Abstract

M₄ muscarinic receptors are highly expressed in the striatum and cortex, brain regions that are involved in diseases such as Parkinson's disease, schizophrenia, and dystonia. Despite potential therapeutic advantages of specifically targeting the M₄ receptor, it has been historically challenging to develop highly selective ligands, resulting in undesired off-target activity at other members of the muscarinic receptor family. Recently, we have reported first-in-class, potent, and selective M₄ receptor antagonists. As an extension of that work, we now report the development and characterization of a radiolabeled M₄ receptor antagonist, [³H]VU6013720, with high affinity (pK_d of 9.5 {plus minus} 0.2 at rat M₄, 9.7 at mouse M₄, and 10 {plus minus} 0.1 at human M₄ with atropine to define nonspecific binding) and selectivity (80-fold over M₂ and >1,000 fold over M₁, M₃ and M₅) for the M₄ receptor versus the other muscarinic subtypes. Binding assays using this radioligand in rodent brain tissues demonstrate loss of specific binding in Chrm4 knockout animals. Dissociation kinetics experiments with various muscarinic ligands show differential effects on the dissociation of [³H]VU6013720 from M₄ receptors, suggesting a binding site that is overlapping but may be distinct from the orthosteric site. Overall, these results demonstrate that [³H]VU6013720 is the first highly selective antagonist radioligand for the M₄ receptor, representing a useful tool for studying the basic biology of M₄ as well for the support of M₄ receptor-based drug discovery.

Significance Statement This manuscript describes the development and characterization of a novel muscarinic acetylcholine subtype 4 receptor antagonist radioligand, [³H]VU6013720. This ligand binds to or overlaps with the acetylcholine binding site, providing a highly selective radioligand for the M₄ receptor that can be used to quantify M₄ protein expression in vivo and probe the selective interactions of acetylcholine with M₄ versus the other members of the muscarinic receptor family.