High-Throughput spectral and lifetime-based FRET screening in living cells to identify small-molecule effectors of SERCA

Tory M. Schaaf; Kurt C. Peterson; Benjamin D. Grant; Prachi Bawaskar; Samantha Yuen; Ji Li; Joseph M Muretta; Gregory D. Gillispie; David D Thomas

doi:10.1177/1087057116680151

High-Throughput spectral and lifetime-based FRET screening in living cells to identify small-molecule effectors of SERCA

Tory M. Schaaf, Kurt C. Peterson, Benjamin D. Grant, Prachi Bawaskar, Samantha Yuen, Ji Li, Joseph M Muretta, Gregory D. Gillispie, David D Thomas

Research output: Contribution to journal › Article › peer-review

46 Scopus citations

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.

Original language	English (US)
Pages (from-to)	262-273
Number of pages	12
Journal	SLAS Discovery
Volume	22
Issue number	3
DOIs	https://doi.org/10.1177/1087057116680151
State	Published - Mar 2017

Bibliographical note

Funding Information:
Jesse E. McCaffrey, Bengt Svensson, Razvan L. Cornea, and J. Michael Autry provided helpful discussions, and Octavian Cornea prepared the manuscript for publication. Simon J. Gruber and Seth L. Robia developed many of the reagents and materials used. Fluorescence microscopy was performed at the UMN Imaging Center, flow cytometry at the UMN Lillehei Heart Institute, compound dispensing at the UMN Institute of Therapeutic Drug Discovery and Development, and spectroscopy at the UMN Biophysical Technology Center. pcDNAD1ER was a gift from Amy Palmer & Roger Tsien (Addgene plasmid 36325). The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by NIH grants R42DA037622 (to G.D.G. and D.D.T.), R01GM27906 (to D.D.T.), and R01HL129814 (to D.D.T.). T.M.S. was supported by the NIH Chemistry-Biology Interface Training Grant (5T32GM008700) and by predoctoral fellowships from 3M and Arnold H. Johnson.

Publisher Copyright:
© 2016 Society for Laboratory.

Keywords

Biosensor
Drug screening
Fluorescence lifetime
Spectral unmixing
Time-resolved FRET

Publisher link

10.1177/1087057116680151

Find It

Find It at U of M Libraries

Cite this

@article{33f1dc8b2ccd4519a83ff53024e10aac,

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{\textquoteright}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{\textquoteright}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.",

keywords = "Biosensor, Drug screening, Fluorescence lifetime, Spectral unmixing, Time-resolved FRET",

author = "Schaaf, {Tory M.} and Peterson, {Kurt C.} and Grant, {Benjamin D.} and Prachi Bawaskar and Samantha Yuen and Ji Li and Muretta, {Joseph M} and Gillispie, {Gregory D.} and Thomas, {David D}",

note = "Funding Information: Jesse E. McCaffrey, Bengt Svensson, Razvan L. Cornea, and J. Michael Autry provided helpful discussions, and Octavian Cornea prepared the manuscript for publication. Simon J. Gruber and Seth L. Robia developed many of the reagents and materials used. Fluorescence microscopy was performed at the UMN Imaging Center, flow cytometry at the UMN Lillehei Heart Institute, compound dispensing at the UMN Institute of Therapeutic Drug Discovery and Development, and spectroscopy at the UMN Biophysical Technology Center. pcDNAD1ER was a gift from Amy Palmer & Roger Tsien (Addgene plasmid 36325). The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by NIH grants R42DA037622 (to G.D.G. and D.D.T.), R01GM27906 (to D.D.T.), and R01HL129814 (to D.D.T.). T.M.S. was supported by the NIH Chemistry-Biology Interface Training Grant (5T32GM008700) and by predoctoral fellowships from 3M and Arnold H. Johnson. Publisher Copyright: {\textcopyright} 2016 Society for Laboratory.",

year = "2017",

month = mar,

doi = "10.1177/1087057116680151",

language = "English (US)",

volume = "22",

pages = "262--273",

journal = "SLAS Discovery",

issn = "2472-5552",

publisher = "Society for Laboratory Automation and Screening (SLAS)",

number = "3",

}

TY - JOUR

T1 - High-Throughput spectral and lifetime-based FRET screening in living cells to identify small-molecule effectors of SERCA

AU - Schaaf, Tory M.

AU - Peterson, Kurt C.

AU - Grant, Benjamin D.

AU - Bawaskar, Prachi

AU - Yuen, Samantha

AU - Li, Ji

AU - Muretta, Joseph M

AU - Gillispie, Gregory D.

AU - Thomas, David D

N1 - Funding Information: Jesse E. McCaffrey, Bengt Svensson, Razvan L. Cornea, and J. Michael Autry provided helpful discussions, and Octavian Cornea prepared the manuscript for publication. Simon J. Gruber and Seth L. Robia developed many of the reagents and materials used. Fluorescence microscopy was performed at the UMN Imaging Center, flow cytometry at the UMN Lillehei Heart Institute, compound dispensing at the UMN Institute of Therapeutic Drug Discovery and Development, and spectroscopy at the UMN Biophysical Technology Center. pcDNAD1ER was a gift from Amy Palmer & Roger Tsien (Addgene plasmid 36325). The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by NIH grants R42DA037622 (to G.D.G. and D.D.T.), R01GM27906 (to D.D.T.), and R01HL129814 (to D.D.T.). T.M.S. was supported by the NIH Chemistry-Biology Interface Training Grant (5T32GM008700) and by predoctoral fellowships from 3M and Arnold H. Johnson. Publisher Copyright: © 2016 Society for Laboratory.

PY - 2017/3

Y1 - 2017/3

N2 - 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.

AB - 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.

KW - Biosensor

KW - Drug screening

KW - Fluorescence lifetime

KW - Spectral unmixing

KW - Time-resolved FRET

UR - http://www.scopus.com/inward/record.url?scp=85021651726&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85021651726&partnerID=8YFLogxK

U2 - 10.1177/1087057116680151

DO - 10.1177/1087057116680151

M3 - Article

C2 - 27899691

AN - SCOPUS:85021651726

SN - 2472-5552

VL - 22

SP - 262

EP - 273

JO - SLAS Discovery

JF - SLAS Discovery

IS - 3

ER -

High-Throughput spectral and lifetime-based FRET screening in living cells to identify small-molecule effectors of SERCA

Abstract

Bibliographical note

Keywords

Publisher link

Find It

Other files and links

Fingerprint

Olympus Fluoview FV1000 IX2 Inverted Confocal with FLIM Detector

University Imaging Centers

Cite this

High-Throughput spectral and lifetime-based FRET screening in living cells to identify small-molecule effectors of SERCA

Abstract

Bibliographical note

Keywords

Publisher link

Find It

Other files and links

Fingerprint

University Assets

Olympus Fluoview FV1000 IX2 Inverted Confocal with FLIM Detector

University Imaging Centers

Cite this