title: High-Throughput Spectral and Lifetime-Based FRET Screening in Living Cells to Identify Small-Molecule Effectors of SERCA.
abstract: A robust high-throughput screening (HTS) strategy has been developed to discover small-molecule effectors targeting the sarco/endoplasmic reticulum calcium ATPase (SERCA), based on a fluorescence microplate reader that records both the nanosecond decay waveform (lifetime mode) and the complete emission spectrum (spectral mode), with high precision and speed. This spectral unmixing plate reader (SUPR) was used to screen libraries of small molecules with a fluorescence resonance energy transfer (FRET) biosensor expressed in living cells. Ligand binding was detected by FRET associated with structural rearrangements of green fluorescent protein (GFP, donor) and red fluorescent protein (RFP, acceptor) fused to the cardiac-specific SERCA2a isoform. The results demonstrate accurate quantitation of FRET along with high precision of hit identification. Fluorescence lifetime analysis resolved SERCA's distinct structural states, providing a method to classify small-molecule chemotypes on the basis of their structural effect on the target. The spectral analysis was also applied to flag interference by fluorescent compounds. FRET hits were further evaluated for functional effects on SERCA's ATPase activity via both a coupled-enzyme assay and a FRET-based calcium sensor. Concentration-response curves indicated excellent correlation between FRET and function. These complementary spectral and lifetime FRET detection methods offer an attractive combination of precision, speed, and resolution for HTS.
authors.affiliation: 1 Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN, USA.
authors.forename: Tory M
authors.initials: TM
authors.lastname: Schaaf
authors.affiliation: 2 Fluorescence Innovations, Inc., Minneapolis, MN, USA.
authors.forename: Kurt C
authors.initials: KC
authors.lastname: Peterson
authors.affiliation: 2 Fluorescence Innovations, Inc., Minneapolis, MN, USA.
authors.forename: Benjamin D
authors.initials: BD
authors.lastname: Grant
authors.affiliation: 1 Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN, USA.
authors.forename: Prachi
authors.initials: P
authors.lastname: Bawaskar
authors.affiliation: 1 Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN, USA.
authors.forename: Samantha
authors.initials: S
authors.lastname: Yuen
authors.affiliation: 1 Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN, USA.
authors.forename: Ji
authors.initials: J
authors.lastname: Li
authors.affiliation: 1 Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN, USA.
authors.forename: Joseph M
authors.initials: JM
authors.lastname: Muretta
authors.affiliation: 2 Fluorescence Innovations, Inc., Minneapolis, MN, USA.
authors.forename: Gregory D
authors.initials: GD
authors.lastname: Gillispie
authors.affiliation: 1 Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN, USA.|3 Photonic Pharma LLC, Minneapolis, MN, USA.
authors.forename: David D
authors.initials: DD
authors.lastname: Thomas
mesh_terms: D015374:Biosensing Techniques; D055808:Drug Discovery / Q000295:instrumentation / Q000379:methods*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005453:Fluorescence; D031541:Fluorescence Resonance Energy Transfer / Q000295:instrumentation / Q000379:methods*; D015870:Gene Expression; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D057166:High-Throughput Screening Assays; D006801:Humans; D019044:Image Cytometry / Q000295:instrumentation / Q000379:methods*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D054852:Small Molecule Libraries / Q000494:pharmacology*; D019284:Thapsigargin / Q000494:pharmacology; D000097573:Red Fluorescent Protein
chemical_list: D004791:Enzyme Inhibitors; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D054852:Small Molecule Libraries; D019284:Thapsigargin; D000097573:Red Fluorescent Protein
keywords: biosensor; drug screening; fluorescence lifetime; spectral unmixing; time-resolved FRET