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• W3023211965 abstract "We have used a “2-color” SERCA (sarco/endo-plasmic reticulum calcium ATPase) biosensor and a high-throughput fluorescence lifetime plate-reader (FLT-PR) to develop a high-precision live-cell assay designed to screen for small molecules that perturb SERCA structure. We used a construct derived from canine cardiac SERCA, in which red fluorescent protein (RFP) was fused to the N terminus and green fluorescent protein (GFP) to an interior loop. This 2-color SERCA was stably expressed in HEK-GnTI- cells, a strain that can be grown in monolayers or in suspension, as needed for automated transfer to multiwell plates. Fluorescence resonance energy transfer (FRET) was measured from GFP to RFP using the FLT-PR, which increases precision by a factor of 30 over a conventional intensity-based plate-reader, without sacrificing throughput. FRET was highly sensitive to both known activators and inhibitors of SERCA. We screened a small (1280-compound) chemical library and identified nine compounds that significantly affect 2-color SERCA FLT. Three of these compounds affected FRET in a dose-dependent manner, and all three were found to inhibit SERCA function. Two of the hits were known SERCA inhibitors and the third was novel (Gruber et al., J. Biol. Screen, in press). This assay is being extended to several human isoforms of SERCA, for therapeutic applications to heart failure, muscular dystrophy, diabetes, and cancer. This assay is ready for a large-scale HTS campaign, and is adaptable to numerous protein targets. Spectroscopy was performed in the Biophysical Spectroscopy Center at the University of Minnesota, with assistance from Fluorescence Innovations, Inc. (Greg Gillispie, President). This work was funded by NIH grants to DDT (R01 GM27906, P30 AR0507220), to SJG (AHA 13PRE13230005), and to SLR (R01 HL106189)." @default.
• W3023211965 created "2020-05-13" @default.
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• W3023211965 date "2014-01-01" @default.
• W3023211965 modified "2023-10-18" @default.
• W3023211965 title "Discovery of Enzyme Modulators via High-Throughput Time-Resolved FRET in Living Cells" @default.
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• W3023211965 doi "https://doi.org/10.1016/j.bpj.2013.11.2409" @default.
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