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

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 ± 0.2 at rat M₄, 9.7 at mouse M₄, and 10 ± 0.1 at human M₄ 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 [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 (M) 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.

Fig. 1.

Structure of VU6013720. Cold VU6013720 was labeled using nonspecific hydrogen/tritium exchange with 99% tritium gas (RC Tritec, Switzerland) with a specific activity of 23.6 Ci/mmol.

Fig. 2.

Binding selectivity of VU6013720 at muscarinic acetylcholine receptor subtypes using [³H]NMS. VU6013720 and atropine competition binding curves at various muscarinic acetylcholine receptor subtypes were performed using the orthosteric muscarinic radioligand, [³H]-N-methylscopolamine. Data are the mean ± S.D. from a representative of four independent experiments performed in triplicate.

Fig. 3.

Among the five muscarinic receptors, [³H]VU6013720 binds specifically to the rat M₄ receptor. Total and specific binding for [³H]VU6013720 was determined using cells expressing each of the rat muscarinic receptors. [³H]VU6013720 bound specifically to the rat M₄ receptor in a saturable manner using either VU6013719 (A) or atropine (B) as nonspecific binding controls, whereas no detectable binding was observed at rat M₁, M₂, M₃, or M₅ using atropine to determine nonspecific binding (C–F). Data shown are the mean ± S.D. of a representative of at least three independent experiments performed in triplicate.

Fig. 4.

Kinetic characterization of the [³H]VU6013720 radioligand. Time course experiments defining association and dissociation of [³H]VU6013720 at rat M₄ membranes were performed at room temperature. (A) Association was initiated by addition of [³H]VU6013720 to membranes at the indicated time points before filtration. (B) Dissociation experiments were performed by allowing [³H]VU6013720 to equilibrate with membranes for 2 hours; at this point, a 10 μM final concentration of VU6013719 (white), atropine (black), or VU0467154 (red) was added at designated times before terminating the reaction by filtration. Data are the mean ± S.D. of three independent experiments performed in triplicate.

Fig. 5.

[³H]VU6013720 binds specifically to rat brain cortical and striatal tissue as well as to cortical membranes from WT but not from Chrm4 knockout mice. Rat cortical (A) and striatal (B) homogenates were incubated with [³H]VU6013720 in the presence and absence of 10 μM atropine to determine total and nonspecific binding. [³H]VU6013720 bound to rat cortical membranes with a pK_d of 9.0 ± 0.2 and a B_max of 260 ± 110 fmol/mg of protein and to rat striatal membranes with a pK_d of 9.3 ± 0.2 and a B_max of 350 ± 60 fmol/mg of protein. Data are mean ± S.D. and representative of three independent experiments performed in triplicate. Cortical homogenates from WT (C) and Chrm4 knockout mice (D) were incubated with [³H]VU6013720 in the presence and absence of 10 μM atropine to determine total and nonspecific binding. [³H]VU6013720 binds to cortical membranes from WT mice with a pK_d of 9.0 ± 0.4 and a B_max of 240 ± 90 fmol/mg of protein, whereas little specific [³H]VU6013720 binding was detected in cortical homogenates from Chrm4 knockout mice. Data are the mean ± S.D. and a representative of three independent experiments performed in duplicate or triplicate.