{"paper_id":"2d6d1f48-8d86-4596-9d9c-6b693e96bc65","body_text":"Abstract\nBackground 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.\nExperimental approach Functional responses at human M1 and M2 receptors expressed in CHO cells were assessed using inositol phosphate accumulation and [35S]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.\nKey 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 M2 receptors, antagonism was largely competitive. Binding studies revealed transient enhancement of agonist binding at M1 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.\nConclusions and implications The simplest model explaining the KH-5 mechanism of action at muscarinic receptors combines two concurrent modes of interaction. From the allosteric site, it positively modulates functional responses to agonists. From the orthosteric site, it exerts competitive antagonism of functional responses. Additionally, molecules of KH-5 bound to allosteric and orthosteric sites exert positive cooperativity.\nHighlights\nKH-5 antagonises muscarinic receptors with a potency exceeding its orthosteric binding affinity\nFunctional antagonism shows probe dependence, indicating an allosteric component\nBinding studies support independent interaction of KH-5 with orthosteric and ectopic sites\nKH-5 does not bind the classical muscarinic allosteric site\nExcept for xanomeline, the operational model of dualsterically modulated agonism explains the complex pharmacology of KH-5 at M1 receptors\nCompeting Interest Statement\nThe authors have declared no competing interest.","source_license":"CC-BY-4.0","license_restricted":false}