Objective/Rationale:
Receptor heteromers must be understood as dimeric or higher order molecular entities that are the result of combinatorial evolution and that are endowed with unique biochemical and functional properties that could be harnessed for therapeutic purposes. Our objective is to find drugs that disrupt dopamine D1-D3 receptor heteromers, which we hypothesized can be involved in the pathogenesis of L-dopa-induced dyskinesia in patients with Parkinson’s disease.
Project Description:
The use of bioluminescence and fluorescence resonance energy transfer (BRET and FRET, respectively) has been fundamental for the demonstration of oligomerization of G-protein-coupled receptors in living cells. RET consists of a nonradioactive transfer of energy from a chromophore in an excited state, the ‘donor’, to a fluorescent ‘acceptor’ molecule. Molecules able to disrupt the D1-D3 receptor heteromer must produce significant changes in the BRET signal and this should be a useful, fast and feasible screening procedure.
A mammalian cell line (HEK 293) with a stable expression of D1 and D3 receptors fused to proteins that allow BRET measurements will be developed. We will then screen numerous compounds synthesized in the National Institute on Drug Abuse, known to bind to D1 or D3 receptors, for their ability to disrupt BRET in the D1-D3 HEK 293 cells.
Relevance to Diagnosis/Treatment of Parkinson’s Disease:
Obtaining positive results would first have an impact in the treatment in Parkinson’s disease and, particularly for L-DOPA-induced dyskinesia. The discovery of compounds able to alter D1-D3 receptor heteromerization will lead to obvious experiments in animal models of L-DOPA-induced dyskinesia. If those compounds are not suitable for clinical application, Medicinal Chemistry will come into play and new compounds with more appropriate pharmacodynamic- pharmacokinetic properties will be sought.
Anticipated Outcome:
The main anticipated outcome is finding a compound that disrupts D1-D3 receptor heteromerization. But, in addition to its obvious importance for the understanding and treatment of L-dopa-induced dyskinesia, this project could provide important implications in the field of G-protein-coupled receptor pharmacology, by drawing attention to receptor heteromers as new relevant targets for drug development.
Final Outcome
We have obtained a mammalian cell clone that expresses modified dopamine D1 and D3 receptors. These modified receptors (fused proteins) allow the identification of direct intermolecular interactions between both receptors (D1-D3 receptor heteromerization) by using a technique called Bioluminescence Resonance Energy Transfer (BRET). We are now in a position to detect compounds that modify D1-D3 receptor interactions. Modifications in BRET values will imply modifications in the tridimensional (quaternary) structure of the D1-D3 receptor heteromer and most probably in its function, which can potentially be beneficial for levodopa-induced dyskinesia in patients with Parkinson’s disease.