Probing the size of a hydrophobic binding pocket within the allosteric site of muscarinic acetylcholine M2-receptors

Hexane-bisammonium-type compounds containing lateral phthalimide moieties are known to have a rather high affinity for the allosteric site of muscarinic 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 for their ability to retard allosterically the dissociation of [H-3]N-methylscopolamine 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 naphthalimide containing agent (half maximum effect at EC50,diss = 60 nM) revealed the highest potency. Apparently, its affinity for the allosteric site in NMS-occupied receptors is 20fold higher compared with the phthalimide containing parent compound W 84. Analysis of quantitative structure-activity relationships yielded a parabolic correlation between the volume of the lateral substituents and the allosteric potency. The maximal volume was determined to be approximately 600 Angstrom(3) suggesting that the allosteric binding site contains a binding pocket of a defined size for the imide moiety.