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. Here, we investigated whether the muscarinic antagonist 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]GTP γ S 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, but 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. 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.