Pharmacological hallmarks of allostery at the M4 muscarinic receptor elucidated through structure and dynamics
Curation statements for this article:-
Curated by eLife
eLife assessment
This fundamental study is important and carefully executed, providing important insights into the allosteric regulation of GPCRs with exceptional strength of evidence. This work will be of interest to a wide audience in drug discovery and receptor biology. The major strengths are the comprehensive structural and pharmacological characterization with only minor weaknesses, most notably a concern regarding the approach used to quantify efficacy.
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Abstract
Allosteric modulation of G protein-coupled receptors (GPCRs) is a major paradigm in drug discovery. Despite decades of research, a molecular-level understanding of the general principles that govern the myriad pharmacological effects exerted by GPCR allosteric modulators remains limited. The M 4 muscarinic acetylcholine receptor (M 4 mAChR) is a validated and clinically relevant allosteric drug target for several major psychiatric and cognitive disorders. In this study, we rigorously quantified the affinity, efficacy, and magnitude of modulation of two different positive allosteric modulators, LY2033298 (LY298) and VU0467154 (VU154), combined with the endogenous agonist acetylcholine (ACh) or the high-affinity agonist iperoxo (Ipx), at the human M 4 mAChR. By determining the cryo-electron microscopy structures of the M 4 mAChR, bound to a cognate G i1 protein and in complex with ACh, Ipx, LY298-Ipx, and VU154-Ipx, and applying molecular dynamics simulations, we determine key molecular mechanisms underlying allosteric pharmacology. In addition to delineating the contribution of spatially distinct binding sites on observed pharmacology, our findings also revealed a vital role for orthosteric and allosteric ligand–receptor–transducer complex stability, mediated by conformational dynamics between these sites, in the ultimate determination of affinity, efficacy, cooperativity, probe dependence, and species variability. There results provide a holistic framework for further GPCR mechanistic studies and can aid in the discovery and design of future allosteric drugs.
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Author Response
We thank the editors and reviewers for their support of our work, as well as their constructive feedback and useful suggestions, which have improved the readability and presentation of the manuscript for a broader audience.
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eLife assessment
This fundamental study is important and carefully executed, providing important insights into the allosteric regulation of GPCRs with exceptional strength of evidence. This work will be of interest to a wide audience in drug discovery and receptor biology. The major strengths are the comprehensive structural and pharmacological characterization with only minor weaknesses, most notably a concern regarding the approach used to quantify efficacy.
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Reviewer #1 (Public Review):
This is an outstanding manuscript that takes a comprehensive approach to studying allosteric modulation at the M4R. I think it is an important addition to the literature and provides important insights into allosteric modulation. Overall the pharmacological approaches are very rigorous and are the types of analyses that need to be performed to move this field forward.
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Reviewer #2 (Public Review):
Overall, the manuscript is clearly written and remarkably comprehensive, presenting a very large amount of data. Experimental methods are well-documented and rigorous, and I have no significant technical concerns about any of the work presented. There are some points where the presentation might be improved by modifications to the text or figures, particularly with the goal of making this important work accessible to a broad audience.
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Reviewer #3 (Public Review):
The authors aimed to study and describe allosteric modulation of the pharmacologically important muscarinic acetylcholine receptor 4 (M4R). Developing orthosteric ligands (agonists and antagonists) has had limited success in the past, due to the conserved binding pocket of acetylcholine across all (five) homologous receptors. The study uses a broad spectrum of experimental results, using binding and signaling assays, structure determination by cryoEM, as well as some mutational studies to study species selectivity. These results were combined with expansive MD simulations, to correlate receptor 'rigidity' with binding affinities, as well as signaling. The main strength of this paper is the sheer breadth of results to study the important aspect of allosteric modulation from any possible angle. I do not see …
Reviewer #3 (Public Review):
The authors aimed to study and describe allosteric modulation of the pharmacologically important muscarinic acetylcholine receptor 4 (M4R). Developing orthosteric ligands (agonists and antagonists) has had limited success in the past, due to the conserved binding pocket of acetylcholine across all (five) homologous receptors. The study uses a broad spectrum of experimental results, using binding and signaling assays, structure determination by cryoEM, as well as some mutational studies to study species selectivity. These results were combined with expansive MD simulations, to correlate receptor 'rigidity' with binding affinities, as well as signaling. The main strength of this paper is the sheer breadth of results to study the important aspect of allosteric modulation from any possible angle. I do not see any noteworthy weaknesses in the manuscript. The work presented here will be an important reference for future drug discovery efforts.
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