To evaluate the agonist or antagonist activity of compounds, we measured the ability of these compounds to either inhibit forskolin-stimulated cAMP accumulation or reverse the inhibition produced by 30 nM of quinpirole in a Chinese Hamster Ovary (CHO) cell line expressing recombinant human dopamine D2L or D2S receptors. D2 receptors in this cell line couple with Gαi/o protein subunits to suppress adenylate cyclase activity. In antagonist assays using the D2-L receptor (Fig. 1A), lumateperone potently (IC50 = 32 nM) and completely antagonized the activity of quinpirole, with an IC50 in the same range as its published affinity for D2 receptors in a ligand-displacement assay (32 nM; Li et al., 2014). We compared this to haloperidol (IC50 = 0.33 nM), brexpiprazole (1.68 nM) and aripiprazole (3.32 nM). In keeping with their partial agonist character, brexpiprazole and aripiprazole only partially antagonized the activity of quinpirole, reaching ~70% and ~45% maximum blockade, respectively.
This assay can also be run in agonist mode. Agents such as aripiprazole, bifeprunox, and brexpiprazole act as partial agonists, partially suppressing adenylate cyclase activity. Depending on receptor abundance and efficiency of receptor coupling to second messengers in the recombinant system, the intrinsic efficacy for aripiprazole can range from 20 to 90% that of dopamine. Figure 1B summarizes the result of a direct comparison of lumateperone with dopamine (full agonist), bifeprunox (partial agonist with high intrinsic efficacy), aripiprazole (partial agonist with medium intrinsic activity), and brexpiprazole (partial agonist with low intrinsic activity) in the D2L-receptor-induced suppression of forskolin-stimulated adenylate cyclase activity. Bifeprunox reached 77% activity relative to quinpirole, with an EC50 of 4.26 nM (Fig. 1B and C). Aripiprazole reached 37% activity, with an EC50 of 4.43 nM. Brexpiprazole has less intrinsic activity than aripiprazole at D2 receptors, with a maximum effect of 11% and an EC50 of 1.32 nM. Lumateperone (up to 3 µM) had no demonstrable agonist activity in this assay.
In vivo, D2L receptors are abundantly found in postsynaptic dopamine-responsive cells. D2S, a splice variant of this receptor lacking exon 6 encoding the third intracellular loop, is most abundant on the presynaptic cells, and has been hypothesized to be the usual form functioning as the presynaptic autoreceptor (reviewed in). Therefore, we asked whether the partial agonist activity of lumateperone would be revealed in assays using this receptor. In CHO cells expressing recombinant human D2S receptors, however, this was not the case (Fig. 1C and D). Once again, while aripiprazole and brexpiprazole acted as functional partial agonists, lumateperone showed only antagonist activity. This may reflect the fact that the G-protein repertoire of CHO cells does not replicate the presynaptic receptor cellular milieu. Prior work with many cell lines expressing recombinant D2 receptors has demonstrated that the cellular milieu, rather than the splice variant, is the major influence determining functional readout from D2 receptors.