Characterization of [ ³ H]MCL-524, a Potent Dopamine D ₂ ^high Receptor Agonist

The D2high 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, D2high. Here, we studied the binding characteristics of [3H]MCL-524 in vitro, with the aim of unambiguously demonstrating its specificity for D2high. 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 Kd of ∼1.34 nM. When binding of [3H]MCL-524 was examined in competition studies using the same in vitro model, the Ki of [3H]MCL-524 against R-(−)-N-n-propylnorapomorphine (NPA) at D2 was 0.14 nM and the Ki 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 D2high and D2low 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.