Xanomeline displays concomitant orthosteric and allosteric binding modes at the M4 mAChR

Nat Commun. 2023 Sep 6;14(1):5440. doi: 10.1038/s41467-023-41199-5.

Wessel A C Burger ^# ¹ ², Vi Pham ^# ¹, Ziva Vuckovic ^# ¹, Alexander S Powers ^# ³ ⁴, Jesse I Mobbs ¹ ², Yianni Laloudakis ³, Alisa Glukhova ¹ ², Denise Wootten ¹ ², Andrew B Tobin ⁵, Patrick M Sexton ¹ ², Steven M Paul ⁶, Christian C Felder ⁶, Radostin Danev ⁷, Ron O Dror ⁸, Arthur Christopoulos ⁹ ¹⁰ ¹¹, Celine Valant ¹², David M Thal ¹³ ¹⁴

Affiliations

1 Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC, 3052, Australia.
2 Australian Research Council Centre for Cryo-Electron Microscopy of Membrane Proteins, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC, 3052, Australia.
3 Department of Chemistry, Stanford University, Stanford, CA, 94305, USA.
4 Departments of Computer Science, Structural Biology, and Molecular and Cellular Physiology, Stanford University, Stanford, CA, 94305, USA.
5 The Advanced Research Centre (ARC), Centre for Translational Science, School of Biomolecular Sciences, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, G12 8QQ, UK.
6 Karuna Therapeutics, Boston, MA, 02110, USA.
7 Graduate School of Medicine, University of Tokyo, N415, 7-3-1 Hongo, Bunkyo-ku, 113-0033, Tokyo, Japan.
8 Departments of Computer Science, Structural Biology, and Molecular and Cellular Physiology, Stanford University, Stanford, CA, 94305, USA. [email protected].
9 Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC, 3052, Australia. [email protected].
10 Australian Research Council Centre for Cryo-Electron Microscopy of Membrane Proteins, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC, 3052, Australia. [email protected].
11 Neuromedicines Discovery Centre, Monash University, Parkville, VIC, 3052, Australia. [email protected].
12 Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC, 3052, Australia. [email protected].
13 Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC, 3052, Australia. [email protected].
14 Australian Research Council Centre for Cryo-Electron Microscopy of Membrane Proteins, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC, 3052, Australia. [email protected].
# Contributed equally.

PMID: 37673901 DOI: 10.1038/s41467-023-41199-5

Abstract

The M₄ Muscarinic Acetylcholine Receptor (M₄ mAChR) has emerged as a drug target of high therapeutic interest due to its expression in regions of the brain involved in the regulation of psychosis, cognition, and addiction. The mAChR agonist, xanomeline, has provided significant improvement in the Positive and Negative Symptom Scale (PANSS) scores in a Phase II clinical trial for the treatment of patients suffering from schizophrenia. Here we report the active state cryo-EM structure of xanomeline bound to the human M₄ mAChR in complex with the heterotrimeric G_i1 transducer protein. Unexpectedly, two molecules of xanomeline were found to concomitantly bind to the monomeric M₄ mAChR, with one molecule bound in the orthosteric (acetylcholine-binding) site and a second molecule in an extracellular vestibular allosteric site. Molecular dynamic simulations supports the structural findings, and pharmacological validation confirmed that xanomeline acts as a dual orthosteric and allosteric ligand at the human M₄ mAChR. These findings provide a basis for further understanding xanomeline's complex pharmacology and highlight the myriad of ways through which clinically relevant ligands can bind to and regulate GPCRs.

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