Allosteric Modulators of Muscarinic ACh Receptors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1 371
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the tissues for 30 min. The antagonistic potency (pKB) was
calculated according to Furchgott’s method.45 Data are presented
as mean ( SEM (n ) 6-8 independent experiments).
[35S]GTP Binding Assay. Cell culture and [35S]GTPγS binding
experiments were carried out as described previously.46 In short,
Chinese hamster ovary (CHO) cells stably transfected with the
human M2 receptor gene (kindly donated by Prof. Dr. G. Lambrecht,
Department of Pharmacology, Biocenter Niederursel, University
of Frankfurt/Main, Germany) were cultured under the conditions
described before and treated for 24 h before harvesting with 5 mM
sodium butyrate in order to boost receptor expression. After
harvesting the cells, a membrane suspension was prepared for use
in the [35S]GTPγS binding experiments (0.07 nM [35S]GTPγS, 10
µM GDP, 10 mM HEPES, 100 mM NaCl, 10 mM MgCl2, pH 7.4;
test compounds at the indicated concentrations). After an incubation
period of 60 min at 30 °C, the incubation medium was filtered
through glass fiber filters and filter bound radioactivity was
measured by scintillation counting. As determined in homologous
competition experiments with [3H]NMS, the density of M2 receptors
amounted to about 6 pmol/mg membrane protein.
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Acknowledgment. The expert technical assistance of Mech-
thild Kepe (Bonn) is gratefully acknowledged. This work was
financially supported by the Universita` degli Studi di Milano
(FIRST 2003 and 2004), and by grants of the Deutsche
Forschungsgemeinschaft to U.H., K. M., and GRK677. In
addition, M.D.A. and U.H. wish to thank the German-Italian
scientific cooperation program Vigoni for supporting this
research.
(22) Dallanoce, C.; Conti, P.; De Amici, M.; De Micheli, C.; Barocelli,
E.; Chiavarini, M.; Ballabeni, V.; Bertoni, S.; Impicciatore, M.
Synthesis and functional characterization of novel derivatives related
to oxotremorine and oxotremorine-M. Bioorg. Med. Chem. 1999, 7,
1539-1547.
Supporting Information Available: Microanalyses and NMR
data are available free of charge via the Internet at http://
pubs.acs.org.
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M.; De Micheli, C.; Impicciatore, M. New analogues of oxotremorine
and oxotremorine-M: estimation of their in vitro affinity and efficacy
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