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

Background and purposeMuscarinic 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 approachFunctional responses at human M1 and M2 receptors expressed in CHO cells were assessed using inositol phosphate accumulation and [35S]GTP{gamma}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 resultsKH-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. Conclusions and implicationsThe 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. HighlightsO_LIKH-5 antagonises muscarinic receptors with a potency exceeding its orthosteric binding affinity C_LIO_LIFunctional antagonism shows probe dependence, indicating an allosteric component C_LIO_LIBinding studies support independent interaction of KH-5 with orthosteric and ectopic sites C_LIO_LIKH-5 does not bind the classical muscarinic allosteric site C_LIO_LIExcept for xanomeline, the operational model of dualsterically modulated agonism explains the complex pharmacology of KH-5 at M1 receptors C_LI