Molecular Mechanisms of Action of M5 Muscarinic Acetylcholine Receptor Allosteric Modulators

TY - JOUR

T1 - Molecular Mechanisms of Action of M5 Muscarinic Acetylcholine Receptor Allosteric Modulators

AU - Berizzi, Alice E

AU - Gentry, Patrick R

AU - Rueda, Patricia

AU - Den Hoedt, Sandra

AU - Sexton, Patrick M

AU - Langmead, Christopher J

AU - Christopoulos, Arthur

N1 - Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

PY - 2016/10

Y1 - 2016/10

N2 - Recently, the first subtype-selective allosteric modulators of the M5 muscarinic acetylcholine receptor (mAChR) have been described, but their molecular mechanisms of action remain unknown. Using radioligand-binding and functional assays of inositol phosphate (IP) accumulation and Ca(2+) mobilization in a recombinant cell line stably expressing the human M5 mAChR, we investigated the effects of the positive allosteric modulator (PAM), ML380, and negative allosteric modulator, ML375. In functional assays, ML380 caused robust enhancements in the potency of the full agonists, acetylcholine (ACh), carbachol, and oxotremorine-M, while significantly increasing the maximal response to the partial agonist, pilocarpine. ML380 also demonstrated direct allosteric agonist activity. In contrast, ML375 displayed negative cooperativity with each of the agonists in a manner that varied with the pathway investigated and progressively reduced the maximal pilocarpine response. Radioligand-binding affinity cooperativity estimates were consistent with values derived from functional assays in some instances but not others, suggesting additional allosteric effects on orthosteric ligand efficacy. For ML375 this was confirmed in IP assays performed after reduction of receptor reserve by the alkylating agent, phenoxybenzamine, as it reduced the maximal ACh response. In contrast, ML380 enhanced only ACh potency after receptor alkylation, with no effect on maximal response, consistent with studies of the M1 mAChR with the prototypical PAM, BQZ12. Interaction studies between ML380 and ML375 also indicated that they most likely used an overlapping allosteric site. Our findings indicate that novel small-molecule modulators of the M5 mAChR display mixed mechanisms of action compared with previously characterized modulators of other mAChRs.

AB - Recently, the first subtype-selective allosteric modulators of the M5 muscarinic acetylcholine receptor (mAChR) have been described, but their molecular mechanisms of action remain unknown. Using radioligand-binding and functional assays of inositol phosphate (IP) accumulation and Ca(2+) mobilization in a recombinant cell line stably expressing the human M5 mAChR, we investigated the effects of the positive allosteric modulator (PAM), ML380, and negative allosteric modulator, ML375. In functional assays, ML380 caused robust enhancements in the potency of the full agonists, acetylcholine (ACh), carbachol, and oxotremorine-M, while significantly increasing the maximal response to the partial agonist, pilocarpine. ML380 also demonstrated direct allosteric agonist activity. In contrast, ML375 displayed negative cooperativity with each of the agonists in a manner that varied with the pathway investigated and progressively reduced the maximal pilocarpine response. Radioligand-binding affinity cooperativity estimates were consistent with values derived from functional assays in some instances but not others, suggesting additional allosteric effects on orthosteric ligand efficacy. For ML375 this was confirmed in IP assays performed after reduction of receptor reserve by the alkylating agent, phenoxybenzamine, as it reduced the maximal ACh response. In contrast, ML380 enhanced only ACh potency after receptor alkylation, with no effect on maximal response, consistent with studies of the M1 mAChR with the prototypical PAM, BQZ12. Interaction studies between ML380 and ML375 also indicated that they most likely used an overlapping allosteric site. Our findings indicate that novel small-molecule modulators of the M5 mAChR display mixed mechanisms of action compared with previously characterized modulators of other mAChRs.

KW - Acetylcholine/pharmacology

KW - Allosteric Regulation/drug effects

KW - Allosteric Site/drug effects

KW - Animals

KW - Atropine/pharmacology

KW - CHO Cells

KW - Cricetinae

KW - Cricetulus

KW - Humans

KW - Imidazoles/chemistry

KW - Indazoles/chemistry

KW - Indoles/chemistry

KW - Inositol Phosphates/metabolism

KW - Phenoxybenzamine/pharmacology

KW - Radioligand Assay

KW - Receptor, Muscarinic M5/metabolism

KW - Sulfonamides/chemistry

U2 - 10.1124/mol.116.104182

DO - 10.1124/mol.116.104182

M3 - Journal article

C2 - 27461343

SN - 0026-895X

VL - 90

SP - 427

EP - 436

JO - Molecular Pharmacology

JF - Molecular Pharmacology

IS - 4

ER -