Development of a radioligand, [(3)H]LY2119620, to probe the human M(2) and M(4) muscarinic receptor allosteric binding sites

Douglas A Schober, Carrie H Croy, Hongling Xiao, Arthur Christopoulos, Christian C Felder

Research output: Contribution to journalArticleResearchpeer-review

Abstract

In this study, we characterized a muscarinic acetylcholine receptor (mAChR) potentiator, LY2119620 (3-amino-5-chloro-N-cyclopropyl-4-methyl-6-[2-(4-methylpiperazin-1-yl)-2-oxoethox y]thieno[2,3-b]pyridine-2-carboxamide) as a novel probe of the human M2 and M4 allosteric binding sites. Since the discovery of allosteric binding sites on G protein-coupled receptors, compounds targeting these novel sites have been starting to emerge. For example, LY2033298 (3-amino-5-chloro-6-methoxy-4-methyl-thieno(2,3-b)pyridine-2-carboxylic acid cyclopropylamid) and a derivative of this chemical scaffold, VU152100 (3-amino-N-(4-methoxybenzyl)-4,6-dimethylthieno[2,3-b]pyridine carboxamide), bind to the human M4 mAChR allosteric pocket. In the current study, we characterized LY2119620, a compound similar in structure to LY2033298 and binds to the same allosteric site on the human M4 mAChRs. However, LY2119620 also binds to an allosteric site on the human M2 subtype. [(3)H]NMS ([(3)H]N-methylscopolamine) binding experiments confirm that LY2119620 does not compete for the orthosteric binding pocket at any of the five muscarinic receptor subtypes. Dissociation kinetic studies using [(3)H]NMS further support that LY2119620 binds allosterically to the M2 and M4 mAChRs and was positively cooperative with muscarinic orthosteric agonists. To probe directly the allosteric sites on M2 and M4, we radiolabeled LY2119620. Cooperativity binding of [(3)H]LY2119620 with mAChR orthosteric agonists detects significant changes in Bmax values with little change in Kd, suggesting a G protein-dependent process. Furthermore, [(3)H]LY2119620 was displaced by compounds of similar chemical structure but not by previously described mAChR allosteric compounds such as gallamine or WIN 62,577 (17-beta-hydroxy-17-alpha-ethynyl-delta-4-androstano[3,2-b]pyrimido[1,2-a]benzimi dazole). Our results therefore demonstrate the development of a radioligand, [(3)H]LY2119620 to probe specifically the human M2 and M4 muscarinic receptor allosteric binding sites.
Original languageEnglish
Pages (from-to)116 - 123
Number of pages8
JournalMolecular Pharmacology
Volume86
Issue number1
DOIs
Publication statusPublished - 2014

Cite this

Schober, Douglas A ; Croy, Carrie H ; Xiao, Hongling ; Christopoulos, Arthur ; Felder, Christian C. / Development of a radioligand, [(3)H]LY2119620, to probe the human M(2) and M(4) muscarinic receptor allosteric binding sites. In: Molecular Pharmacology. 2014 ; Vol. 86, No. 1. pp. 116 - 123.
@article{2f5b0305612b42b49a85c3dcda540205,
title = "Development of a radioligand, [(3)H]LY2119620, to probe the human M(2) and M(4) muscarinic receptor allosteric binding sites",
abstract = "In this study, we characterized a muscarinic acetylcholine receptor (mAChR) potentiator, LY2119620 (3-amino-5-chloro-N-cyclopropyl-4-methyl-6-[2-(4-methylpiperazin-1-yl)-2-oxoethox y]thieno[2,3-b]pyridine-2-carboxamide) as a novel probe of the human M2 and M4 allosteric binding sites. Since the discovery of allosteric binding sites on G protein-coupled receptors, compounds targeting these novel sites have been starting to emerge. For example, LY2033298 (3-amino-5-chloro-6-methoxy-4-methyl-thieno(2,3-b)pyridine-2-carboxylic acid cyclopropylamid) and a derivative of this chemical scaffold, VU152100 (3-amino-N-(4-methoxybenzyl)-4,6-dimethylthieno[2,3-b]pyridine carboxamide), bind to the human M4 mAChR allosteric pocket. In the current study, we characterized LY2119620, a compound similar in structure to LY2033298 and binds to the same allosteric site on the human M4 mAChRs. However, LY2119620 also binds to an allosteric site on the human M2 subtype. [(3)H]NMS ([(3)H]N-methylscopolamine) binding experiments confirm that LY2119620 does not compete for the orthosteric binding pocket at any of the five muscarinic receptor subtypes. Dissociation kinetic studies using [(3)H]NMS further support that LY2119620 binds allosterically to the M2 and M4 mAChRs and was positively cooperative with muscarinic orthosteric agonists. To probe directly the allosteric sites on M2 and M4, we radiolabeled LY2119620. Cooperativity binding of [(3)H]LY2119620 with mAChR orthosteric agonists detects significant changes in Bmax values with little change in Kd, suggesting a G protein-dependent process. Furthermore, [(3)H]LY2119620 was displaced by compounds of similar chemical structure but not by previously described mAChR allosteric compounds such as gallamine or WIN 62,577 (17-beta-hydroxy-17-alpha-ethynyl-delta-4-androstano[3,2-b]pyrimido[1,2-a]benzimi dazole). Our results therefore demonstrate the development of a radioligand, [(3)H]LY2119620 to probe specifically the human M2 and M4 muscarinic receptor allosteric binding sites.",
author = "Schober, {Douglas A} and Croy, {Carrie H} and Hongling Xiao and Arthur Christopoulos and Felder, {Christian C}",
year = "2014",
doi = "10.1124/mol.114.091785",
language = "English",
volume = "86",
pages = "116 -- 123",
journal = "Molecular Pharmacology",
issn = "1521-0111",
publisher = "ACS Books",
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}

Development of a radioligand, [(3)H]LY2119620, to probe the human M(2) and M(4) muscarinic receptor allosteric binding sites. / Schober, Douglas A; Croy, Carrie H; Xiao, Hongling; Christopoulos, Arthur; Felder, Christian C.

