Development of a High-Throughput Assay using R-CEPIA1er Fluorescence to Assess Small-Molecule Modulators of SERCA Activity in Living Cells

Abstract

The sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) is an essential component of Ca2+ signaling in most eukaryotic cells. Dysregulation of intracellular Ca2+ is associated with specific health problems, thus attracting attention for SERCA as a promising therapeutic target for skeletal and cardiac myopathies (e.g., Duchenne muscular dystrophy, sarcopenia, heart failure and atrial fibrillation). Here, we report on the development of a high-throughput assay to characterize novel drug-like small molecules that modulate SERCA-Ca2+ transport. This assay is based on microplate fluorescence measurement of ER luminal Ca2+ concentration ([Ca]ER) in HEK293 cells that co-express human SERCA2a and the ER-targeted Ca2+ biosensor R-CEPIA1er (genetically encoded, organelle-entrapped Ca2+ biosensor). We validated the cellular localization, biosensor response, and sensitivity limit of the R-CEPIA assays using fluorescence microscopy and plate-reader spectroscopy, including effects of the well-known Ca2+ pump inhibitors thapsigargin (TG) and cyclopiazonic acid (CPA). Microplate measurement and fluorescence intensity analysis revealed highly reproducible results for R-CEPIA1er detection of [Ca]ER inhibition by TG and CPA. Co-expression of the SERCA inhibitory peptide phospholamban (PLB) in HEK cells decreased [Ca]ER, demonstrating PLB-specific cardiac-type inhibition that was relieved by the known PLB antagonist, ellagic acid. We also analyzed the effect of additional small-molecule modulators of SERCA function and obtained results that are consistent with Ca2+-ATPase and Ca2+-transport assays using isolated sarcoplasmic reticulum vesicles. Thus, our new microplate-compatible, live-cell fluorescence assay offers a powerful combination of sensitivity and speed for high-throughput analysis of pre-clinical hit compounds that control SERCA function.