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

Juan Zhen, Tamara Antonio, Solav Ali, Kim Neve, Aloke K. Dutta, Maarten E A Reith

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

Background: Cell-based drug screening assays are essential tools for drug discovery and development targeting G protein-coupled receptors, which include dopamine D3 receptors. D3 is notorious for its poor coupling to G protein in most heterologous cell lines, and therefore D3 agonist-stimulated binding of [35S]GTPγS to G protein cannot be observed in many "non-functional" D3 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 [3H]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 [3H]spiperone binding assays for the assessment of agonists' potencies (at D2 and D3) and efficacies (at D3). Compared with GTPγ35S binding assays, the new method removes the cumbersome need of functional D3 cell lines and limited project duration due to short half-life of isotope 35S. Conclusion: The new method allows the estimation of potency (D2 and D3) and efficacy (D3) at the level of receptor and G protein activation in a simple fashion from shifts in monophasic-inhibition curves. Moreover, it does not require [35S]GTPγS binding assays with functional D3 cells. This method will have wide applicability for D3-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.

Original languageEnglish (US)
Pages (from-to)7-15
Number of pages9
JournalJournal of Neuroscience Methods
Volume248
DOIs
StatePublished - Jun 5 2015
Externally publishedYes

Fingerprint

Dopamine D3 Receptors
Dopamine D2 Receptors
GTP-Binding Proteins
Spiperone
Guanosine Triphosphate
Cell Line
Essential Drugs
Preclinical Drug Evaluations
Drug Discovery
G-Protein-Coupled Receptors
Isotopes
Half-Life

Keywords

  • Affinity
  • Binding assay
  • D<inf>2</inf> receptors
  • D<inf>3</inf> receptors
  • Dopamine receptors
  • Efficacy
  • Functional assay
  • GTP shift
  • [<sup>35</sup>S] GTP<inf>γ</inf>S binding
  • [<sup>3</sup>H]spiperone binding

ASJC Scopus subject areas

  • Neuroscience(all)

Cite this

Use of radiolabeled antagonist assays for assessing agonism at D2 and D3 dopamine receptors : Comparison with functional GTPγS assays. / Zhen, Juan; Antonio, Tamara; Ali, Solav; Neve, Kim; Dutta, Aloke K.; Reith, Maarten E A.

In: Journal of Neuroscience Methods, Vol. 248, 05.06.2015, p. 7-15.

Research output: Contribution to journalArticle

Zhen, Juan ; Antonio, Tamara ; Ali, Solav ; Neve, Kim ; Dutta, Aloke K. ; Reith, Maarten E A. / Use of radiolabeled antagonist assays for assessing agonism at D2 and D3 dopamine receptors : Comparison with functional GTPγS assays. In: Journal of Neuroscience Methods. 2015 ; Vol. 248. pp. 7-15.
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AU - Antonio, Tamara

AU - Ali, Solav

AU - Neve, Kim

AU - Dutta, Aloke K.

AU - Reith, Maarten E A

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N2 - Background: Cell-based drug screening assays are essential tools for drug discovery and development targeting G protein-coupled receptors, which include dopamine D3 receptors. D3 is notorious for its poor coupling to G protein in most heterologous cell lines, and therefore D3 agonist-stimulated binding of [35S]GTPγS to G protein cannot be observed in many "non-functional" D3 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 [3H]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 [3H]spiperone binding assays for the assessment of agonists' potencies (at D2 and D3) and efficacies (at D3). Compared with GTPγ35S binding assays, the new method removes the cumbersome need of functional D3 cell lines and limited project duration due to short half-life of isotope 35S. Conclusion: The new method allows the estimation of potency (D2 and D3) and efficacy (D3) at the level of receptor and G protein activation in a simple fashion from shifts in monophasic-inhibition curves. Moreover, it does not require [35S]GTPγS binding assays with functional D3 cells. This method will have wide applicability for D3-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.

AB - Background: Cell-based drug screening assays are essential tools for drug discovery and development targeting G protein-coupled receptors, which include dopamine D3 receptors. D3 is notorious for its poor coupling to G protein in most heterologous cell lines, and therefore D3 agonist-stimulated binding of [35S]GTPγS to G protein cannot be observed in many "non-functional" D3 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 [3H]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 [3H]spiperone binding assays for the assessment of agonists' potencies (at D2 and D3) and efficacies (at D3). Compared with GTPγ35S binding assays, the new method removes the cumbersome need of functional D3 cell lines and limited project duration due to short half-life of isotope 35S. Conclusion: The new method allows the estimation of potency (D2 and D3) and efficacy (D3) at the level of receptor and G protein activation in a simple fashion from shifts in monophasic-inhibition curves. Moreover, it does not require [35S]GTPγS binding assays with functional D3 cells. This method will have wide applicability for D3-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.

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