Targeting protein-protein interactions for therapeutic discovery via FRET-based high-throughput screening in living cells

Abstract

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”.

Viral expression of a SERCA2a-activating PLB mutant improves calcium cycling and synchronicity in dilated cardiomyopathic hiPSC-CMs

“…28 We varied the ratio between the donor molecule (GFP-SERCA2a) and acceptor molecule (RFP-PLBWT), and found that the maximal energy transfer efficiency E (fractional decrease of the fluorescence lifetime) in this live-cell based system saturates at 0.10, as previously reported. 28 We transfected cells expressing GFP-SERCA2a and RFP-PLBWT with either untagged PLBWT or PLBM containing TM mutations (L31A, I40A, or L31A/I40A). Displacement of the RFP-PLBWT from its interaction with GFP-SERCA2a was observed as a decrease in the FRET efficiency relative to that measured in control cells expressing only the GFP-SERCA/RFP-PLBWT donor-acceptor pair (Fig.…”

“…5 We demonstrated previously that the interaction between the cardiac Ca 2+ pump, SERCA2a, and its principal inhibitor, PLB, can be measured via FRET in HEK293 cells. 28 In the present study, we applied this FRET assay to measure the competition of non-inhibitory PLBM and RFP-labeled PLBWT for binding to GFP-SERCA2a. We used this assay to measure the relative affinity of non-inhibitory PLBM and identified a double mutant (L31A/I40A) that binds with affinity greater than PLBWT.…”

“…2A, black) increases with Ca 2+ at physiological Ca 2+ concentrations (e.g., between pCa 7 and pCa 6) and saturates at higher Ca 2+ concentrations (pCa 5), consistent with previous measurements. 28 Concomitant expression of PLBWT ( Fig. 2A,B, blue) decreases Ca 2+ affinity (increases pKCa) of SERCA2a, and the additional expression of PLBWT (purple) or L31A-PLBM (cyan dashes) does not significantly decrease the apparent Ca 2+ affinity; indicating that SERCA2a is fully inhibited and that L31A-PLBM is not sufficient to relieve PLBWT inhibition under these experimental conditions.…”

“…We previously validated the performance of this FLT plate reader with known fluorescence standards, as well as with a FRET-based highthroughput screening strategy that that targeted 2-color SERCA. 28,29 FLT waveforms from donor-and donor/acceptor-labeled samples were analyzed as described in our previous publications and the supplemental methods. 28,29…”

“…28,29 FLT waveforms from donor-and donor/acceptor-labeled samples were analyzed as described in our previous publications and the supplemental methods. 28,29…”