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J. Biol. Chem., Vol. 283, Issue 43, 29312-29321, October 24, 2008

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A Novel Mechanism of G Protein-coupled Receptor Functional Selectivity

MUSCARINIC PARTIAL AGONIST McN-A-343 AS A BITOPIC ORTHOSTERIC/ALLOSTERIC LIGAND^*

Celine Valant, Karen J. Gregory¹, Nathan E. Hall, Peter J. Scammells^¶, Michael J. Lew^||, Patrick M. Sexton², and Arthur Christopoulos, A senior research fellow of the NHMRC³

From the Drug Discovery Biology Laboratory, and the ^¶Department of Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, 3052 Victoria, the Department of Pharmacology, Monash University, 3800 Victoria, and the ^||Department of Pharmacology, University of Melbourne, 3010 Victoria, Australia

Many G protein-coupled receptors (GPCRs) possess allosteric binding sites distinct from the orthosteric site utilized by their cognate ligands, but most GPCR allosteric modulators reported to date lack signaling efficacy in their own right. McN-A-343 (4-(N-(3-chlorophenyl)carbamoyloxy)-2-butynyltrimethylammonium chloride) is a functionally selective muscarinic acetylcholine receptor (mAChR) partial agonist that can also interact allosterically at the M₂ mAChR. We hypothesized that this molecule simultaneously utilizes both an allosteric and the orthosteric site on the M₂ mAChR to mediate these effects. By synthesizing progressively truncated McN-A-343 derivatives, we identified two, which minimally contain 3-chlorophenylcarbamate, as pure allosteric modulators. These compounds were positive modulators of the orthosteric antagonist N-[³H]methylscopolamine, but in functional assays of M₂ mAChR-mediated ERK1/2 phosphorylation and guanosine 5'-3-O-([³⁵S]thio)triphosphate binding, they were negative modulators of agonist efficacy. This negative allosteric effect was diminished upon mutation of Y177A in the second extracellular (E2) loop of the M₂ mAChR that is known to reduce prototypical allosteric modulator potency. Our results are consistent with McN-A-343 being a bitopic orthosteric/allosteric ligand with the allosteric moiety engendering partial agonism and functional selectivity. This finding suggests a novel and largely unappreciated mechanism of "directed efficacy" whereby functional selectivity may be engendered in a GPCR by utilizing an allosteric ligand to direct the signaling of an orthosteric ligand encoded within the same molecule.

Received for publication, May 19, 2008 , and in revised form, August 22, 2008.

^* This work was supported in part by National Health and Medical Research Council (NHMRC) of Australia Grant 400134 and Australian Research Council Discovery Grant DP0877497. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1 and 2 as well as other information.

¹ Recipient of an NHMRC Dora Lush Postgraduate Research Scholarship and a Dowd Foundation Scholarship in the neurosciences.

² A principal research fellow of the NHMRC.

³ To whom correspondence should be addressed: Drug Discovery Biology Laboratory, Dept. of Pharmacology, Monash University, Clayton, 3800 Victoria, Australia. Tel.: 613-9905-3817; Fax: 613-9905-5953; E-mail: arthur.christopoulos{at}med.monash.edu.au.

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This article has been cited by other articles:

				C. Valant, P. M. Sexton, and A. Christopoulos Orthosteric/Allosteric Bitopic Ligands: Going Hybrid at GPCRs Mol. Interv., June 1, 2009; 9(3): 125 - 135. [Abstract] [Full Text] [PDF]