The M1–M5 muscarinic acetylcholine receptors (mAChRs) are G protein-coupled receptors activated by acetylcholine and implicated in central nervous system disorders including Alzheimer's disease, schizophrenia, and substance abuse. Developing selective drugs targeting mAChRs has been challenging due to their highly conserved acetylcholine-binding site. Off-target activation of peripheral mAChRs often leads to clinical failure. This challenge is exemplified by Xanomeline, an M1/M4-preferring agonist that showed promise in treating patients suffering from schizophrenia but initially failed clinical trials due to side effects resulting from peripheral M2/M3 activation. Subsequent reformulation with Trospium, a peripherally-restricted muscarinic antagonist, effectively mitigated these adverse effects. The resulting combination therapy (KarXT) received FDA approval for schizophrenia treatment in 2024, representing the first novel mechanistic approach to schizophrenia pharmacotherapy in over four decades (1). This clinical success validates mAChRs as therapeutic targets while underscoring the critical importance of achieving subtype selectivity.
A more promising approach involves targeting allosteric sites, which are binding sites that are distinct from but conformationally linked to the endogenous binding sites. Allosteric sites are typically less conserved and therefore easier to selectivity target. While subtype-selective allosteric ligands exist for most mAChR subtypes, their binding locations and mechanisms of selective modulation remained unknown. To address this, we determined high-resolution cryo-EM structures (1.9-2.5Å) of M1, M4, and M5 mAChRs bound to subtype-selective allosteric ligands. These structures revealed diverse allosteric binding sites and explained the mechanism of selective modulation. Through targeted mutation studies, swapping non-conserved residues between the M1 and M4 mAChRs, we identified four key residues that determine allosteric modulator selectivity. Exchanging these residues completely reversed the selectivity profiles of M1/M4-selective ligands. Our findings illuminate both the location and mechanism of allosteric modulation in mAChRs, providing a structural framework for developing future subtype-selective therapeutics with broad implications for GPCR drug design and allosteric modulation across the superfamily.