Use of radiolabeled antagonist assays for assessing agonism at D2 and D3 dopamine receptors: comparison with functional GTPγS assays.

Abstract Background Cell-based drug screening assays are essential tools for drug discovery and development targeting G protein-coupled receptors, which include dopamine D 3 receptors. D 3 is notorious for its poor coupling to G protein in most heterologous cell lines, and therefore D 3 agonist-stimulated binding of [ 35 S]GTP γ S to G protein cannot be observed in many “non-functional” D 3 expressing cell lines. New method The present work explores the use of an alternate method for assessing agonist activity, consisting of measuring the difference in agonist competition between [ 3 H]spiperone bound to low-affinity states of the receptor and that with radioligand bound to high-affinity states (GTP shift assay). Comparison with existing method The current study describes the determination of GTP shifts in [ 3 H]spiperone binding assays for the assessment of agonists’ potencies (at D 2 and D 3 ) and efficacies (at D 3 ). Compared with GTP γ 35 S binding assays, the new method removes the cumbersome need of functional D 3 cell lines and limited project duration due to short half-life of isotope 35 S. Conclusion The new method allows the estimation of potency (D 2 and D 3 ) and efficacy (D 3 ) at the level of receptor and G protein activation in a simple fashion from shifts in monophasic-inhibition curves. Moreover, it does not require [ 35 S]GTP γ S binding assays with functional D 3 cells. This method will have wide applicability for D 3 -selective agonist screening. It may also be useful for other GPCRs circumventing the need for functional assays and offering the ability to detect agonist activity regardless of the particular signaling pathway.