Quantitative pharmacology of the Dopamine DRD1 receptor using baculovirus and controlled expression

Jamie Cartland, Jeff Jerman, Paul Wright. MRC Technology, Mill Hill, London, UK

The ability to control receptor levels within assay systems confers numerous advantages to early phase drug discovery. Generation of cells expressing receptors at high levels lends itself to high throughput hit detection, whereas levels more akin to the (patho) physiological situation aid modelling of native pharmacology.

Our aim was to use the BacMam (Life Technologies, USA) baculovirus system to titrate gene expression levels and monitor receptor binding and function. Using the Dopamine Receptor D1 (DRD1), well characterised ligands were used to compare pharmacology. DRD1 has been extensively studied and has been implicated in a plethora of disease states including Parkinson’s disease, cognition and schizophrenia.

U2OS cells were transduced using DRD1 baculovirus (1) and subsequently radioligand binding, second messenger and whole cell phenotypic assays were performed. Binding was measured using the tritiated antagonist, [3H]-SCH23390, in a Scintillation Proximity Assay (SPA) (2). Functional outputs were measured using a homogenous time resolved fluorescence (HTRF) cAMP assay (Cisbio, UK) in whole cells (3). Cellular phenotypic changes were measured using dynamic mass redistribution (DMR) on the BT EPIC (Corning, USA) label free system (4). In each assay dopamine and SKF83822 (full agonists) and SKF83959 (partial agonist) were studied.

Binding data indicated a near fivefold increased BMAX with increased baculoviral load without a commensurate change in affinity. Of note was an indication of (very low) specific binding in non-transfected U2OS cells. cAMP assays indicated a significant increase in intrinsic activity (5) with receptor expression of full agonists. These agonists demonstrated an increasing trend of potency to receptor expression that was significant when comparing to non-transduced. In contrast, the intrinsic activity of SKF 83959 was significantly increased but potency was not (Table 1). These data are in keeping with receptor theory. Both full agonists elicited a cAMP response in host cells suggesting an endogenous dopamine receptor. Although preliminary in nature, responses in phenotypic assays were dependent on expression and provided an alternative (arguably) holistic measure of receptor function.

Taken collectively these data highlight the dependency of both compound and assay system on apparent potency/efficacy and the presence of an un-described endogenous dopamine receptor in U2OS cells.

Table 1.

%IA is expressed relative to dopamine at 1% v/v. Data are mean + SD; n≥3

*p≤0.05 compared to 1% (t test)

(1) Davenport EA et al, Methods in Molecular Biology 552:199, (2009)

(2) Sun S et al, Metabolic Engineering 7:38, (2005)

(3) Tardieu J-L, Nature Methods 5, (2008)

(4) Lee PH et al, ASSAY and Drug Development Technologies 6:83,(2008)

(5) Ariens EJ, Archives internationales de pharmacodynamie et de thérapie. 99: 32-49 (1954)