Dataset Information

Identification of a Novel Allosteric Site at the M₅ Muscarinic Acetylcholine Receptor.

ABSTRACT: The M₅ muscarinic acetylcholine receptor (mAChR) has emerged as an exciting therapeutic target for the treatment of addiction and behavioral disorders. This has been in part due to promising preclinical studies with the M₅ mAChR selective negative allosteric modulator (NAM), ML375. The binding site of ML375 remains unknown, however, making it difficult to develop improved M₅ mAChR selective modulators. To determine the possible location of the ML375 binding site, we used radioligand binding and functional assays to show that ML375 does not interact with the well-characterized "common" mAChR allosteric site located in the receptor's extracellular vestibule, nor a previously proposed second allosteric site recognized by the modulator, amiodarone. Molecular docking was used to predict potential allosteric sites within the transmembrane (TM) domain of the M₅ mAChR. These predicted sites were assessed using M₅-M₂ mAChR receptor chimeras and further targeted with site-directed mutagenesis, which enabled the identification of a putative binding site for ML375 at the interface of TMs 2-4. Collectively, these results identify a third allosteric site at the M₅ mAChR and highlight the ability of allosteric modulators to selectively target highly conserved proteins.

SUBMITTER: Burger WAC

PROVIDER: S-EPMC7616173 | biostudies-literature | 2021 Aug

REPOSITORIES: biostudies-literature

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Publications

Identification of a Novel Allosteric Site at the M5 Muscarinic Acetylcholine Receptor.

Burger Wessel A C WAC Gentry Patrick R PR Berizzi Alice E AE Vuckovic Ziva Z van der Westhuizen Emma T ET Thompson Geoff G Yeasmin Mahmuda M Lindsley Craig W CW Sexton Patrick M PM Langmead Christopher J CJ Tobin Andrew B AB Christopoulos Arthur A Valant Celine C Thal David M DM

ACS chemical neuroscience 20210805 16

The M5 muscarinic acetylcholine receptor (mAChR) has emerged as an exciting therapeutic target for the treatment of addiction and behavioral disorders. This has been in part due to promising preclinical studies with the M5 mAChR selective negative allosteric modulator (NAM), ML375. The binding site of ML375 remains unknown, however, making it difficult to develop improved M5 mAChR selective modulators. To determine the possible location of the ML375 binding site, ...[more]

PMID: 34351123

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Project description:Background and objectiveMuscarinic acetylcholine receptors (mAChRs) are 7-transmembrane, G protein-coupled receptors that regulate a variety of physiological processes and represent potentially important targets for therapeutic intervention. mAChRs can be stimulated by full and partial orthosteric and allosteric agonists, however the relative abilities of such ligands to induce conformational changes in the receptor remain unclear. To gain further insight into the actions of mAChR agonists, we have developed a fluorescently tagged M(1) mAChR that reports ligand-induced conformational changes in real-time by changes in Förster resonance energy transfer (FRET).MethodsVariants of CFP and YFP were inserted into the third intracellular loop and at the end of the C-terminus of the mouse M(1) mAChR, respectively. The optimized FRET receptor construct (M(1)-cam5) was expressed stably in HEK293 cells.ResultsThe variant CFP/YFP-receptor chimera expressed predominantly at the plasma membrane of HEK293 cells and displayed ligand-binding affinities comparable with those of the wild-type receptor. It also retained an ability to interact with G?(q/11) proteins and to stimulate phosphoinositide turnover, ERK1/2 phosphorylation and undergo agonist-dependent internalization. Addition of the full agonist methacholine caused a reversible decrease in M(1) FRET (F(EYFP)/F(ECFP)) that was prevented by atropine pre-addition and showed concentration-dependent amplitude and kinetics. Partial orthosteric agonists, arecoline and pilocarpine, as well as allosteric agonists, AC-42 and 77-LH-28-1, also caused atropine-sensitive decreases in the FRET signal, which were smaller in amplitude and significantly slower in onset compared to those evoked by methacholine.ConclusionThe M(1) FRET-based receptor chimera reports that allosteric and orthosteric agonists induce similar conformational changes in the third intracellular loop and/or C-terminus, and should prove to be a valuable molecular reagent for pharmacological and structural investigations of M(1) mAChR activation.

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Identification of a Novel Allosteric Site at the M₅ Muscarinic Acetylcholine Receptor.

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Identification of a Novel Allosteric Site at the M<sub>5</sub> Muscarinic Acetylcholine Receptor.

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Dataset Information

Identification of a Novel Allosteric Site at the M5 Muscarinic Acetylcholine Receptor.

Publications

Identification of a Novel Allosteric Site at the M&lt;sub&gt;5&lt;/sub&gt; Muscarinic Acetylcholine Receptor.

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Identification of a Novel Allosteric Site at the M₅ Muscarinic Acetylcholine Receptor.

Identification of a Novel Allosteric Site at the M<sub>5</sub> Muscarinic Acetylcholine Receptor.