Fluorescence lifetime FRET assay for live-cell high-throughput screening of the cardiac SERCA pump yields multiple classes of small-molecule allosteric modulators

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Abstract

We have used FRET-based biosensors in live cells, in a robust high-throughput screening (HTS) platform, to identify small-molecules that alter the structure and activity of the cardiac sarco/endoplasmic reticulum calcium ATPase (SERCA2a). Our primary aim is to discover drug-like small-molecule activators that improve SERCA’s function for the treatment of heart failure. We have previously demonstrated the use of an intramolecular FRET biosensor, based on human SERCA2a, by screening two different small validation libraries using novel microplate readers that detect the fluorescence lifetime or emission spectrum with high speed, precision, and resolution. Here we report results from FRET-HTS of 50,000 compounds using the same biosensor, with hit compounds functionally evaluated using assays for Ca2+-ATPase activity and Ca2+-transport. We focused on 18 hit compounds, from which we identified eight structurally unique scaffolds and four scaffold classes as SERCA modulators, approximately half of which are activators and half are inhibitors. Five of these compounds were identified as promising SERCA activators, one of which activates Ca2+-transport even more than Ca2+-ATPase activity thus improving SERCA efficiency. While both activators and inhibitors have therapeutic potential, the activators establish the basis for future testing in heart disease models and lead development, toward pharmaceutical therapy for heart failure.

Original languageEnglish (US)
Article number10673
JournalScientific reports
Volume13
Issue number1
DOIs
StatePublished - Dec 2023

Bibliographical note

Funding Information:
Prachi Bawaskar, Ben Grant, Simon J. Gruber, Evan W. Kleinboehl, Ang Li, Ji Li, Kurt Peterson, Seth L. Robia, and Tory M. Schaaf contributed to the conception of this project. Jesse E. McCaffrey, Bengt Svensson, Sarah Blakely Anderson, and J. Michael Autry provided helpful discussions. Marzena Brinkmann provided helpful advice on the chemical scaffold nomenclature. Bengt Svensson also provided technical assistance with illustrations. 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. This work was supported by NIH grants R01HL139065 (to DDT and RLC) and R37AG26160 (to DDT).

Funding Information:
Prachi Bawaskar, Ben Grant, Simon J. Gruber, Evan W. Kleinboehl, Ang Li, Ji Li, Kurt Peterson, Seth L. Robia, and Tory M. Schaaf contributed to the conception of this project. Jesse E. McCaffrey, Bengt Svensson, Sarah Blakely Anderson, and J. Michael Autry provided helpful discussions. Marzena Brinkmann provided helpful advice on the chemical scaffold nomenclature. Bengt Svensson also provided technical assistance with illustrations. 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. This work was supported by NIH grants R01HL139065 (to DDT and RLC) and R37AG26160 (to DDT).

Publisher Copyright:
© 2023, The Author(s).

PubMed: MeSH publication types

  • Journal Article
  • Research Support, N.I.H., Extramural

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