1 Vol.:(0123456789) Scientific Reports | (2023) 13:10673 | https://doi.org/10.1038/s41598-023-37704-x www.nature.com/scientificreports Fluorescence lifetime FRET assay for live‑cell high‑throughput screening of the cardiac SERCA pump yields multiple classes of small‑molecule allosteric modulators Osha Roopnarine 1,4* , Samantha L. Yuen 1,4 , Andrew R. Thompson 1 , Lauren N. Roelike 1 , Robyn T. Rebbeck 1 , Philip A. Bidwell 2 , Courtney C. Aldrich 3 , Razvan L. Cornea 1 & David D. Thomas 1* 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 Ca 2+ ‑ATPase activity and Ca 2+ ‑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 Ca 2+ ‑transport even more than Ca 2+ ‑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. Sarco/endoplasmic reticulum calcium ATPase (SERCA), integral to the sarcoplasmic reticulum (SR, muscle) or endoplasmic reticulum (ER, non-muscle) membrane in most mammalian cells, uses Ca 2+ -dependent hydrolysis of ATP to fuel active transport (uptake) of cytosolic Ca 2+ into the SR or ER. e activity of SERCA1a (skeletal isoform) or SERCA2a (cardiac isoform) is essential for muscle relaxation (diastole), restoring SR Ca 2+ following its release via Ca 2+ channels (ryanodine receptors, RyR) for muscle contraction (systole). Decreased SERCA activity and excessive RyR leak results in failure to maintain the high gradient of [Ca 2+ ] between the cytoplasm (sub-μM) and the SR (mM) during diastole and are associated with heart failure (HF) in humans and animals 1 . Decreased SERCA activity is related to multiple factors, including reduced SERCA gene expression, increased post-translational modifications, and altered interaction with regulatory proteins 1 . Overall, decreased SERCA activity and increased Ca 2+ -leak can lead to a pathophysiological state of the cardiac myocyte 2 (HF, cardiac hypertrophy, diabetic hypertrophy), skeletal myofiber (Brody’s disease and myotonic dystrophy) 3 , or non-muscle cells (Darier’s disease, diabetes, Alzheimer’s disease) 4 . Altered SERCA interactions with regulatory proteins (regulins), e.g., phospholamban (PLB), have been linked to HF 5 . Of the seven known regulins 6 , the dwarf open OPEN 1 Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN, USA. 2 Department of Medicine, Cardiovascular Division, University of Minnesota, Minneapolis, MN 55455, USA. 3 Department of Medicinal Chemistry, University of Minnesota, Minneapolis, MN 55455, USA. 4 These authors contributed equally: Osha Roopnarine and Samantha L. Yuen. * email: roopn001@umn.edu; ddt@umn.edu