Current Chemical Genomics and Translational Medicine

2013, 7 : 1-8
Published online 2013 April 3. DOI: 10.2174/2213988501307010001
Publisher ID: CCGTM-7-1

RESEARCH ARTICLE
 A Multiplexed Fluorescent Calcium and NFAT Reporter Gene Assay to Identify GPCR Agonists

Heeral Sheth 1 , Colleen Gorey 2 , Nicole Roush 3 , Shelly Smallman 3 , Elizabeth Collantes 3 , Maxine Santoro 3 , Barbara Olson 3 , Laura Fitzgerald 3 , Paul H Lee 4 and Xiqiang John Shen 3, *
1 Lawrence Livermore National Laboratory, Livermore, CA 94550, USA
2 Department of Chemical and Environmental Engineering, University of Toledo, Toledo OH 43606, USA
3 Pfizer Global Research and Development, Groton Laboratories, Groton, CT 06340, USA
4 Amgen, Inc., Lead Discovery, Thousand Oaks, CA 91320, USA

* Address correspondence to this author at the Therapeutics for Rare and Neglected Diseases (TRND), National Center for Advancing Translational Sciences (NCATS), National Institutes of Health (NIH), 9800 Medical Center Drive, Rockville, MD 20850, USA; Tel: +1-301-827-2512; Fax: +1-301-827-2534; E-mail: john.shen@nih.gov

ABSTRACT

Intracellular calcium response and resulting calcium signaling to an agonist-GPCR interaction are important for the measurement of compound activity in the GPCR drug development. The increase in cytosol calcium concentration can be measured by the fluorescent calcium indicator dye such as Fluo-4 in a quick assay (in 3-5 minutes) using the fluorescence imaging plate reader. The calcium signaling through the transcription factors such as NFAT that induces gene expression can be measured by the reporter gene assay that links to the expression of reporter enzyme such as the beta-lactamase that requires 5-hour incubation. We have evaluated a multiplexed assay that sequentially measures the calcium response to a GPCR agonist in a rapid fluorescent calcium dye assay, followed by a NFAT beta-lactamase assay, and compared them in the single assay format. We found that the agonist activity determined in the multiplexed assay were comparable with these determined in the single assay format and the Z’ factors were all > 0.5. Five active compounds were identified that were active in both calcium dye assay and beta-lactamase assay. Therefore, our results demonstrated the utility of this multiplexed calcium assay for screening of GPCR compounds that can cross validate the primary hits and help to eliminate the false positive compounds.

Keywords:

FLIPR assay, fluorescent calcium assay, beta-lactamase reporter gene assay, GPCR, high throughput screening, multiplex assay.