Sarcoplasmic Reticulum from Horse Gluteal Muscle Is Poised for Enhanced Calcium Transport

et al. 2023

Sci Rep

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.

Section: Resultsmentioning

confidence: 99%

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

et al. 2023

Sci Rep

“…We de ne "divergent" to indicate that the compound induces opposing effects at two different [Ca] (an increase at one [Ca] and a decrease at the other) in one assay. These effects indicate induced changes in the coupling ratio (CR), which is optimally two Ca 2+ ions transported per molecule of ATP hydrolyzed [27][28][29][30] . This change was determined from the ratio of V MAX (Ca 2+ -transport) to V MAX (Ca 2+ -ATPase) (Table 1), as discussed below in SERCA Activators.…”

Section: Validation Of Hit Compounds Using Flt Crcmentioning

confidence: 99%

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

et al. 2023

Preprint

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 a small validation library using novel microplate readers that can detect the fluorescence lifetime or emission spectrum with high speed, precision, and resolution. Here we report results from a 50,000-compound screen using the same biosensor, with hit compounds functionally evaluated using Ca2+-ATPase and Ca2+-transport assays. We focused on 18 hit compounds, from which we identified eight structurally unique compounds and four compound classes as SERCA modulators, approximately half of which are activators and half are inhibitors. 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.

“…We define "divergent" to indicate that the compound induces opposing effects at two different [Ca] (an increase at one [Ca] and a decrease at the other) in one assay. These effects indicate induced changes in the coupling ratio (CR), which is optimally two Ca 2+ ions transported per molecule of ATP hydrolyzed [27][28][29][30] . This change was determined from the ratio of VMAX (Ca 2+ -transport) to VMAX (Ca 2+ -ATPase) (Table 1), as discussed below in SERCA Activators.…”

Section: Effects Of Hit Compounds On Serca2a Activity Using Ca 2+ -At...mentioning

confidence: 99%

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

et al. 2023

Preprint

Self Cite

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 a small validation library using novel microplate readers that can detect the fluorescence lifetime or emission spectrum with high speed, precision, and resolution. Here we report results from a 50,000-compound screen using the same biosensor, with hit compounds functionally evaluated using Ca2+-ATPase and Ca2+-transport assays. We focused on 18 hit compounds, from which we identified eight structurally unique compounds and four compound classes as SERCA modulators, approximately half of which are activators and half are inhibitors. 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.