The Tetrahydroisoquinoline Derivative SB269,652 Is an Allosteric Antagonist at Dopamine D3 and D2 Receptors

Abstract

In view of the therapeutic importance of dopamine D₃ and D₂ receptors, there remains considerable interest in novel ligands. Herein, we show that the tetrahydroisoquinoline 1H-indole-2-carboxylic acid {4-[2-(cyano-3,4-dihydro-1H-isoquinolin-2-yl)-ethyl]-cyclohexyl}-amide (SB269,652) behaves as an atypical, allosteric antagonist at D₃ and D₂ receptors. Accordingly, SB269,652 potently (low nanomolar range) abolished specific binding of [³H]nemanopride and [³H]spiperone to Chinese hamster ovary-transfected D₃ receptors when radioligands were used at 0.2 and 0.5 nM, respectively. However, even at high concentrations (5 μM), SB269,652 only submaximally inhibited the specific binding of these radioligands when they were employed at 10-fold higher concentrations. By analogy, although SB269,652 potently blocked D₃ receptor-mediated activation of Gα_i3 and phosphorylation of extracellular-signal-regulated kinase (ERK)1/2, when concentrations of dopamine were increased by 10-fold, from 1 μM to 10 μM, SB269,652 only submaximally inhibited dopamine-induced stimulation of Gα_i3. SB269,652 (up to 10 μM) only weakly and partially (by approximately 20–30%) inhibited radioligand binding to D₂ receptors. Likewise, SB269,652 only submaximally suppressed D₂ receptor-mediated stimulation of Gα_i3 and Gα_qi5 (detected with the aequorin assay) and phosphorylation of ERK1/2 and Akt. Furthermore, SB269,652 only partially (35%) inhibited the dopamine-induced recruitment of β-arrestin2 to D₂ receptors. Finally, Schild analysis using Gα_i3 assays, and studies of radioligand association and dissociation kinetics, supported allosteric actions of SB269,652 at D₃ and D₂ receptors.