| Summary: | SB269,652 has been described as the first negative allosteric modulator (NAM) of the dopamine D<sub>2</sub> receptor (D<sub>2</sub>R), 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 D<sub>2</sub>R. Here, we observed a surprisingly low potency of SB269,652 to negatively modulate the D<sub>2</sub>R-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 D<sub>2</sub>R and may be useful for the development of novel D<sub>2</sub>R ligands, such as antipsychotic drug candidates.
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