Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2008 Jul 11:1:70-8.
doi: 10.2174/1875397300801010070.

Comparison on functional assays for Gq-coupled GPCRs by measuring inositol monophospate-1 and intracellular calcium in 1536-well plate format

Ke Liu  1 , Steve Titus, Noel Southall, Pingjun Zhu, James Inglese, Christopher P Austin, Wei Zheng
Affiliations

Comparison on functional assays for Gq-coupled GPCRs by measuring inositol monophospate-1 and intracellular calcium in 1536-well plate format

Ke Liu et al. Curr Chem Genomics. .

Abstract

Cell-based functional assays used for compound screening and lead optimization play an important role in drug discovery for G-protein coupled receptors (GPCRs). Cell-based assays can define the role of a compound as an agonist, antagonist or inverse agonist and can provide detailed information about the potency and efficacy of a compound. In addition, cell-based screens can be used to identify allosteric modulators that interact with sites other than the binding site of the endogenous ligand. Intracellular calcium assays which use a fluorescent calcium binding dye (such as Fluo-3, Fluo-4 or Fura-2) have been used in compound screening campaigns to measure the activity of Gq-coupled GPCRs. However, such screening methodologies require a special instrumentation to record the rapid change in intracellular free calcium concentration over time. The radioactive inositol 1,4,5- triphosphate (IP(3)) assay measures (3)H-inositol incorporation and is another traditional assay for the assessment of Gq-coupled GPCR activity, but it is not suitable for screening of large size compound collections because it requires a cell wash step and generates radioactive waste. To avoid these limitations, we have optimized and miniaturized a TR-FRET based IP-One assay that measures inositol monophosphate in a 1536-well plate format. This assay is homogenous, non-radioactive and does not require a kinetic readout. It has been tested with the cell lines expressing M(1) acetylcholine, FFAR1, vasopressin V1b, or Neuropeptide S receptors. The activities of antagonists determined in the IP-One assay correlated well with these measured in the intracellular calcium assay while the correlation of agonist activities might vary from cell line to cell line. This IP-One assay offers an alternative method for high throughput screening of Gq-coupled GPCRs without using costly kinetic plate readers.

PubMed Disclaimer

Figures

Fig. (1)
Fig. (1)
Schematic representation of the principles for the intracellular calcium assay and the IP-One assay.
Fig. (2)
Fig. (2)
Comparison between the “wash” and “no wash” protocols for the IP-One assay. (a) The fluorescence intensity of the fluorescence donor and acceptor when assayed with the “wash” or “no wash” protocol. (b) HTRF ratios determined with the “wash” or “no wash” protocol.
Fig. (3)
Fig. (3)
Comparison between the adherent cell mode and the suspension cell mode for the IP-One assay. (a) Carbachol dose-response curves for the IP-One assay were determined with different densities of adherent cells. (b) Carbachol dose-response curves for the IP-One assay were determined with different densities of suspension cells.
Fig. (4)
Fig. (4)
Effects of serum and DMSO on the performance of the IP-One assay. (a) Effect of FBS concentration on the assay performance. (b) Effect of DMSO concentration on the assay performance.
Fig. (5)
Fig. (5)
Comparison of agonist and antagonist activities determined in IP-One and intracellular calcium assays. (a) Concentration responses of carbachol. (b) Concentration-responses of muscarinic acetylcholine antagonists. The EC80 amount of agonist, carbachol, was used in both assays (5 μM for the IP-One assay and 0.1 μM for the intracellular calcium assay).
Fig. (6)
Fig. (6)
DMSO plate tests in a M1-CHO cell line for the intracellular calcium and IP-One assays. (a) Scatter plot of the results from IP-One assay. All wells were stimulated the EC80 amount of carbachol (4 μM). Column 2 was received 50 μM atropine (IC100). (b) Scatter plot of the results from intracellular calcium assay. All wells in this plate were stimulated with 0.1 μM carbachol (in EC80). Column 1 was treated with 50 μM atropine (IC100) and column 2 was added with the EC100 amount of carbachol.
Fig. (7)
Fig. (7)
Correlation of known cholinergic antagonist activities between the IP-One and intracellular calcium assays. The LOPAC collection was screened in the qHTS format with both the IP-One assay and the calcium assay. The IC50 values of 30 know cholinergic antagonists determined from both screens were correlated.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Hopkins AL, Groom CR. The druggable genome. Nat Rev Drug Discov. 2002;1(9):727–30. - PubMed
    1. Pierce KL, Premont RT, Lefkowitz RJ. Seven-transmembrane receptors. Nat Rev Mol Cell Biol. 2002;3(9):639–50. - PubMed
    1. Eglen RM, Bosse R, Reisine T. Emerging concepts of guanine nucleotide-binding protein-coupled receptor (GPCR) function and implications for high throughput screening. Assay Drug Dev Technol. 2007;5(3):425–51. - PubMed
    1. Inglese J, Johnson RL, Simeonov A, Xia M, Zheng W, Austin CP, Auld DS. High-throughput screening assays for the identification of chemical probes. Nat Chem Biol. 2007;3(8):466–79. - PubMed
    1. Prystay L, Gagne A, Kasila P, Yeh LA, Banks P. Homogeneous cell-based fluorescence polarization assay for the direct detection of cAMP. J Biomol Screen. 2001;6(2):75–82. - PubMed

LinkOut - more resources