title: A Multiplexed Fluorescent Calcium and NFAT Reporter Gene Assay to Identify GPCR Agonists.
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.
authors.affiliation: Lawrence Livermore National Laboratory, Livermore, CA 94550, USA.
authors.forename: Heeral
authors.initials: H
authors.lastname: Sheth
authors.affiliation: Department of Chemical and Environmental Engineering, University of Toledo, Toledo OH 43606, USA.
authors.forename: Colleen
authors.initials: C
authors.lastname: Gorey
authors.affiliation: Pfizer Global Research and Development, Groton Laboratories, Groton, CT 06340, USA.
authors.forename: Nicole
authors.initials: N
authors.lastname: Roush
authors.affiliation: Pfizer Global Research and Development, Groton Laboratories, Groton, CT 06340, USA.
authors.forename: Shelly
authors.initials: S
authors.lastname: Smallman
authors.affiliation: Pfizer Global Research and Development, Groton Laboratories, Groton, CT 06340, USA.
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Collantes
authors.affiliation: Pfizer Global Research and Development, Groton Laboratories, Groton, CT 06340, USA.
authors.forename: Maxine
authors.initials: M
authors.lastname: Santoro
authors.affiliation: Pfizer Global Research and Development, Groton Laboratories, Groton, CT 06340, USA.
authors.forename: Barbara
authors.initials: B
authors.lastname: Olson
authors.affiliation: Pfizer Global Research and Development, Groton Laboratories, Groton, CT 06340, USA.
authors.forename: Laura
authors.initials: L
authors.lastname: Fitzgerald
authors.affiliation: Amgen, Inc., Lead Discovery, Thousand Oaks, CA 91320, USA.
authors.forename: Paul H
authors.initials: PH
authors.lastname: Lee
authors.affiliation: Pfizer Global Research and Development, Groton Laboratories, Groton, CT 06340, USA.
authors.forename: Xiqiang John
authors.initials: XJ
authors.lastname: Shen
keywords: FLIPR assay; GPCR; beta-lactamase reporter gene assay; fluorescent calcium assay; high throughput screening; multiplex assay.