M. J. Stocks et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7458–7461
7461
M. R.; Belmonte, K. J. Med. Chem. 2009, 52, 5241; (e) Jin, J.; Budzik, B.; Wang, Y.;
Shi, D.; Wang, F.; Xie, H.; Wan, Z.; Zhu, C.; Foley, J. J.; Webb, E. F.; Berlanga, M.;
Burman, M.; Sarau, H. M.; Morrow, D. M.; Moore, M. L.; Rivero, R. A.; Palovich,
M.; Salmon, M.; Belmonte, K. E.; Laine, D. I. J. Med. Chem. 2008, 51, 5915; (f)
Starck, J.-P.; Provins, L.; Christophe, B.; Gillard, M.; Jadot, S.; Lo Brutto, P.;
Quere, L.; Talaga, P.; Guyaux, M. Bioorg. Med. Chem. Lett. 2008, 18, 2675.
8. The affinity (pIC50) of compounds binding to the M3 receptor was determined
by competition binding of [3H]N-methyl scopolamine (NMS) to CHO-K1
(Chinese Hamster Ovary) cell membranes expressing the human muscarinic
acetylcholine M3 or M2 receptor (M3-ACh) in a scintillation proximity assay
(SPA) format. SPA beads were pre-coated with membranes and then incubated
at 2 mg of beads per well with serial dilutions of compounds, [3H]NMS at half
KD (experimentally determined dissociation constant) and assay buffer (20 mM
HEPES pH 7.4 containing 5 mM MgCl2). The assay was conducted in a final
Acknowledgements
The authors thank Dr. Simon Teague and Dr. Tim Luker for
helpful discussions.
References and notes
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