A high-throughput FRET screen of 20,000 compounds identified six novel allosteric activators that significantly increased SERCA activity by up to 60% and enhanced cardiomyocyte contractility.
A high-throughput FRET assay successfully identified novel allosteric SERCA activators that enhance cardiomyocyte contractility, providing potential leads for heart failure therapy.
Using fluorescence resonance energy transfer (FRET), we performed a high-throughput screen (HTS) in a reconstituted membrane system, seeking compounds that reverse inhibition of sarcoplasmic reticulum Ca-ATPase (SERCA) by its cardiac regulator, phospholamban (PLB). Such compounds have long been sought to correct aberrant Ca(2+) regulation in heart failure. Donor-SERCA was reconstituted in phospholipid membranes with or without acceptor-PLB, and FRET was measured in a steady-state fluorescence microplate reader. A 20 000-compound library was tested in duplicate. Compounds that decreased FRET by more than three standard deviations were considered hits. From 43 hits (0.2%), 31 (72%) were found to be false-positives upon more thorough FRET testing. The remaining 12 hits were tested in assays of Ca-ATPase activity, and six of these activated SERCA significantly, by as much as 60%, and several also enhanced cardiomyocyte contractility. These compounds directly activated SERCA from heart and other tissues. These results validate our FRET approach and set the stage for medicinal chemistry and preclinical testing. We were concerned about the high rate of false-positives, resulting from the low precision of steady-state fluorescence. Preliminary studies with a novel fluorescence lifetime plate reader show 20-fold higher precision. This instrument can dramatically increase the quality of future HTS.
Cornea et al. (Sat,) conducted a other in Heart failure. 20,000-compound library was evaluated on Decrease in FRET by more than three standard deviations. A high-throughput FRET screen of 20,000 compounds identified six novel allosteric activators that significantly increased SERCA activity by up to 60% and enhanced cardiomyocyte contractility.