Juan Zhen1, Tamara Antonio2, Solav Ali3, Kim A Neve4, Aloke K Dutta5, Maarten E A Reith6. 1. Department of Psychiatry, New York University School of Medicine, New York, NY, USA. Electronic address: juan.zhen@nyumc.org. 2. Department of Psychiatry, New York University School of Medicine, New York, NY, USA. 3. Department of Neuroscience and Physiology, New York University School of Medicine, New York, NY, USA. 4. Research Service, Department of Veterans Affairs Medical Center, Portland, OR, USA. 5. Department of Pharmaceutical Sciences, Wayne State University, Detroit, MI, USA. 6. Department of Psychiatry, New York University School of Medicine, New York, NY, USA; Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, NY, USA.
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 [(35)S]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 [(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 D2 and D3) and efficacies (at D3). Compared with GTPγ(35)S 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 (35)S. 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 [(35)S]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.
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 [(35)S]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 [(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 D2 and D3) and efficacies (at D3). Compared with GTPγ(35)S 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 (35)S. 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 [(35)S]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.
Authors: Banibrata Das; Seenuvasan Vedachalam; Dan Luo; Tamara Antonio; Maarten E A Reith; Aloke K Dutta Journal: J Med Chem Date: 2015-11-30 Impact factor: 7.446
Authors: Thayssa Tavares da Silva Cunha; Rafaela Ribeiro Silva; Daniel Alencar Rodrigues; Pedro de Sena Murteira Pinheiro; Thales Kronenberger; Carlos Maurício R Sant'Anna; François Noël; Carlos Alberto Manssour Fraga Journal: Biomolecules Date: 2022-08-12
Authors: Mu-Fa Zou; Thomas M Keck; Vivek Kumar; Prashant Donthamsetti; Mayako Michino; Caitlin Burzynski; Catherine Schweppe; Alessandro Bonifazi; R Benjamin Free; David R Sibley; Aaron Janowsky; Lei Shi; Jonathan A Javitch; Amy Hauck Newman Journal: J Med Chem Date: 2016-04-01 Impact factor: 7.446