Live-Cell FRET Biosensors for High-Throughput Screening Targeting the SERCA2a/Plb Complex

Abstract

We have engineered cell-based high-throughput screening (HTS) methods using time-resolved fluorescence energy transfer (TR-FRET) sensitive to binding and structural dynamics of the SERCA2a/PLB complex. The membrane protein complex between the sarcoplasmic reticulum Ca-ATPase 2a (SERCA2a) and its regulator phospholamban (PLB) is a validated therapeutic target for reversing contractile dysfunction caused by aberrant calcium handling in the heart. Unfortunately, efforts to discover compounds with specificity for this complex have yet to yield an efficacious drug. GFP-SERCA2a (donor) was co-expressed in the endoplasmic reticulum of HEK293 cells with and without RFP-PLB (acceptor), and FRET was measured in a unique fluorescence lifetime microplate reader (FLT-PR), which increases the throughput of high-precision FLT measurements by several orders of magnitude. A triplicate screen against a small-molecule library containing 1500 compounds (LOPAC = Library of Pharmaceutically Active Compounds) identified ten hit compounds that reproducibly changed FRET by >3 SD and alter SERCA's ATPase activity. One compound increases SERCA2a Ca affinity and activity at physiological calcium levels in cardiac SR, but not in skeletal SR, suggesting that the compound is acting specifically on the SERCA2a/PLB complex (Stroik et al., Nature Scientific Reports 8:12560). These properties characterize a drug that could reverse calcium mishandling, as required for treatment of heart disease. We will present recent developments in the development and applications of live-cell FRET biosensors. This work was supported by NIH grants (GM27906, HL129814, AR07612, and DA037622).