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

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.