Dualsteric and dual-acting modulation of muscarinic receptors by antagonist KH-5

Background and purpose Muscarinic acetylcholine receptors are key therapeutic targets, and ligands engaging both orthosteric and allosteric sites may offer improved selectivity and efficacy. The muscarinic antagonist KH-5 displays functional antagonistic potency exceeding its binding affinity, suggesting a non-classical mechanism of action. Here, we investigated whether KH-5 acts as a dualsteric antagonist and defined its mode of interaction with muscarinic receptors. Experimental approach Functional responses at human M 1 and M 2 receptors expressed in CHO cells were assessed using inositol phosphate accumulation and [ 35 S]GTPγS binding, respectively. Radioligand binding studies employed orthosteric antagonists and agonists in combination with KH-5 and classical allosteric modulators. Data were analysed using competitive, allosteric, and dualsteric binding and operational models. Molecular docking, molecular dynamics simulations, and site-directed mutagenesis were used to identify structural determinants of KH-5 binding. Key results KH-5 antagonised responses to multiple agonists in a saturable and probe-dependent manner consistent with an allosteric interaction. However, KH-5 did not decrease maximal response to agonists, contradicting simple allosteric antagonism. At M 2 receptors, antagonism was largely competitive. Binding studies revealed transient enhancement of agonist binding at M 1 receptors at nanomolar concentrations of KH-5, best described by a dualsteric binding model involving independent orthosteric and ectopic site interactions. KH-5 did not bind to the classical muscarinic allosteric site at the second extracellular loop but interacted with an extracellular vestibule site, supported by molecular modelling and mutation of key residues. Conclusions and implications At M 1 receptors, the most parsimonious model among those tested combines orthosteric competition with an ectopic/allosteric component. At M 2 receptors, KH-5 behaves predominantly as an orthosteric antagonist under the present conditions, although a weak or probe-specific allosteric component cannot be excluded. Summary What is already known Muscarinic receptors are therapeutic targets with conserved orthosteric binding sites. Allosteric or dualsteric ligands may improve receptor-subtype selectivity. What this study adds KH-5 shows dualsteric, dual-acting modulation at M 1 receptors. At M 2 receptors, KH-5 behaves mainly as a competitive antagonist. Clinical significance Dualsteric muscarinic antagonists may enable subtype-selective anticholinergic drug development. KH-5 provides a framework for designing mixed orthosteric/allosteric ligands.