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 implicationsAt M1 receptors, the most parsimonious model among those tested combines orthosteric competition with an ectopic/allosteric component. At M2 receptors, KH-5 behaves predominantly as an orthosteric antagonist under the present conditions, although a weak or probe-specific allosteric component cannot be excluded. SummaryO_ST_ABSWhat is already knownC_ST_ABSMuscarinic receptors are therapeutic targets with conserved orthosteric binding sites. Allosteric or dualsteric ligands may improve receptor-subtype selectivity. What this study addsKH-5 shows dualsteric, dual-acting modulation at M1 receptors. At M2 receptors, KH-5 behaves mainly as a competitive antagonist. Clinical significanceDualsteric muscarinic antagonists may enable subtype-selective anticholinergic drug development. KH-5 provides a framework for designing mixed orthosteric/allosteric ligands.