RT Journal Article
SR Electronic
T1 Probing the Molecular Mechanism of Interaction between 4-<em>n</em>-Butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl]-piperidine (AC-42) and the Muscarinic M<sub>1</sub> Receptor: Direct Pharmacological Evidence That AC-42 Is an Allosteric Agonist
JF Molecular Pharmacology
JO Mol Pharmacol
FD American Society for Pharmacology and Experimental Therapeutics
SP 236
OP 246
DO 10.1124/mol.105.017814
VO 69
IS 1
A1 Christopher J. Langmead
A1 Victoria A. H. Fry
A1 Ian T. Forbes
A1 Clive L. Branch
A1 Arthur Christopoulos
A1 Martyn D. Wood
A1 Hugh J. Herdon
YR 2006
UL http://molpharm.aspetjournals.org/content/69/1/236.abstract
AB 4-n-Butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl]-piperidine hydrogen chloride (AC-42) is a selective agonist of the muscarinic M1 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 M1 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 [3H]N-methylscopolamine ([3H]NMS) binding studies, in that both AC-42 and the prototypical modulator gallamine failed to fully inhibit specific [3H]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 [3H]NMS dissociation from CHO-hM1 cell membranes, conclusively demonstrating their ability to bind to a topographically distinct site to change M1 receptor conformation. These data provide the first direct evidence that AC-42 is an allosteric agonist that activates M1 receptors in the absence of the orthosteric agonist.