Probing the size of a hydrophobic binding pocket within the allosteric site of muscarinic acetylcholine M-2-receptors

Hexane-bisammonium-type compounds containing lateral phthalimide moieties a re known to have a rather high affinity for the allosteric site of muscarin ic M-2 receptors. In order to get more insight into the contribution of the lateral substituents for alloster binding affinity, a series of compounds with unilaterally varying imide substituents were synthesized and tested fo r their ability to retard allosterically the dissociation of [H-3]N-methyls copolamine fi om the receptor protein (control t(1/2) = 2 min; 3 mM MgHCO4, 50 mM Tris, pH 7.3, 37 degrees C). Among the test compounds, the naphthali mide containing agent (half maximum effect at EC50,diss = 60 nM) revealed t he highest potency. Apparently, its affinity for the allosteric site in NMS -occupied receptors is 20fold higher compared with the phthalimide containi ng parent compound W 84. Analysis of quantitative structure-activity relati onships yielded a parabolic correlation between the volume of the lateral s ubstituents and the allosteric potency. The maximal volume was determined t o be approximately 600 Angstrom(3) suggesting that the allosteric binding s ite contains a binding pocket of a defined size for the imide moiety.