Lockington, Hannah J.
(2025)
The molecular basis of atypicality in antipsychotic drug action.
PhD thesis, University of Nottingham.
Abstract
Dopamine D2 receptor (D2R) antagonism is thought to be the pharmacological mechanism behind the clinical efficacy of antipsychotic drugs (APDs) in treating the positive symptoms of schizophrenia. Unfortunately, D2R antagonism is also associated with extrapyramidal symptoms (EPS) that encompass a range of motor side effects. More recently developed APDs are termed ‘atypical’ based on their low propensity to cause EPS. Despite several suggested hypotheses, no single mechanism has yet to account for all cases of atypicality in clinically prescribed APDs. This thesis explored two promising molecular mechanisms recently proposed to account for APD atypicality: (1) the reduced rebinding of atypical APDs to D2R resulting in surmountable D2R antagonism and (2) the low efficacy of atypical APDs as pharmacological chaperones of D2R, resulting in reduced D2R upregulation to the cell surface.
First, in-solution reads of fluorescent APDs with differing binding kinetics at D2R, spiperone-d2 and clozapine-Cy5, were optimised using the advanced spectroscopy technique, fluorescence correlation spectroscopy (FCS). In addition, the variable expression of SNAP-D2R in a CHO tetracycline-inducible SNAP-D2R cell line was characterised using confocal imaging and FCS. FCS was also used to investigate the membrane dynamics of SNAP-D2R within CHO tetracycline-inducible SNAP-D2R cells which revealed that a small percentage of receptors may form clusters on the plasma membrane.
Subsequently, the rebinding effects of these fluorescent APDs at D2R were investigated by measuring their concentration above cells with a range of SNAP-D2R expression levels. A high concentration of spiperone-d2 was found near the upper membrane of D2R-expressing cells which decreased further away from cells into the bulk aqueous solution. Interestingly, spiperone-d2 formed concentration gradients above cells that were dependent on the level of SNAP-D2R expression at the plasma membrane. These concentration gradients were indicative of spiperone-d2 rebinding to D2R and provided evidence of drug rebinding in vitro. In contrast, clozapine-Cy5, which has a slower association rate, showed negligible concentrating effects above D2R-expressing cells. When an added concentration of 0.2 x Kd of each fluorescent APD was used, the concentrating effect at 3 μm above the membrane of high D2R- expressing cells was 95-fold greater for spiperone-d2 in comparison to clozapine-Cy5. This is consistent with the association rate of APDs at D2R being the driver for APD rebinding, resulting in sustained D2R antagonism leading to higher EPS risks.
The APD-induced trafficking of D2R to various cellular compartments was next investigated using a bystander BRET-based D2R trafficking assay and confocal imaging. Most notably, APDs showed differing efficacies as pharmacological chaperones of D2R by increasing D2R trafficking from the endoplasmic reticulum to the plasma membrane. Interestingly, APDs that showed high efficacy for D2R chaperoning were also associated with high EPS risks. However, the APD-induced upregulation of D2R at the plasma membrane was also shown to be acutely reversible in vitro.
Finally, the optimisation of the primary culture of striatal neurones from SNAP-D2R mice was carried out with the aim of extending studies into more therapeutically relevant cells. Determining the molecular basis behind the atypicality of APDs would enable better prediction of the EPS risks of future APDs, ultimately leading to the improved treatment of schizophrenia and other psychiatric disorders where psychosis is a primary symptom.
| Item Type: |
Thesis (University of Nottingham only)
(PhD)
|
| Supervisors: |
Lane, Robert J.
Briddon, Stephen J.
Canals, Meritxell |
| Keywords: |
Antipsychotic drugs, Dopamine D2 Receptor, Drug rebinding, Pharmacological chaperones |
| Subjects: |
R Medicine > RM Therapeutics. Pharmacology |
| Faculties/Schools: |
UK Campuses > Faculty of Medicine and Health Sciences > School of Life Sciences |
| Item ID: |
80591 |
| Depositing User: |
Lockington, Hannah
|
| Date Deposited: |
24 Jul 2025 04:40 |
| Last Modified: |
24 Jul 2025 04:40 |
| URI: |
https://eprints.nottingham.ac.uk/id/eprint/80591 |
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