In: Molecular Pharmacology, Vol. 86, No. 1, 2014, p. 116 - 123.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Development of a radioligand, [(3)H]LY2119620, to probe the human M(2) and M(4) muscarinic receptor allosteric binding sites

AU - Schober, Douglas A

AU - Croy, Carrie H

AU - Xiao, Hongling

AU - Christopoulos, Arthur

AU - Felder, Christian C

PY - 2014

Y1 - 2014

N2 - In this study, we characterized a muscarinic acetylcholine receptor (mAChR) potentiator, LY2119620 (3-amino-5-chloro-N-cyclopropyl-4-methyl-6-[2-(4-methylpiperazin-1-yl)-2-oxoethox y]thieno[2,3-b]pyridine-2-carboxamide) as a novel probe of the human M2 and M4 allosteric binding sites. Since the discovery of allosteric binding sites on G protein-coupled receptors, compounds targeting these novel sites have been starting to emerge. For example, LY2033298 (3-amino-5-chloro-6-methoxy-4-methyl-thieno(2,3-b)pyridine-2-carboxylic acid cyclopropylamid) and a derivative of this chemical scaffold, VU152100 (3-amino-N-(4-methoxybenzyl)-4,6-dimethylthieno[2,3-b]pyridine carboxamide), bind to the human M4 mAChR allosteric pocket. In the current study, we characterized LY2119620, a compound similar in structure to LY2033298 and binds to the same allosteric site on the human M4 mAChRs. However, LY2119620 also binds to an allosteric site on the human M2 subtype. [(3)H]NMS ([(3)H]N-methylscopolamine) binding experiments confirm that LY2119620 does not compete for the orthosteric binding pocket at any of the five muscarinic receptor subtypes. Dissociation kinetic studies using [(3)H]NMS further support that LY2119620 binds allosterically to the M2 and M4 mAChRs and was positively cooperative with muscarinic orthosteric agonists. To probe directly the allosteric sites on M2 and M4, we radiolabeled LY2119620. Cooperativity binding of [(3)H]LY2119620 with mAChR orthosteric agonists detects significant changes in Bmax values with little change in Kd, suggesting a G protein-dependent process. Furthermore, [(3)H]LY2119620 was displaced by compounds of similar chemical structure but not by previously described mAChR allosteric compounds such as gallamine or WIN 62,577 (17-beta-hydroxy-17-alpha-ethynyl-delta-4-androstano[3,2-b]pyrimido[1,2-a]benzimi dazole). Our results therefore demonstrate the development of a radioligand, [(3)H]LY2119620 to probe specifically the human M2 and M4 muscarinic receptor allosteric binding sites.

AB - In this study, we characterized a muscarinic acetylcholine receptor (mAChR) potentiator, LY2119620 (3-amino-5-chloro-N-cyclopropyl-4-methyl-6-[2-(4-methylpiperazin-1-yl)-2-oxoethox y]thieno[2,3-b]pyridine-2-carboxamide) as a novel probe of the human M2 and M4 allosteric binding sites. Since the discovery of allosteric binding sites on G protein-coupled receptors, compounds targeting these novel sites have been starting to emerge. For example, LY2033298 (3-amino-5-chloro-6-methoxy-4-methyl-thieno(2,3-b)pyridine-2-carboxylic acid cyclopropylamid) and a derivative of this chemical scaffold, VU152100 (3-amino-N-(4-methoxybenzyl)-4,6-dimethylthieno[2,3-b]pyridine carboxamide), bind to the human M4 mAChR allosteric pocket. In the current study, we characterized LY2119620, a compound similar in structure to LY2033298 and binds to the same allosteric site on the human M4 mAChRs. However, LY2119620 also binds to an allosteric site on the human M2 subtype. [(3)H]NMS ([(3)H]N-methylscopolamine) binding experiments confirm that LY2119620 does not compete for the orthosteric binding pocket at any of the five muscarinic receptor subtypes. Dissociation kinetic studies using [(3)H]NMS further support that LY2119620 binds allosterically to the M2 and M4 mAChRs and was positively cooperative with muscarinic orthosteric agonists. To probe directly the allosteric sites on M2 and M4, we radiolabeled LY2119620. Cooperativity binding of [(3)H]LY2119620 with mAChR orthosteric agonists detects significant changes in Bmax values with little change in Kd, suggesting a G protein-dependent process. Furthermore, [(3)H]LY2119620 was displaced by compounds of similar chemical structure but not by previously described mAChR allosteric compounds such as gallamine or WIN 62,577 (17-beta-hydroxy-17-alpha-ethynyl-delta-4-androstano[3,2-b]pyrimido[1,2-a]benzimi dazole). Our results therefore demonstrate the development of a radioligand, [(3)H]LY2119620 to probe specifically the human M2 and M4 muscarinic receptor allosteric binding sites.

UR - http://molpharm.aspetjournals.org/content/86/1/116.full.pdf+html

U2 - 10.1124/mol.114.091785

DO - 10.1124/mol.114.091785

M3 - Article

VL - 86

SP - 116

EP - 123

JO - Molecular Pharmacology

JF - Molecular Pharmacology

SN - 1521-0111

IS - 1

ER -