Molecular rigidity and potency of bispyridinium type allosteric modulators at muscarinic M-2-receptors

Several bispyridinium compounds have been shown to be potent allosteric modulators of ligand binding to muscarinic M-2-receptors. ''Uno compounds'' are benzyl derivatives of the bispyridinium ''TMB4'' (trimethylene-bis-[4-hydroxy-iminomethylpyridinium]). To gain more insight into structure activity relationships, eleven derivatives with varying structure of the oxime-linked aromatic substituent were tested for their ability to inhibit the equilibrium-binding of [H-3]N-methylscopolamine ([H-3]NMS) in guinea pig cardiac membranes and to retard [H-3]NMS-dissociation allosterically. At a concentration of 3 mu M, all compounds reduced [H-3]NMS-binding to about 40 % of the control level, indicating a similar potency to inhibit the association of [H-3]NMS. Allosteric retardation of [H-3]NMS-dissociation required higher concentrations. Comparing the effects of the compounds at 30 and 300 mu M, respectively, revealed considerable differences in potency. Therefore, the concentration-dependency of the delay of [H-3]NMS-dissociation was determined for selected compounds. The results indicate that introduction of a benzyl-moiety into TMB4 leads to a 20-fold increase in allosteric potency. A further increment by a factor of 10 is obtained with the 2,6-dichlorobenzyl-substitution and with the naphthyl-derivative. The other compounds were less potent. An inverse correlation was found between the rotational freedom of the aromatic substituent and the allosteric potency. In conclusion, the aromatic moiety of non-symmetric bispyridinium-type modulators does not seem to be part of the pharmacophore involved in the inhibitory effect on the association of [H-3]NMS. In contrast, a rigid aromatic lateral moiety appears to be essential for the interaction with the recognition site mediating the allosteric delay of [H-3]NMS dissociation from muscarinic M-2-receptors.