Probing the Molecular Mechanism of Interaction between 4-n-Butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl]-piperidine (AC-42) and the Muscarinic M₁Receptor: Direct Pharmacological Evidence That AC-42 Is an Allosteric Agonist

Abstract

4-n-Butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl]-piperidine hydrogen chloride (AC-42) is a selective agonist of the muscarinic M 1 receptor previously suggested to interact with an "ectopic" site on this receptor. However, the pharmacological properties of this site (i.e., whether it overlaps to any extent with the classic orthosteric site or represents a novel allosteric site) remain undetermined. In the present study, atropine or pirenzepine significantly inhibited the ability of either carbachol or AC-42 to stimulate inositol phosphate accumulation or intracellular calcium mobilization in Chinese hamster ovary (CHO) cells stably expressing the human M 1 receptor. However, the interaction between either of these antagonists and AC-42 was characterized by Schild slopes significantly less than unity. Increasing the concentrations of atropine revealed that the Schild regression was curvilinear, consistent with a negative allosteric interaction. More direct evidence for an allosteric mode of action of AC-42 was obtained in [3 H]N-methylscopolamine ([ 3 H]NMS) binding studies, in that both AC-42 and the prototypical modulator gallamine failed to fully inhibit specific [ 3 H]NMS binding in a manner that was quantitatively described by an allosteric model applied to both modulator data sets. Furthermore, AC-42 and gallamine significantly retarded the rate of [ 3 H]NMS dissociation from CHO-hM 1 cell membranes, conclusively demonstrating their ability to bind to a topographically distinct site to change M 1 receptor conformation. These data provide the first direct evidence that AC-42 is an allosteric agonist that activates M 1 receptors in the absence of the orthosteric agonist.

Characterization of a CNS penetrant, selective M1muscarinic receptor agonist, 77‐LH‐28‐1

“…for each concentration of agonist was determined on a TopCount plate counter (Perkin Elmer). Agonist concentration-response curves were analysed using a four-parameter logistic fit; pirenzepine and scopolamine datasets were analysed according to models of both competitive and allosteric interactions (Langmead et al, 2006; Prism 4, GraphPad).…”

“…Functional studies using chimeric receptors indicated that the N-terminus-transmembrane (TM)1 and extracellular loop 3-TM7 regions of the M 1 receptor are required for selective activation by AC-42: regions that are distinct from the orthosteric site of the M 1 mAChR deeper in the TM bundle. Subsequently, it was demonstrated that AC-42 is an allosteric agonist of the M 1 mAChR (Langmead et al, 2006), a property that is likely to account for its good selectivity profile.M 1 mAChRs are located centrally in regions such as the cortex, hippocampus and amygdala (Levey, 2003;Wess, 2003). This pattern of distribution suggests that this mAChR subtype may play a role in cognition.…”

“…for each concentration of agonist was determined on a TopCount plate counter (Perkin Elmer). Agonist concentration-response curves were analysed using a four-parameter logistic fit; pirenzepine and scopolamine datasets were analysed according to models of both competitive and allosteric interactions (Langmead et al, 2006; Prism 4, GraphPad).Rat hippocampal CA1 cell firing studies Rats (Sprague-Dawley, Charles River, Margate, UK; 180-230 g) were killed and 400 mm sagittal brain slices containing the hippocampus were prepared. The hippocampal CA3 region of each slice was removed and the remaining slice transferred to a recording chamber.…”

Novel N-Substituted Benzimidazolones as Potent, Selective, CNS-Penetrant, and Orally Active M1 mAChR Agonists

“…Virtual screening of the corporate compound collection against the allosteric binding site 11,12 of M 1 mAChR using an M 1 mAChR homology model, which was built on the basis of the crystal structure of bovine rhodopsin, 20 yielded about 1000 putative hits. Evaluation of these compounds in our human M 1 fluorometric imaging plate reader (FLIPR) assay, which measures compound induced Ca 2þ mobilization in Chinese hamster ovary (CHO) cells that stably expressed human M 1 mAChR, led to the identification of compound 1 as an M 1 mAChR agonist with moderate potency 21 (Figure 1 It is worth noting that compound 1 was also identified as a hit via high throughput screening (HTS) of the corporate compound collection that was carried out later.…”

Cooperativity Has Empirical and Ultimate Levels of Explanation

“…A more extreme form of this latter idea might explain why the muscarinic antagonist, atropine, causes a maximal 10 000-fold shift in the concentration-response curve of the ectopic agonist, AC-42 {4-n-Butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl]-piperidine}, in a manner consistent with the prototypical limiting effect of allosteric antagonism [11]. Given the structure of AC-42, it might behave as a bivalent ligand that derives considerable affinity through the interaction of its 1-(1-oxobutyl) -2-methylbenzene moiety with an ectopic site on the muscarinic receptor, while its 4-n-butylpiperidine residue interacts with the orthosteric site to compete with atropine.…”