Design and Synthesis of Dualsteric Muscarinic M5 Receptor Ligands Reveal G Protein Bias

The M5 muscarinic acetylcholine receptor (M5R) is selectively expressed in dopaminergic neurons where it plays a key role in modulating dopaminergic activity, making it a promising target for neuropsychiatric disorders. Selectively targeting the M5R remains challenging due to high orthosteric site conservation across all five muscarinic acetylcholine receptor (mAChR or MR) subtypes. Here, we report a series of potential dualsteric M5R agonists that combine the orthosteric, but unselective agonist iperoxo with the M5R-selective positive allosteric modulator VU0238429. Using systematic modifications of linker length, rigidity, and topology, respectively, we designed and synthesized a panel of ligands with diverse signaling profiles. All compounds were assessed using BRET-based assays for G protein activation (GEMTA/TRUPATH) and β-arrestin2 recruitment (ebBRET). Functional characterization revealed moderate M5R preference and a moderate, but consistent bias toward G protein activation over β-arrestin2 recruitment across all compounds. These ligands present valuable tools for dissecting M5R function in the context of G protein-biased GPCR targeting.