Distinctive binding properties of the negative allosteric modulator, [³H]SB269,652, at recombinant dopamine D₃...

Recently, employing radioligand displacement and functional coupling studies, we demonstrated that SB269,652 (N-[(1r,4r)-4-[2-(7-cyano-1,2,3,4-tetrahydroisoquinolin-2-yl)ethyl]cyclohexyl]-1H-indole-2-carboxamide) interacts in an atypical manner with dopamine D₃ receptor displaying a unique profile reminiscent of a negative allosteric ligand. Here, we characterized the binding of radiolabelled [³H]SB269,652 to human dopamine D₃ receptor stably expressed in Chinese Hamster Ovary cells. Under saturating conditions, SB269,652 showed a KD value of ≈ 1nM. Consistent with high selectivity for human dopamine D₃ receptor, [³H]SB269,652 binding was undetectable in cells expressing human dopamine D₁, D_2L or D₄ receptors and absent in synaptosomes from dopamine D₃ receptor knockout vs. wild-type mice. In contrast to saturation binding experiments, the dissociation kinetics of [³H]SB269,652 from human dopamine D₃ receptors initiated with an excess of unlabelled ligand were best fitted by a bi-exponential binding model. Supporting the kinetic data, competition experiments with haloperidol, S33084 (a dopamine D₃ receptor antagonist) or dopamine, were best described by a two-site model. In co-transfection experiments binding of SB269,652 to dopamine D₃ receptor was able to influence the functional coupling of dopamine D₂ receptor, supporting the notion that SB269,652 is a negative allosteric modulator across receptor dimers. However, because SB269,652 decreases the rate of [³H]nemonapride dissociation, the present data suggest that SB269,652 behaves as a bitopic antagonist at unoccupied dopamine D₃ receptor, binding simultaneously to both orthosteric and allosteric sites, and as a pure negative allosteric modulator when receptors are occupied and it can solely bind to the allosteric site.