B. M. Fox et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2460–2467
2467
22. Hervieu, G. J.; Cluderay, J. E.; Harrison, D.; Meakin, J.; Maycox, P.; Nasir, S.;
Leslie, R. A. Eur. J. Neurosci. 2000, 12, 1194.
23. Chen, Y.; Hu, C.; Hsu, C.-K.; Zhang, Q.; Bi, C.; Asnicar, M.; Hsiung, H. M.; Fox, N.;
Slieker, L. J.; Yang, D. D.; Heiman, M. L.; Shi, Y. Endocrinology 2002, 143(7),
2469.
24. Marsh, D. J.; Weingarth, D. T.; Novi, D. E.; Chen, H. Y.; Trumbauer, M. E.; Chen,
A. S.; Guan, X.-M.; Jiang, M. M.; Feng, Y.; Camacho, R. E.; Shen, Z.; Frazier, E. G.;
Yu, H.; Metzger, J. M.; Kuca, S. J.; Shearman, S. J.; Gopal-Truter, S.; MacNeil, D. J.;
Strack, A. M.; MacIntyre, D. E.; Van der Ploeg, L. H. T.; Qiang, S. Proc. Nat. Acad.
Sci. USA 2002, 99(5), 3240.
double digit nanomolar MCHR1 IC50 values and micromolar hERG
inhibition.
Acknowledgments
The authors thank Dr. Mario Cardozo for information obtained
from the hERG pharmacophore model.
25. Padwa, A.; Dent, W. J. Org. Chem. 1987, 52, 235.
26. Laguzza, B. C.; Ganem, G. Tetrahedron Lett. 1981, 22, 1483.
27. Ohira, S. Synth. Comm. 1989, 19, 561.
References and notes
1. Vaughan, J. M.; Fischer, W. H.; Hoeger, C.; Rivier, J.; Vale, W. Endocrinology
1989, 125(3), 1660.
28. Müller, S.; Liepold, B.; Roth, G. J.; Bestmann, H. J. Synlett 1996, 6,
521.
2. Presse, F.; Nahon, J. L.; Fischer, W. H.; Vale, W. Mol. Endocrinol. 1990, 4(4), 632.
3. Nahon, J. L.; Presse, F.; Bittencourt, J. C.; Sawchenko, P. E.; Vale, W.
Endocrinology 1989, 125(4), 2056.
4. Viale, A.; Zhixing, Y.; Breton, C.; Pedeutour, F.; Coquerel, A.; Jordan, D.; Nahon, J.
L. Brain Res. Mol. Brain Res. 1997, 46(1–2), 243.
29. Evans, D. A.; Ellman, J. A. JACS 1989, 111, 1063.
30. The mean and standard deviation of a common standard included in each run
was acceptable. A description of the aequorin assay can be found in the
following references. (a) An, S.; Thieu, B.; Yuhua, Z.; Goetzl, E. J. Mol. Pharm.
1998, 54, 88; (b) Bandoh, K.; Aoki, J.; Hosono, H.; Kobayashi, S.; Kobayashi, T.;
Murakami-Murofushi, K.; Tsujimoto, M.; Arai, H.; Inoue, K. J. Biol. Chem. 1999,
274, 27776.
5. Bittencourt, J. C.; Presse, F.; Arias, C.; Peto, C.; Vaughan, J.; Nahon, J. L.; Vale, W.;
Sawchenko, P. E. J. Comp. Neurol. 1992, 319(2), 218.
6. Skofitsch, G.; Jacobowitz, D. M.; Zamir, N. Brain Res. Bull. 1985, 15(6), 635.
7. Nahon, J. L. Crit. Rev. Neurobiol. 1994, 8(4), 221.
8. Elias, C. F.; Saper, C. B.; Maratos-Flier, E.; Tritos, N. A.; Lee, C.; Kelly, J.; Tatro, J.
B.; Hoffman, G. E.; Ollmann, M. M.; Barsh, G. S.; Sakurai, T.; Yanagisawa, M.;
Elmquist, J. K. J. Comp. Neurol. 1998, 402(4), 442.
31. The mean and standard deviation of a common standard included in each run
was acceptable. Material and solutions: Binding-buffer: 50 mM Tris pH 7.4,
10 mM MgCl2, 2.5 mM EDTA; 0.2% BSA (w/v). Washing-buffer: 25 mM Hepes
pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 0.5% BSA (w/v), 0.5 M NaCl. MulitiscreenÒ-FB
plates (Cat# MAFBN0B50, Millipore USA) were, prior to use, treated with
9. Ludwig, D. S.; Mountjoy, K. G.; Tatro, J. B.; Gillette, J. A.; Frederich, R. C.; Flier, J.
S.; Maratos-Flier, E. Am. J. Physiol. Endocrinol. Metab. 1998, 274, 627.
10. Della-Zuana, O.; Presse, F.; Ortola, C.; Duhault, J.; Nahon, J. L.; Levens, N. Int. J.
Obesity 2002, 26, 1289.
11. Gomori, A.; Ishihara, A.; Ito, M.; Mashiko, S.; Matsushita, H.; Yumoto, M.; Ito,
M.; Tanaka, T.; Tokita, S.; Moriya, M.; Iwaasa, H.; Kanatani, A. Am. J. Physiol.
