Characterization of [³H]MCL-524, a Potent Dopamine D₂ ^high Receptor Agonist

The D₂ ^high receptor, functional high-affinity state of the dopamine D₂ receptor, has been shown to be upregulated in the context of neurological disorders. We had synthesized a specific D₂ receptor agonist, the aporphine derivative [³H]-MCL-524. Previous, preliminary data indicated that [¹⁸F]-MCL-524 specifically binds to the activated form of the D₂ receptor, D₂ ^high. Here, we studied the binding characteristics of [³H]-MCL-524 in vitro, with the aim of unambiguously demonstrating its specificity for D₂ ^high. In saturation binding studies using Chinese Hamster Ovary (CHO) cell membranes expressing the human D₂ long receptor, two components were identified in the presence of 100 nM R-(-)-N-n-propylnorapomorphine (NPA), a D₂/D₃ agonist; a saturable specific component represents the total specific binding to both high- and low-affinity D₂ receptors, and a nonsaturable, nonspecific component which corresponds to a known aporphine site to which both NPA and MCL-524 bind. [³H]-MCL-524 was found to have a K _d of ∼1.34 nM. When binding of [³H]-MCL-524 was examined in competition studies using the same in vitro model, the K _i of [³H]-MCL-524 against R-(-)-N-n-propylnorapomorphine (NPA) at D₂ was 0.14 nM and the K _i against the D₂/₃ antagonist raclopride was 0.13 nM. Co-incubation with guanylylimidodiphosphate completely inhibited binding to D₂, indicating specificity of MCL-524 for the high-affinity state of the D₂ receptor. The data indicate that [³H]-MCL-524 is specific for the D₂ receptor and is able to distinguish between D₂ ^high and D_2low binding sites. Thus, this agonist shows potential for research, and, in its [¹⁸F]-labeled form, toward diagnosis or treatment of neurological disorders exhibiting a dysregulation of D₂ receptors such as Parkinson's disease or schizophrenia.