Student Posters 51st Lorne Proteins Conference 2026

Structural and Pharmacological Basis of Allosteric Modulation at the M5 Muscarinic Acetylcholine Receptor (#315)

Bhavika Rana 1 2 , Jesse Mobbs 1 2 , Celine Valant 1 2 , David Thal 1 2
  1. Monash University, Melbourne, STATE / PROVINCE, Australia
  2. Drug discovery biology(DDB), MIPS, Monash University, Melbourne, victoria, Australia

G protein-coupled receptors (GPCRs) are preeminent drug targets accounting for a third of approved medicines. However, discovering new drugs that selectively target GPCRs remains challenging since many GPCRs are activated by similar ligands. GPCRs contain allosteric sites, which are binding sites distinct from, but conformationally linked to the orthosteric binding site. Allosteric modulators bind to less conserved sites, potentially Thus, circumventing the challenges associated with targeting conserved orthosteric sites found on closely related receptors. The muscarinic acetylcholine receptors (mAChRs) are a five-membered (M1-M5) subfamily of GPCRs exemplifying therapeutically relevant targets for selective allosteric ligands. mAChR subtypes play critical roles in neurological functioning, and M5R knockout mice demonstrate their physiological role in CNS disorders. Despite strong data supporting the M5 mAChR as a therapeutic target, clinical research has been limited by a lack of selective ligands, prompting focus on allosteric ligands.

Here, we report a 2.6 Å cryo-EM structure of the human M5 mAChR bound to acetylcholine (ACh) and the positive allosteric modulator ML129 (Isatin PAM). ML129 covalently interacts with Cys2145.59 at a previously unidentified allosteric site on transmembrane helix 5 (TM5). and with another cysteine residue Cys4847.42 on TM7. We propose that ML129 forms a transient interactions with TM7 cysteines: C4847.42 and C4947.52. We designed alanine point mutations of key Cys residues to validate this site. Our previous study with a different PAM, VU6007678, revealed an intracellular allosteric site located at the receptor–lipid interface. In contrast, the structurally distinct ML129 binds to a separate region of the receptor, highlighting the structural and topological diversity of allosteric modulation at the M5 mAChR. Overall, our study has identified a new mAChR allosteric site that may be useful for the design of selective allosteric modulators.

  1. Discovery of the First Highly M5-Preferring Muscarinic Acetylcholine Receptor Ligand, an M5 Positive Allosteric Modulator Derived from a Series of 5-Trifluoromethoxy N-Benzyl Isatins https://pubs.acs.org/doi/10.1021/jm900286j
  2. Development and characterization of a highly selective M5 PAM probe molecule with improved potency https://www.ncbi.nlm.nih.gov/books/NBK148851/
  3. Burger, W.A.C., Mobbs, J.I., Rana, B. et al. Cryo-EM reveals an extrahelical allosteric binding site at the M5 mAChR. Nat Commun 16, 7046 (2025). https://doi.org/10.1038/s41467-025-62212-z
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