We have developed a structure-based high-throughput screening (HTS) method, using time-resolved fluorescence resonance energy transfer (TR-FRET) that is sensitive to protein-protein interactions in living cells. The membrane protein complex between the cardiac sarcoplasmic reticulum Ca-ATPase (SERCA2a) and phospholamban (PLB), its Ca-dependent regulator, is a validated therapeutic target for reversing cardiac contractile dysfunction caused by aberrant calcium handling. However, efforts to develop compounds with SERCA2a-PLB specificity have yet to yield an effective drug. We co-expressed GFP-SERCA2a (donor) in the endoplasmic reticulum membrane of HEK293 cells with RFP-PLB (acceptor), and measured FRET using a fluorescence lifetime microplate reader. We screened a small-molecule library and identified 21 compounds (Hits) that changed FRET by >3SD. 10 of these Hits reproducibly alter SERCA2a-PLB structure and function. One compound increases SERCA2a calcium affinity in cardiac membranes but not in skeletal, suggesting that the compound is acting specifically on the SERCA2a-PLB complex, as needed for a drug to mitigate deficient calcium transport in heart failure. The excellent assay quality and correlation between structural and functional assays validate this method for large-scale HTS campaigns. This approach offers a powerful pathway to drug discovery for a wide range of protein-protein interaction targets that were previously considered “undruggable”.
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Joseph M. Autry provided helpful discussion, and Octavian Cornea, Destiny Ziebol and Sarah Blakely Anderson provided administrative support. Fluorescence measurements were performed using facilities provided by the Biophysical Technology Center, University of Minnesota, and by Fluorescence Innovations, Inc. (Minneapolis, MN), where assistance was provided by Benjamin D. Grant and Kurt C. Peterson. DRS was supported by NIH Training Grant T32 AR07612. TMS was supported by NIH Institutional Research and Academic Career Development Award K12 GM119955. This work was supported by grants from NIH to DDT (R01 GM27906, R01 HL129814, and R37 AG26160), RJH (R01 HL129814), and to RLC (R01 HL092097 and R01 HL138539).