Development of a Selective and High Affinity Radioligand, [3H]VU6013720, for the M4 Muscarinic ReceptorS

M-4 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 M-4 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 M-4 receptor antagonists. As an extension of that work, we now report the development and characterization of a radiolabeled M-4 receptor antagonist, [H-3]VU6013720, with high affinity (pK(d) of 9.5 +/- 0.2 at rat M-4, 9.7 at mouse M-4, and 10 +/- 0.1 at human M-4 with atropine to define nonspecific binding) and no significant binding at 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 [H-3]VU6013720 from M-4 receptors, suggesting a binding site that is overlapping but may be distinct from the orthosteric site. Overall, these results demonstrate that [H-3]VU6013720 is the first highly selective antagonist radioligand for the M-4 receptor, representing a useful tool for studying the basic biology of M-4 as well for the support of M-4 receptor-based drug discovery.