Probe dependence in the allosteric modulation of a G protein-coupled receptor: implications for detection and validation of allosteric ligand effects

Research output: Contribution to journalArticleResearchpeer-review

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 M(4) muscarinic acetylcholine (ACh) receptors (mAChRs) on the basis of its ability to preferentially potentiate the actions of ACh at the M(4) mAChR subtype. In the current study, we show that LY2033298 can also bind to the M(2) 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 [(35)S]guanosine 5 -O-(3-thio)triphosphate or extracellular signal-regulated kinase 1/2 phosphorylation assay at the M(2) 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 Trp99(3.28) (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, Tyr104(3.33) and Tyr403(6.51), 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.
Original languageEnglish
Pages (from-to)41 - 52
Number of pages12
JournalMolecular Pharmacology
Volume81
Issue number1
DOIs
Publication statusPublished - 2012

Cite this

@article{b6c98cb22b214c3c98c11cdbb229eb91,
title = "Probe dependence in the allosteric modulation of a G protein-coupled receptor: implications for detection and validation of allosteric ligand effects",
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 M(4) muscarinic acetylcholine (ACh) receptors (mAChRs) on the basis of its ability to preferentially potentiate the actions of ACh at the M(4) mAChR subtype. In the current study, we show that LY2033298 can also bind to the M(2) 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 [(35)S]guanosine 5 -O-(3-thio)triphosphate or extracellular signal-regulated kinase 1/2 phosphorylation assay at the M(2) 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 Trp99(3.28) (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, Tyr104(3.33) and Tyr403(6.51), 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.",
author = "Celine Valant and Felder, {Christian C} and Sexton, {Patrick M} and Arthur Christopoulos",
year = "2012",
doi = "10.1124/mol.111.074872",
language = "English",
volume = "81",
pages = "41 -- 52",
journal = "Molecular Pharmacology",
issn = "1521-0111",
publisher = "ACS Books",
number = "1",

}

TY - JOUR

T1 - Probe dependence in the allosteric modulation of a G protein-coupled receptor: implications for detection and validation of allosteric ligand effects

AU - Valant, Celine

AU - Felder, Christian C

AU - Sexton, Patrick M

AU - Christopoulos, Arthur

PY - 2012

Y1 - 2012

N2 - 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 M(4) muscarinic acetylcholine (ACh) receptors (mAChRs) on the basis of its ability to preferentially potentiate the actions of ACh at the M(4) mAChR subtype. In the current study, we show that LY2033298 can also bind to the M(2) 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 [(35)S]guanosine 5 -O-(3-thio)triphosphate or extracellular signal-regulated kinase 1/2 phosphorylation assay at the M(2) 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 Trp99(3.28) (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, Tyr104(3.33) and Tyr403(6.51), 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.

AB - 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 M(4) muscarinic acetylcholine (ACh) receptors (mAChRs) on the basis of its ability to preferentially potentiate the actions of ACh at the M(4) mAChR subtype. In the current study, we show that LY2033298 can also bind to the M(2) 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 [(35)S]guanosine 5 -O-(3-thio)triphosphate or extracellular signal-regulated kinase 1/2 phosphorylation assay at the M(2) 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 Trp99(3.28) (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, Tyr104(3.33) and Tyr403(6.51), 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.

UR - http://molpharm.aspetjournals.org/content/81/1/41.full.pdf

U2 - 10.1124/mol.111.074872

DO - 10.1124/mol.111.074872

M3 - Article

VL - 81

SP - 41

EP - 52

JO - Molecular Pharmacology

JF - Molecular Pharmacology

SN - 1521-0111

IS - 1

ER -