cells was 800–1200 arbitrary units
and the fluorescence peak upon maximal response was
5000–7000 U. To assess the ability of each of the
compounds to induce calcium mobilization, they were
tested at a concentration of 10 lM in triplicate, in at least
2 independent experiments. In each case, the change in
fluorescence upon addition of the compound was com-
pared with the basal fluorescence ouput measured with the
control (addition of buffer A only). The maximum
fluorescent output was equivalent to that achieved when
the cells were stimulated with 0.1 lM ghrelin. For com-
pounds behaving as agonists and displaying a high affinity
binding for hGHS-R1a in radiolabelled binding experi-
ments, EC50 (the molar concentration of the agonist
producing 50% of the maximal possible effect of that
agonist) was determined using a dose-response curve. In
the case of high affinity antagonists, IC50 and Kb were
determined using antagonist inhibition curves in the
presence of 0.1 lM Ghrelin (submaximal concentration).
The IC50 was calculated as the molar concentration of
antagonist that reduced the maximal response of ghrelin
by 50% and an estimation of the Kb was made using the
Cheng-Prusoff Equation.13
Heitz, A.; Deghenghi, R.; Locatelli, V.; Torsello, A.; Ghe,
C.; Catapano, F.; Muccioli, G.; Galleyrand, J. C.;
Fehrentz, J. A.; Martinez, J. J. Med. Chem. 2003, 46, 1191.
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J. A. Org. Lett. 2003, 5, 4465.
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10. Receptor Binding Studies. Isolated plasma membranes
from LLC PK-1 cells (10 lg protein) were incubated in HB
for 60 min at 25 °C (steady state conditions) with 60 pM
125I-His9-ghrelin (Amersham) in the presence or absence
of competing compounds. Non-specific binding was
defined using an excess (1 lM) of ghrelin and was always
less than 20% of total binding. The binding reaction was
stopped by addition of 4 mL of ice-cold HB followed by
rapid filtration over Whatman GF/C filters presoaked
with 0.5% polyethyleneimine to prevent excessive binding
of radioligand to the filters. Filters were rinsed 3 times
with 3 mL of ice-cold Wash Buffer (50 mM Tris (pH 7.3),
10 mM MgCl2, 2.5 mM EDTA and 0.015% (w/v) Triton
X-100) and the radioactivity bound to membranes was
measured in a gamma counter.
11. Mousseaux, D.; Le Gallic, L.; Ryan, J.; Oiry, C.; Gagne,
D.; Fehrentz, J. A.; Galleyrand, J. C.; Martinez, J. Br. J.
Pharmacol. 2006, 148, 350.
12. Intracellular Calcium Mobilization Assay. The calcium
experiments were performed using the benchtop scanning
fluorometer FlexStation II machine (Pharmacologie &
13. Cheng, Y.-C.; Prusoff, W. H. Biochem. Pharmacol. 1973,
22, 3099.
´ ´
Screening Plateform of the Institut Federatif de Recherche
3, Montpellier, France). CHO cells were transiently
transfected with the hGHS-1a receptor, using electropor-
ation, and were then plated into 96-well black-bottom
plates (80,000 cells/well). Twenty-four hours later the cells
were washed with 150 lL Buffer A (Hanks’ balanced salt
solution, 0.5% BSA, 20 mM CaCl2, 2.5 mM probenecid,
pH 7.4) and were then loaded with 1 lM of the fluorescent
calcium indicator Fluo-4AM prepared in Buffer A, con-
taining 0.06% pluronic acid (a mild-ionic detergent which
facilitates Fluo-4AM ester loading). The cells were incu-
bated for 1 h in the dark at 37 °C. Following the
incubation, excess Fluo-4AM was removed from the cells
by washing twice with 100 lL Buffer A and 50 lL of the
same buffer was then added to each well. The cells were
left at room temperature for 30 min to allow complete de-
esterification of intracellular Fluo-4AM esters. The black-
bottom plate containing the cells, as well as the plate
containing the compounds to be tested, were then placed
14. Patchett, A. A.; Nargund, R. P.; Tata, J. R.; Chen, M. H.;
Barakat, K. J.; Johnston, D. B.; Cheng, K.; Chan, W. W.;
Butler, B.; Hickey, G. Proc. Natl. Acad. Sci. U.S.A. 1995,
92, 7001.
15. Carpino, P. A.; Lefker, B. A.; Toler, S. M.; Pan, L. C.;
Hadcock, J. R.; Cook, E. R.; DiBrino, J. N.; Campeta, A.
M.; DeNinno, S. L.; Chidsey-Frink, K. L.; Hada, W. A.;
Inthavongsay, J.; Mangano, F. M.; Mullins, M. A.;
Nickerson, D. F.; Ng, O.; Pirie, C. M.; Ragan, J. A.;
Rose, C. R.; Tess, D. A.; Wright, A. S.; Yu, L.;
Zawistoski, M. P.; DaSilva-Jardine, P. A.; Wilson, T. C.;
Thompson, D. D. Bioorg. Med. Chem. 2003, 11, 581.
16. Moulin, A.; Demange, L.; Berge, G.; Gagne, D.; Ryan, J.;
Mousseaux, D.; Heitz, A.; Perrissoud, D.; Locatelli, V.;
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lished results.
17. Unpublished results.
Moulin, Aline
