Efficacy of hybrid tetrahydrobenzo[d]thiazole based aryl piperazines D-264 and D-301 at D₂ and D₃ receptors

In elucidating the role of pharmacodynamic efficacy at D₃ receptors in therapeutic effectiveness of dopamine receptor agonists, the influence of study system must be understood. Here two compounds with D₃ over D₂ selectivity developed in our earlier work, D-264 and D-301, are compared in dopamine receptor-mediated G-protein activation in striatal regions of wild-type and D₂ receptor knockout mice and in CHO cells expressing D₂ or D₃ receptors. In caudate-putamen of D₂ knockout mice, D-301 was ~3-fold more efficacious than D-264 in activating G-proteins as assessed by [³⁵S]GTPγS binding; in nucleus accumbens, D-301 stimulated G-protein activation whereas D-264 did not. In contrast, the two ligands exerted similar efficacy in both regions of wild-type mice, suggesting both ligands activate D₂ receptors with similar efficacy. In D₂ and D₃ receptor-expressing CHO cells, D-264 and D-301 appeared to act in the [³⁵S]GTPγS assay as full agonists because they produced maximal stimulation equal to dopamine. Competition for [³H]spiperone binding was then performed to determine K_i/EC₅₀ ratios as an index of receptor reserve for each ligand. Action of D-301, but not D-264, showed receptor reserve in D₃ but not in D₂ receptor-expressing cells, whereas dopamine showed receptor reserve in both cell lines. Gα_o1 is highly expressed in brain and is important in D₂-like receptor-G protein coupling. Transfection of Gα_o1 in D₃- but not D₂-expressing CHO cells led to receptor reserve for D-264 without altering receptor expression levels. D-301 and dopamine exhibited receptor reserve in D₃-expressing cells both with and without transfection of Gα_o1. Altogether, these results indicate that D-301 has greater intrinsic efficacy to activate D₃ receptors than D-264, whereas the two compounds act on D₂ receptors with similar intrinsic efficacy. These findings also suggest caution in interpreting E_max values from functional assays in receptor-transfected cell models without accounting for receptor reserve.