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

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 dopamineD 3 receptor displaying a unique profile reminiscent of a negative allosteric ligand. Here, we characterized the binding of radiolabelled [ 3 H]SB269,652 to human dopamineD 3 receptor stably expressed in Chinese Hamster Ovary cells. Under saturating conditions, SB269,652 showed a KD value of ≈ 1 nM. Consistent with high selectivity for human dopamineD 3 receptor, [ 3 H]SB269,652 binding was undetectable in cells expressing human dopamineD 1 , D 2L or D 4 receptors and absent in synaptosomes from dopamineD 3 receptor knockout vs . wild-type mice. In contrast to saturation binding experiments, the dissociation kinetics of [ 3 H]SB269,652 from human dopamineD 3 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 dopamineD 3 receptor antagonist) or dopamine, were best described by a two-site model. In co-transfection experiments binding of SB269,652 to dopamineD 3 receptor was able to influence the functional coupling of dopamineD 2 receptor, supporting the notion that SB269,652 is a negative allosteric modulatoracross receptor dimers. However, because SB269,652 decreases the rate of [ 3 H] nemonapridedissociation, the present data suggest that SB269,652 behaves as a bitopic antagonist at unoccupied dopamineD 3 receptor, binding simultaneously to both orthosteric and allosteric sites, and as a pure negative allosteric modulatorwhen receptors are occupied and it can solely bind to the allosteric site.