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Research ArticleArticle

Orthosteric and Allosteric Modes of Interaction of Novel Selective Agonists of the M1 Muscarinic Acetylcholine Receptor

Vimesh A. Avlani, Christopher J. Langmead, Elizabeth Guida, Martyn D. Wood, Ben G. Tehan, Hugh J. Herdon, Jeannette M. Watson, Patrick M. Sexton and Arthur Christopoulos
Molecular Pharmacology July 2010, 78 (1) 94-104; DOI: https://doi.org/10.1124/mol.110.064345
Vimesh A. Avlani
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Christopher J. Langmead
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Elizabeth Guida
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Martyn D. Wood
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Ben G. Tehan
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Hugh J. Herdon
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Jeannette M. Watson
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Patrick M. Sexton
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Arthur Christopoulos
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Abstract

Recent years have witnessed the discovery of novel selective agonists of the M1 muscarinic acetylcholine (ACh) receptor (mAChR). One mechanism invoked to account for the selectivity of such agents is that they interact with allosteric sites. We investigated the molecular pharmacology of two such agonists, 1-[3-(4-butyl-1-piperidinyl)propyl]-3,4-dihydro-2(1H)-quinolinone (77-LH-28-1) and 4-n-butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl] piperidine hydrogen chloride (AC-42), at the wild-type M1 mAChR and three mutant M1 mAChRs. Both agonists inhibited the binding of the orthosteric antagonist [3H]N-methyl scopolamine ([3H]NMS) in a manner consistent with orthosteric competition or high negative cooperativity. Functional interaction studies between 77-LH-28-1 and ACh also indicated a competitive mechanism. Dissociation kinetics assays revealed that the agonists could bind allosterically when the orthosteric site was prelabeled with [3H]NMS and that 77-LH-28-1 competed with the prototypical allosteric modulator heptane-1,7-bis-[dimethyl-3′-phthalimidopropyl]-ammonium bromide under these conditions. Mutation of the key orthosteric site residues Y381A (transmembrane helix 6) and W101A (transmembrane helix 3) reduced the affinity of prototypical orthosteric agonists but increased the affinity of the novel agonists. Divergent effects were also noted on agonist signaling efficacies at these mutants. We identified a novel mutation, F77I (transmembrane helix 2), which selectively reduced the efficacy of the novel agonists in mediating intracellular Ca2+ elevation and phosphorylation of extracellular signal regulated kinase 1/2. Molecular modeling suggested a possible “bitopic” binding mode, whereby the agonists extend down into the orthosteric site as well as up toward extracellular receptor regions associated with an allosteric site. It is possible that this bitopic mode may explain the pharmacology of other selective mAChR agonists.

Footnotes

  • This work was funded by the National Health and Medical Research Council (NHMRC) of Australia [Program Grant 519461]. A.C. is a Senior Research Fellow of the NHMRC, and P.S. is a Principal Research Fellow of the NHMRC.

  • Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.

    doi:10.1124/mol.110.064345.

  • ABBREVIATIONS:

    mAChR
    muscarinic acetylcholine receptor
    C7/3-phth
    heptane-1,7-bis-[dimethyl-3′-phthalimidopropyl]-ammonium bromide
    AC-42
    4-n-butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl] piperidine hydrogen chloride
    NMS
    N-methylscopolamine
    TM
    transmembrane domain
    McN-A-343
    4-I-[3-chlorophenyl]carbamoyloxy)-2-butynyltrimethylammnonium chloride
    77-LH-28-1
    1-[3-(4-butyl-1-piperidinyl)propyl]-3,4-dihydro-2(1H)-quinolinone
    CHO
    Chinese hamster ovary
    FBS
    fetal bovine serum
    ERK
    extracellular signal-regulated kinase
    QNB
    quinuclidinyl benzilate
    WT
    wild type
    pERK1/2
    phosphorylated ERK1/2.

  • Received February 23, 2010.
  • Accepted April 21, 2010.
  • Copyright © 2010 The American Society for Pharmacology and Experimental Therapeutics
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Molecular Pharmacology: 78 (1)
Molecular Pharmacology
Vol. 78, Issue 1
1 Jul 2010
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Research ArticleArticle

Orthosteric and Allosteric Modes of Interaction of Novel Selective Agonists of the M1 Muscarinic Acetylcholine Receptor

Vimesh A. Avlani, Christopher J. Langmead, Elizabeth Guida, Martyn D. Wood, Ben G. Tehan, Hugh J. Herdon, Jeannette M. Watson, Patrick M. Sexton and Arthur Christopoulos
Molecular Pharmacology July 1, 2010, 78 (1) 94-104; DOI: https://doi.org/10.1124/mol.110.064345

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Research ArticleArticle

Orthosteric and Allosteric Modes of Interaction of Novel Selective Agonists of the M1 Muscarinic Acetylcholine Receptor

Vimesh A. Avlani, Christopher J. Langmead, Elizabeth Guida, Martyn D. Wood, Ben G. Tehan, Hugh J. Herdon, Jeannette M. Watson, Patrick M. Sexton and Arthur Christopoulos
Molecular Pharmacology July 1, 2010, 78 (1) 94-104; DOI: https://doi.org/10.1124/mol.110.064345
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