Molecular basis of atypicality in antipsychotic drug action

Antagonism of the dopamine D2 receptor (D2R) is required for the efficacy of all clinically used antipsychotic drugs (APDs) for the treatment of schizophrenia. Unfortunately, antagonism of the D2R is associated with extrapyramidal side effects (EPS) such as Parkinsonism, and tardive dyskinesia. Some newer APDs (termed atypical APDs) are thought to have a lower side-effect risk but the mechanism behind these differences is unclear. We have recently shown that the binding association rate of APDs predicts their ability to cause EPS although we lack direct experimental evidence of this phenomenon. Furthermore, in another recent study we have revealed that some but not all APDs can act as pharmacological chaperones to increase cell surface expression of D2Rs. Increases in brain D2R after chronic treatment with APDs have been suggested as the reason for iatrogenic psychoses and for resistance of schizophrenia patients to pharmacotherapy over time. APD-induced upregulation of D2R has also been implicated in tardive dyskinesia, suggesting that chronic D2R upregulation can produce permanent adverse alterations in D2R-expressing neurons. However, neither the inverse agonist properties or the chaperone activity have been studied in brain cells relevant to EPS. Hypothesis: Understanding the molecular basis of drug rebinding and pharmacological chaperoning activity of APDs at the D2R will reveal differences in antipsychotic drug action that predict their side effect profile. This project will use a wide range of imaging and biophysical techniques in combination with classical pharmacology and biochemistry approaches to address this hypothesis. We have used Fluorescence Correlation Spectroscopy (FCS) to show that drug interactions with the cell membrane and receptors drives a higher local concentration of drug proximal to the cell membrane and developed a series of fluorescently-labeled D2R antagonists whose binding kinetics properties span the range of those exhibited by APDs. The student will investigate the molecular determinants of drug rebinding. Using FCS in model cell lines expressing different levels of SNAP-D2R in combination with fluorescent D2R ligands with known binding kinetics, we will assess how different levels of cell surface D2R expression can drive local concentrations of the drug. Then the student will extend these studies to primary cultures of striatal and cortical neurons as well as pituitary lactotrophs derived from the SNAP-D2R mouse to understand how these phenomena might impact drug rebinding at endogenously expressed D2Rs in disease-relevant tissues. In parallel, the student will use Tr-FRET binding to measure the binding kinetics of selected unlabelled APDs at natively expressed D2Rs in the above primary neuronal cultures. To investigate the ability of different APDs to drive D2R cell surface expression the student will use BRET to quantify D2R trafficking. These experiments will be complemented with classical confocal, TIRF microscopy and high content imaging. Then we will extend these studies to primary cell cultures expressing the SNAP-D2R to investigate the impact of the acute and longer-term application of APDs on D2R expression and trafficking in these disease-relevant cells.