Investigation of pharmacochaperone actions of antipsychotic drugs at the D2-dopamine receptor
Biomedical and Pharmaceutical Sciences
D2-dopamine receptor; antipsychotic drugs; pharmacochaperone
The D2-Dopamine Receptor (D2R) is a membrane-inserted receptor protein that is important clinically as it is a target for pharmacotherapy in psychosis, depression, brain injury, attention deficit hyperactivity disorder and Parkinsonism. D2R is particularly important in schizophrenia since all clinically approved antipsychotic drugs (APDs) are competitive antagonists of dopamine at D2R. The classical definition of an antagonist is a molecule that simply sits in a receptor and competitively prevents the natural ligand from binding; without altering the function of the receptor. The suggestion that APDs are acting as more than just antagonists at D2R—as pharmacochaperones that bind to the receptor intracellularly and aid in its folding—has been proposed by the Kovoor lab, and this theory can help explain why different APDs have shown differential efficacies and side effects at the clinical level. My hypothesis explored this pharmacochaperone mechanism by proposing two possibilities: either the APDs are acting in conjunction with protein folding machinery already present in mammalian cells, or the APDs contain intrinsic pharmacochaperone activity specific to the D2R-APD complex. I designed an experiment utilizing protein solubility in detergent as a function of how D2R folds in the presence and absence of APDs. I began by over-expressing D2R in bacteria, causing it to segregate into dense organelles called inclusion bodies by which I could purify using lysis and centrifugation protocols. I then completely unfolded the pure D2R using a denaturing solvent and evaluated the detergent solubility of the receptor as I dialyzed away the denaturing solvent and it refolded in the presence and absence of different APDs. After extensively repeating and optimizing the experiment, I concluded that there was no effect of treatment of APDs on the detergent solubility of D2R as it was allowed to refold through the denaturing assay. These results suggest that if APDs are acting as pharmacochaperones at D2R, then they may require additional chaperone proteins that are not present in the bacteria system I used to purify the receptor.