Dequalinium-based bitopic ligands uncover distinct pharmacological modulation of muscarinic receptors

Bitopic ligands represent a powerful strategy to modulate G protein-coupled receptors by simultaneously engaging orthosteric and allosteric binding sites. In this study, we designed and synthesized two series of dequalinium-based hybrid ligands targeting muscarinic acetylcholine receptors (mAChRs), in which a dequalinium fragment was linked through polymethylene spacers of variable length either to the orthosteric superagonist iperoxo (Deq-X-Iper, 5a–c) or to a molecular portion of the allosteric inverse agonist W84 (Deq-X-W84, 6a–c). Equilibrium and kinetic binding studies at human mAChR subtypes (hM1–hM5) revealed good to high affinity for all compounds and a consistent bitopic mode of action, supported by a spacer-length-dependent enhancement of both orthosteric and allosteric interactions. Functional assays showed that Deq-X-W84 derivatives act as unselective muscarinic antagonists, whereas Deq-X-Iper ligands behave as agonists. Notably, the latter displayed probe-dependent negative allosteric modulation of acetylcholine-induced ERK1/2 phosphorylation at the hM4 receptor, revealing an unexpected functional profile. Docking and molecular dynamics simulations provided a structural rationale for these findings, highlighting stable bitopic binding poses and linker-dependent optimization of receptor contacts. Overall, these results demonstrate that dequalinium-based bitopic ligands can encode distinct functional outcomes at muscarinic receptors, offering new insights into the design of ligands with tailored signaling profiles.