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P2082
A Functional Assay for Gq-coupled GPCRs by Measuring Inositol Monophospate-1 in 1536-well Plate format
Presenter Ke Liu, NIH Chemical Genomics Center
Additional Authors: Steve Titus, Noel Southall, Pingjun Zhu, James Inglese, Christopher P. Austin, and Wei Zheng
The cell-based screen can provide the detailed information about a compound for its properties such as agonist, inverse agonist or antagonist and efficacy on a receptor of interest. In addition, it can be used to identify the allosteric modulator that interacts with a different site compared to the binding site of a known ligand on the receptor. The intracellular calcium assay with fluorescence dye (Fluo-3 or Fluo-4) has been used in the compound screen to measure the changes in intracellular calcium for the Gq-coupled GPCRs, which requires a special instrument for the kinetic recording of the rapid change in intracellular free calcium concentration. Inositol triphosphates (IP3) assay using 3H-inositol incorporation is another traditional assay for the assessment of Gq-coupled GPCRs but it is not suitable for screening of large size compound collections because it requires cell wash and generates radioactive waste. We have optimized and miniaturized a TR-FRET based IP-One assay in 1536-well plate format with the cell lines expressing M1 acetylcholine and other three receptors. While the agonists activities were generally more potent in the intracellular calcium assay, the antagonists activities were similar in both intracellular calcium and IP-One assays. Thus, the IP-One assay in TR-FRET detection format offers an alternative method for screening of Gq-coupled GPCRs without using the costly kinetic plate reader.