Quantification of drug rebinding at dopamine D2 receptor-expressing cell membranes with implications for extrapyramidal side effects

The mechanisms behind the superior side effect profile of clozapine in comparison to other dopamine D2 receptor (D2R) antagonist antipsychotic drugs (APDs) remain incompletely understood. Faster binding association rates of APDs at the D2R correlate with a higher risk of extrapyramidal side effects. This may be explained through APD rebinding to D2R within a diffusion-limited synaptic space resulting in an increased concentration of APD proximal to the D2R. Here, we provide evidence for such a mechanism, using fluorescence correlation spectroscopy to show that that the antagonist spiperone-d2 (fast association rate) forms concentration gradients above D2R-expressing cells, whereas clozapine-Cy5 (slow association rate) does not. Proximal to the upper cell membrane, spiperone-d2 concentrations were 250-fold higher than the added concentration, indicative of profound rebinding. Such drug rebinding will influence affinity and potency measurements with a broader impact on drug discovery.