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.