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

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.

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.

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 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.

At 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.