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, D2L 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. Hovever, 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.
Distinctive binding properties of the negative allosteric modulator, [3H]SB269,652, at recombinant dopamine D3 receptors
Rossi, Mario;Scarselli, Marco;
2018-01-01
Abstract
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, D2L 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. Hovever, 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.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