Endocrinol. Metab. 2003, 284, 583.
12. Ito, M.; Gomori, A.; Ishihara, A.; Oda, Z.; Masahiko, S.; Matsushita, H.; Yumoto,
M.; Ito, M.; Sano, H.; Tokita, S.; Moriya, M.; Iwaasa, H.; Kanatani, A. Am. J.
Physiol. Endocrinolo. Metab. 2003, 284, 940.
13. Ludwig, D. S.; Tritos, N. A.; Mastaitis, J. W.; Kulkarni, R.; Kokkotou, E.; Elmquist,
J.; Lowell, B.; Flier, J. S.; Maratos-Flier, E. J. Clin. Inv. 2001, 107(3), 379.
14. Qu, D.; Ludwig, D. S.; Gammeltoft, S.; Piper, M.; Pelleymounter, M. A.; Cullen,
M. J.; Mathes, W. F.; Przypek, J.; Kanarek, R.; Maratos-Flier, E. Nature 1996, 380,
243.
100
solution was removed by vacuum filtration and the wells were washed 2 times
with 100 l of binding-buffer. Methods: Fifty microliters of compound-dilution,
made in binding-buffer, was added to 50 l of binding buffer containing 1 unit
MCHR1 membrane preparation (Cat# ES-370 M, Euroscreen; Belgium) and
0.02 Ci/mL, PerkinElmer, USA).
Ci 125I-MCH (2200 Ci (81.4TBq)/mmol; 100
The 100 l reaction mixture was incubated in a Corning 96-well plate (Cat#
3363, Corning USA) at 37 °C for 2 h on a shaking platform. Bound and unbound
ligand was separated by vacuum filtration by transferring 80 l of the reaction
mixture into
corresponding well of the MulitiscreenÒ-FB plates. After
filtration the wells were washed 4 times with 100 of washing-buffer
before 100 l of Scintillation cocktail (PerkinElmer, USA) was added. Plates
were read on a MicroBetaÒ Trilux (PerkinElmer, USA).
ll of 0.5% BSA (w/v)/H20 solution. After 2 h at room temperature the
l
l
l
l
l
l
a
ll
l
32. The mean and standard deviation of a common standard included in each run
was acceptable. Finlayson, K.; Turnbull, L.; January, C. T.;Sharkey, J.; Kelly, J. S.
Eur. J. Pharm.2001, 430, 147. A stable HEK293 cell line expressing the hERG
channel was established in house. Compounds were tested in the [3H]
dofetilide binding assay with cell membranes prepared from this cell line
using method of Finlayson et al. (2001) with some modifications. Briefly,
15. Shimada, M.; Tritos, N. A.; Lowell, B. B.; Flier, J. S.; Maratos-Flier, E. Nature 1998,
396, 670.
16. Chambers, J.; Ames, R. S.; Bergsma, D.; Muir, A.; Fitzgerald, L. R.; Hervieu, G.;
Dytko, G. M.; Foley, J. J.; Martin, J.; Liu, W.-S.; Park, J.; Ellis, C.; Ganguly, S.;
Konchar, S.; Cluderay, J.; Leslie, R.; Wilson, S.; Sarau, H. M. Nature 1999, 400,
261.
filtration assays were carried out in 194
pH 7.4, 60 mM KCl, 70 mM NaCl, 1 mM CaCl2, 2 mM MgCl2) with 10
membrane (based on membrane protein) and [3H]-dofetilide (8 nM), 6
compound dissolved in 100% DMSO. Non-specific binding was determined by
l
L of binding buffer (10 mM HEPES,
g/well
L of
l
17. Saito, Y.; Nothacker, H.-P.; Wang, Z.; Lin, S. H. S.; Leslie, F.; Civelli, O. Nature
1999, 400, 265.
l
18. Bächner, D.; Kreienkamp, H.-J.; Weise, C.; Buck, F.; Richter, D. FEBS Lett. 1999,
457, 522.
using 10
lM cold dofetilide (ꢀ1000-fold molar excess over hot ligand). The
entire assay was conducted in 96-well WhatmanÒ Unifilter plates at room
temperature for 90 min. The binding assay was terminated by washing the
19. Kolakowski, L. F., Jr.; Jung, B. P.; Nguyen, T.; Johnson, M. P.; Lynch, K. R.; Cheng,
R.; Heng, H. H. Q.; George, S. R.; O’Dowd, B. F. FEBS Lett. 1996, 398, 253.
20. Lakaye, B.; Minet, A.; Zorzi, W.; Grisar, T. Biochim. Biophys. Acta 1998, 1401, 216.
21. Hawes, B. E.; Kil, E.; Green, B.; O’Neill, K.; Fried, S.; Graziano, M. P. Endocrinology
2000, 14(12), 4524.
plates four times on a MilliporeÒ Vacuum filtration manifold with 100
lL/well
of ice cold wash buffer (130 mM NaCl, 1 mM CaCl2, 2 mM MgCl2, 10 mM HEPES,
pH 7.4). The bound radioisotope was quantified using a Packard TopCountÒ
NTS liquid scintillation counter with scintillation fluid.