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Research ArticleArticle

Structure-Function Studies of Allosteric Agonism at M2 Muscarinic Acetylcholine Receptors

Lauren T. May, Vimesh A. Avlani, Christopher J. Langmead, Hugh J. Herdon, Martyn D. Wood, Patrick M. Sexton and Arthur Christopoulos
Molecular Pharmacology August 2007, 72 (2) 463-476; DOI: https://doi.org/10.1124/mol.107.037630
Lauren T. May
Drug Discovery Biology Laboratory, Department of Pharmacology, Monash University, Clayton, Victoria, Australia (L.T.M., V.A.A., P.M.S., A.C.) and Psychiatry Centre of Excellence for Drug Discovery, GlaxoSmithKline, Harlow, Essex, United Kingdom (C.J.L., H.J.H., M.D.W.)
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Vimesh A. Avlani
Drug Discovery Biology Laboratory, Department of Pharmacology, Monash University, Clayton, Victoria, Australia (L.T.M., V.A.A., P.M.S., A.C.) and Psychiatry Centre of Excellence for Drug Discovery, GlaxoSmithKline, Harlow, Essex, United Kingdom (C.J.L., H.J.H., M.D.W.)
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Christopher J. Langmead
Drug Discovery Biology Laboratory, Department of Pharmacology, Monash University, Clayton, Victoria, Australia (L.T.M., V.A.A., P.M.S., A.C.) and Psychiatry Centre of Excellence for Drug Discovery, GlaxoSmithKline, Harlow, Essex, United Kingdom (C.J.L., H.J.H., M.D.W.)
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Hugh J. Herdon
Drug Discovery Biology Laboratory, Department of Pharmacology, Monash University, Clayton, Victoria, Australia (L.T.M., V.A.A., P.M.S., A.C.) and Psychiatry Centre of Excellence for Drug Discovery, GlaxoSmithKline, Harlow, Essex, United Kingdom (C.J.L., H.J.H., M.D.W.)
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Martyn D. Wood
Drug Discovery Biology Laboratory, Department of Pharmacology, Monash University, Clayton, Victoria, Australia (L.T.M., V.A.A., P.M.S., A.C.) and Psychiatry Centre of Excellence for Drug Discovery, GlaxoSmithKline, Harlow, Essex, United Kingdom (C.J.L., H.J.H., M.D.W.)
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Patrick M. Sexton
Drug Discovery Biology Laboratory, Department of Pharmacology, Monash University, Clayton, Victoria, Australia (L.T.M., V.A.A., P.M.S., A.C.) and Psychiatry Centre of Excellence for Drug Discovery, GlaxoSmithKline, Harlow, Essex, United Kingdom (C.J.L., H.J.H., M.D.W.)
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Arthur Christopoulos
Drug Discovery Biology Laboratory, Department of Pharmacology, Monash University, Clayton, Victoria, Australia (L.T.M., V.A.A., P.M.S., A.C.) and Psychiatry Centre of Excellence for Drug Discovery, GlaxoSmithKline, Harlow, Essex, United Kingdom (C.J.L., H.J.H., M.D.W.)
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Abstract

The M2 muscarinic acetylcholine receptor (mAChR) possesses at least one binding site for allosteric modulators that is dependent on the residues 172EDGE175, Tyr177, and Thr423. However, the contribution of these residues to actions of allosteric agonists, as opposed to modulators, is unknown. We created mutant M2 mAChRs in which the charge of the 172EDGE175 sequence had been neutralized and each Tyr177 and Thr423 was substituted with alanine. Radioligand binding experiments revealed that these mutations had a profound inhibitory effect on the prototypical modulators gallamine, alcuronium, and heptane-1,7-bis-[dimethyl-3′-phthalimidopropyl]-ammonium bromide (C7/3-phth) but minimal effects on the orthosteric antagonist [3H]N-methyl scopolamine. In contrast, the allosteric agonists 4-I-[3-chlorophenyl]carbamoyloxy)-2-butynyltrimethylammnonium chloride (McN-A-343), 4-n-butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl] piperidine hydrogen chloride (AC-42), and the novel AC-42 derivative 1-[3-(4-butyl-1-piperidinyl)propyl]-3,4-dihydro-2(1H)-quinolinone (77-LH-28-1) demonstrated an increased affinity or proportion of high-affinity sites at the combined EDGE-YT mutation, indicating a different mode of binding to the prototypical modulators. Subsequent functional assays of extracellular signal-regulated kinase (ERK)1/2 phosphorylation and guanosine 5′-(γ-[35S]thio)triphosphate ([35S]GTPγS) binding revealed minimal effects of the mutations on the orthosteric agonists acetylcholine (ACh) and pilocarpine but a significant increase in the efficacy of McN-A-343 and potency of 77-LH-28-1. Additional mutagenesis experiments found that these effects were predominantly mediated by Tyr177 and Thr423, rather than the 172EDGE175 sequence. The functional interaction between each of the allosteric agonists and ACh was characterized by high negative cooperativity but was consistent with an increased allosteric agonist affinity at the combined EDGE-YT mutant M2 mAChR. This study has thus revealed a differential role of critical allosteric site residues on the binding and function of allosteric agonists versus allosteric modulators of M2 mAChRs.

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Molecular Pharmacology: 72 (2)
Molecular Pharmacology
Vol. 72, Issue 2
1 Aug 2007
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Research ArticleArticle

Structure-Function Studies of Allosteric Agonism at M2 Muscarinic Acetylcholine Receptors

Lauren T. May, Vimesh A. Avlani, Christopher J. Langmead, Hugh J. Herdon, Martyn D. Wood, Patrick M. Sexton and Arthur Christopoulos
Molecular Pharmacology August 1, 2007, 72 (2) 463-476; DOI: https://doi.org/10.1124/mol.107.037630

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Research ArticleArticle

Structure-Function Studies of Allosteric Agonism at M2 Muscarinic Acetylcholine Receptors

Lauren T. May, Vimesh A. Avlani, Christopher J. Langmead, Hugh J. Herdon, Martyn D. Wood, Patrick M. Sexton and Arthur Christopoulos
Molecular Pharmacology August 1, 2007, 72 (2) 463-476; DOI: https://doi.org/10.1124/mol.107.037630
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