Evidence for Two Modes of Binding of the Negative Allosteric Modulator SB269,652 to the Dopamine D2 Receptor

SB269,652 has been described as the first negative allosteric modulator (NAM) of the dopamine D2 receptor (D2R), however, the binding mode and allosteric mechanism of action of this ligand remain incompletely understood. SB269,652 comprises an orthosteric, primary pharmacophore and a secondary (or allosteric) pharmacophore joined by a hydrophilic cyclohexyl linker and is known to form corresponding interactions with the orthosteric binding site (OBS) and the secondary binding pocket (SBP) in the D2R. Here, we observed a surprisingly low potency of SB269,652 to negatively modulate the D2R-mediated activation of G protein-coupled inward-rectifier potassium channels (GIRK) and decided to perform a more detailed investigation of the interaction between dopamine and SB269,652. The results indicated that the SB269,652 inhibitory potency is increased 6.6-fold upon ligand pre-incubation, compared to the simultaneous co-application with dopamine. Mutagenesis experiments implicated both S193 in the OBS and E95 in the SBP in the effect of pre-application. The present findings extend previous knowledge about how SB269,652 competes with dopamine at the D2R and may be useful for the development of novel D2R ligands, such as antipsychotic drug candidates.