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New Insights into the Function of M₄ Muscarinic Acetylcholine Receptors Gained Using a Novel Allosteric Modulator and a DREADD (Designer Receptor Exclusively Activated by a Designer Drug)

Drug Discovery Biology Laboratory, Monash Institute of Pharmaceutical Sciences and Department of Pharmacology, Monash University, Melbourne, Australia (V.N., K.L., N.S., R.E.L., P.M.S., A.C.), Neuroscience, Lilly Corporate Center, Indianapolis, Indiana (C.C.F.); Millipore Inc., St. Louis, Missouri (B.N.A.); and Departments of Pharmacology and Medicinal Chemistry, University of North Carolina Chapel Hill Medical School, North Carolina (B.L.R.)

The M₄ muscarinic acetylcholine (ACh) receptor (mAChR) is a potential therapeutic target but characterized by a lack of subtype-selective ligands. We recently generated "designer receptors exclusively activated by a designer drug" (DREADDs), which contained mutations of two conserved orthosteric-site residues (Y¹¹³C/A²⁰³G in the M₄ mAChR) that caused a loss of ACh activity but a gain in responsiveness to clozapine-N-oxide (CNO). The current study characterized the interactions of the wild type and the M₄ DREADD with a range of agonists, antagonists, and the recently discovered M₄ mAChR allosteric potentiator, 3-amino-5-chloro-6-methoxy-4-methyl-thieno[2,3-b]pyridine-2-carboxylic acid cyclopropylamide (LY2033298). LY2033298 displayed positive binding cooperativity with ACh, neutral cooperativity with the antagonist, [³H]quinuclidinyl benzilate, and agonism for activation of phosphorylated extracellular signal-regulated kinase (ERK) 1/2 at the wild-type M₄ mAChR. LY2033298's cooperativity with clozapine or CNO was weakly positive with respect to binding but profoundly negative with respect to LY2033298 signaling. Although the DREADD mutations increased the binding and function of clozapine-like compounds, all other agonists lost the ability to activate the mutant; for the orthosteric agonists ACh and pilocarpine, this was due partly to a reduced affinity, whereas the affinity of LY2033298 or the atypical agonist 4-I-[3-chlorophenyl]carbamoyloxy)-2-butynyltrimethylammnonium chloride was unaltered. The interaction between LY2033298 and clozapine-like compounds reverted to neutral cooperativity on the DREADD, whereas LY2033298 caused a striking functional rescue of ACh potency and efficacy at the DREADD. These results provide conclusive evidence for the retention of a functional allosteric site on the M₄ DREADD and highlight a role for residues Tyr¹¹³ and Ala²⁰³ in the transmission of cooperativity.

Address correspondence to: Prof. Arthur Christopoulos, Drug Discovery Biology Laboratory, Department of Pharmacology, Monash University, Clayton, Victoria 3800, Australia. E-mail: arthur.christopoulos{at}med.monash.edu.au