Functional and structural studies of cholinergic receptors using physostigmine and acridines: Mapping of the recognition sites of acridine araphanes

Abstract

This work was undertaken to clarify the actions of physostigmine (Phy) and the bridged bis-aminoacridine molecules with various numbers of alkyl carbons, named acridine araphanes, on cholinergic receptors. Electrophysiological studies showed that, in addition to its ability to block cholinesterase, Phy blocks the nicotinic acetylcholine receptor ion-channel complex (AChR) in the open state in a voltage- and concentration-dependent manner. Phy activates the ion channel of the AChR. These results from Phy lead to the discovery of a second pathway of activation of the AChR-channel. Tetrahydro-aminoacridine (THA) and acridine araphanes appeared to be open channel blockers of the AChR. In the acridine araphane series, as the number of carbons (n) in the alkyl chain was decreased from 6 to 2, the ability of these compounds to desensitize the AChR was reduced. The length of the alkyl chain also affected the binding of these compounds to the agonist sites of nicotinic receptors. Functional studies on the atrium, ileum and vas deferens revealed that acridine araphanes antagonize the actions of agonists on several muscarinic receptor sub-types. Binding assays demonstrated that as n increased from 2 to 4, the affinities of the compounds for muscarinic receptors were enhanced by about 10-fold, such that the compound with n = 4 was the most potent in this series. Increasing n to 7 decreased the affinity about 30 to 50-fold. When n = 9 the affinity suddenly returned to a value similar to that of n = 2. Acridine araphanes had 100- to 300-fold higher affinity for muscarinic receptors than did mono-acridines. Protonation of acridine araphanes occurs at the N{dollar}\sb{lcub}\rm ar{rcub}{dollar} (nitrogen in acridine ring). The acridine rings are parallel when n = 2-4 before protonation. After protonation, the rings move apart due to a double bond formation between N{dollar}\sb{lcub}\rm c{rcub}{dollar} (nitrogen in alkyl chain) and C{dollar}\sb9{dollar} of each acridine ring. The acridine rings tend to become coplanar as n increases from 2 to 7 and the inter-N{dollar}\sb{lcub}\rm ar{rcub}{dollar} distance extends from 8.6 to 14 A. Beyond n = 9, the rings fold together. Phy alone can activate the AChR-channel. Phy and acridine derivatives including THA are open channel blockers. Acridine araphanes tend to interact with the ligand-binding site, non-competitive binding site and desensitization site of the AChR. The length of the alkyl chain affects the compounds' interaction with the AChR. The hydrophobic regions are optimal for the specific cholinergic antagonist potency of the acridine araphanes which appear as a new class of cholinergic probes. (Abstract shortened with permission of author.)