title: 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).
abstract: 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.
authors.affiliation: Drug Discovery Biology Laboratory, Monash Institute of Pharmaceutical Sciences, Department of Pharmacology, Monash University, Clayton, Victoria 3800, Australia.
authors.forename: Vindhya
authors.initials: V
authors.lastname: Nawaratne
authors.forename: Katie
authors.initials: K
authors.lastname: Leach
authors.forename: Nur
authors.initials: N
authors.lastname: Suratman
authors.forename: Richard E
authors.initials: RE
authors.lastname: Loiacono
authors.forename: Christian C
authors.initials: CC
authors.lastname: Felder
authors.forename: Blaine N
authors.initials: BN
authors.lastname: Armbruster
authors.forename: Bryan L
authors.initials: BL
authors.lastname: Roth
authors.forename: Patrick M
authors.initials: PM
authors.lastname: Sexton
authors.forename: Arthur
authors.initials: A
authors.lastname: Christopoulos
mesh_terms: D000109:Acetylcholine / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D000494:Allosteric Regulation / Q000502:physiology; D000495:Allosteric Site / Q000502:physiology; D000818:Animals; D016466:CHO Cells; D003024:Clozapine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D006224:Cricetinae; D003412:Cricetulus; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D008954:Models, Biological; D015394:Molecular Structure; D009154:Mutation; D009539:Nicotinic Acids / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology*; D010766:Phosphorylation / Q000187:drug effects; D011813:Quinuclidinyl Benzilate / Q000378:metabolism / Q000494:pharmacology; D011869:Radioligand Assay; D043588:Receptor, Muscarinic M4 / Q000819:agonists / Q000037:antagonists & inhibitors / Q000502:physiology*; D015398:Signal Transduction; D013876:Thiophenes / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology*
chemical_list: C531529:3-amino-5-chloro-6-methoxy-4-methyl-thieno(2,3-b)pyridine-2-carboxylic acid cyclopropylamide; D009539:Nicotinic Acids; D043588:Receptor, Muscarinic M4; D013876:Thiophenes; D011813:Quinuclidinyl Benzilate; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D003024:Clozapine; C079149:clozapine N-oxide; D000109:Acetylcholine