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J Biomol Screen. 2014 Feb;19(2):215-22. doi: 10.1177/1087057113510740.

Discovery of enzyme modulators via high-throughput time-resolved FRET in living cells.

Author information

1
1Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN, USA.

Abstract

We have used a "two-color" SERCA (sarco/endoplasmic reticulum calcium ATPase) biosensor and a unique high-throughput fluorescence lifetime plate reader (FLT-PR) to develop a high-precision live-cell assay designed to screen for small molecules that perturb SERCA structure. A SERCA construct, in which red fluorescent protein (RFP) was fused to the N terminus and green fluorescent protein (GFP) to an interior loop, was stably expressed in an HEK cell line that grows in monolayer or suspension. Fluorescence resonance energy transfer (FRET) from GFP to RFP was measured in the FLT-PR, which increases precision 30-fold over intensity-based plate readers without sacrificing throughput. FRET was highly sensitive to known SERCA modulators. We screened a small chemical library and identified 10 compounds that significantly affected two-color SERCA FLT. Three of these compounds reproducibly lowered FRET and inhibited SERCA in a dose-dependent manner. This assay is ready for large-scale HTS campaigns and is adaptable to many other targets.

KEYWORDS:

FRET; HEK; LOPAC; SERCA2a; enzyme activation; screening

PMID:
24436077
PMCID:
PMC4013825
DOI:
10.1177/1087057113510740
[Indexed for MEDLINE]
Free PMC Article

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