Subtype-Selective Positive Cooperative Interactions between Brucine Analogues and Acetylcholine at Muscarinic Receptors: Radioligand Binding Studies

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Abstract
We studied the interactions of strychnine, brucine, and three of theN-substituted analogues of brucine with [3H]N-methylscopolamine (NMS) and unlabeled acetylcholine at m1–m5 muscarinic receptors using equilibrium and nonequilibrium radioligand binding studies. The results were consistent with a ternary allosteric model in which both the primary and allosteric ligands bind simultaneously to the receptor and modify the affinities of each other. The compounds hadKd values in the submillimolar range, inhibited [3H]NMS dissociation, and showed various patterns of positive, neutral, and negative cooperativity with [3H]NMS and acetylcholine, but there was no predictive relationship between the effects. Acetylcholine affinity was increased ∼2-fold by brucine at m1 receptors, ∼3-fold byN-chloromethyl brucine at m3 receptors, and ∼1.5-fold by brucine-N-oxide at m4 receptors. The existence of neutral cooperativity, in which the compound bound to the receptor but did not modify the affinity of acetylcholine, provides the opportunity for a novel form of drug selectivity that we refer to as absolute subtype selectivity: an agent showing positive or negative cooperativity with the endogenous ligand at one receptor subtype and neutral cooperativity at the other subtypes would exert functional effects at only the one subtype, regardless of the concentration of agent or its affinities for the subtypes. Our results demonstrate the potential for developing allosteric enhancers of acetylcholine affinity at individual subtypes of muscarinic receptor and suggest that minor modification of a compound showing positive, neutral, or low negative cooperativity with acetylcholine may yield compounds with various patterns of cooperativity across the receptor subtypes.