Abstract
We recently described 3-amino-5-chloro-6-methoxy-4-methylthieno[2,3-b]pyridine-2-carboxylic acid cyclopropylamide (LY2033298) as a novel allosteric modulator of M4 muscarinic acetylcholine (ACh) receptors (mAChRs) on the basis of its ability to preferentially potentiate the actions of ACh at the M4 mAChR subtype. In the current study, we show that LY2033298 can also bind to the M2 mAChR and mediate robust positive or negative allosteric effects, depending on the orthosteric ligand used as a probe of receptor activity. This finding of striking “probe dependence” indicates that the previously described selectivity of the modulator does not arise as a consequence of selective affinity for a poorly conserved allosteric site but rather is due to subtype-selective cooperativity with ACh upon interaction with a common allosteric binding site. Moreover, a comparison of the effects of the modulator on orthosteric ligand affinity relative to signaling through a [35S]guanosine 5′-O-(3-thio)triphosphate or extracellular signal-regulated kinase 1/2 phosphorylation assay at the M2 mAChR revealed that, although the effects on binding were positive in all instances, the effects on signaling were either positive or strongly negative, depending on the agonist and the pathway. Mutational analysis identified residues Tyr177 and Trp993.28 (Ballesteros and Weinstein numbers are provided in superscript to indicate relative position of residues within the transmembrane domain) as contributing to the binding of LY2033298, whereas the orthosteric site residues, Tyr1043.33 and Tyr4036.51, contributed to the ability of the ligand to impose pathway-biased modulation. Taken together, these findings have important implications for the detection and validation of allosteric modulators of G protein-coupled receptors (GPCRs), because they highlight the potential for ligand misclassification or lack of appreciation of off-target allosteric activities.
Footnotes
↵ The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.
This work was supported by the National Health and Medical Research Council of Australia [Program Grant 519461]. A.C. is a Senior Research Fellow and P.M.S. is a Principal Research Fellow of the National Health and Medical Research Council of Australia.
A.C. is a Consultant for Johnson and Johnson, Kai Pharmaceuticals, and Alchemia.
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
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ABBREVIATIONS:
- mAChR
- muscarinic acetylcholine receptor
- ACh
- acetylcholine
- GPCR
- G protein-coupled receptor
- LY2033298
- 3-amino-5-chloro-6-methoxy-4-methyl-thieno[2,3-b]pyridine-2-carboxylic acid cyclopropylamide
- QNB
- quinuclidinyl benzilate
- NMS
- N-methylscopolamine
- GTPγS
- guanosine 5′-O-(3-thio)triphosphate
- ERK1/2
- extracellular signal-regulated kinases 1/2
- p
- phospho
- CHO
- Chinese hamster ovary
- WT
- wild-type
- McN-A-343
- 4-I-[3-chlorophenyl]carbamoyloxy)-2-butynyltrimethylammnonium chloride
- TMA
- tetramethylammonium
- TM
- transmembrane domain
- E2L
- second extracellular loop
- C7/3-phth
- heptane-1,7-bis-(dimethyl-3′-phthalimidopropyl) ammonium bromide
- Org27569
- 5-chloro-3-ethyl-1H-indole-2-carboxylic acid [2-(4-piperidin-1-yl-phenyl)-ethyl]-amide.
- Received July 19, 2011.
- Accepted October 7, 2011.
- Copyright © 2012 The American Society for Pharmacology and Experimental Therapeutics
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