title: Discovery of enzyme modulators via high-throughput time-resolved FRET in living cells.
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.
authors.affiliation: 1Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN, USA.
authors.forename: Simon J
authors.initials: SJ
authors.lastname: Gruber
authors.forename: Razvan L
authors.initials: RL
authors.lastname: Cornea
authors.forename: Ji
authors.initials: J
authors.lastname: Li
authors.forename: Kurt C
authors.initials: KC
authors.lastname: Peterson
authors.forename: Tory M
authors.initials: TM
authors.lastname: Schaaf
authors.forename: Gregory D
authors.initials: GD
authors.lastname: Gillispie
authors.forename: Russell
authors.initials: R
authors.lastname: Dahl
authors.forename: Krisztina M
authors.initials: KM
authors.lastname: Zsebo
authors.forename: Seth L
authors.initials: SL
authors.lastname: Robia
authors.forename: David D
authors.initials: DD
authors.lastname: Thomas
mesh_terms: D000818:Animals; D015374:Biosensing Techniques / Q000379:methods*; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins / Q000737:chemistry; D057809:HEK293 Cells; D022781:Hepatocytes / Q000378:metabolism; D057166:High-Throughput Screening Assays; D006801:Humans; D008164:Luminescent Proteins / Q000737:chemistry; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D054852:Small Molecule Libraries; D000097573:Red Fluorescent Protein
chemical_list: D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D054852:Small Molecule Libraries; D000097573:Red Fluorescent Protein
keywords: FRET; HEK; LOPAC; SERCA2a; enzyme activation; screening