High-throughput FRET assay yields allosteric SERCA activators.

Area of Science:

• Biochemistry
• Cardiovascular Biology
• Pharmacology

Background:

• Aberrant calcium (Ca2+) regulation is a hallmark of heart failure.
• Sarcoplasmic reticulum Ca-ATPase (SERCA) activity is inhibited by phospholamban (PLB) in the heart.
• Compounds that reverse PLB inhibition of SERCA are potential therapeutics for heart failure.

Purpose of the Study:

• To identify compounds that disrupt the SERCA-PLB interaction and activate SERCA using a high-throughput screen (HTS).
• To validate a fluorescence resonance energy transfer (FRET) assay for screening SERCA modulators.
• To assess the efficacy of identified compounds in activating SERCA and improving cardiomyocyte function.

Main Methods:

• A FRET-based HTS was employed using a reconstituted membrane system with donor-SERCA and acceptor-PLB.
• A library of 20,000 compounds was screened for their ability to decrease FRET, indicating disrupted SERCA-PLB interaction.
• Hits were validated through secondary FRET assays and direct measurement of SERCA Ca-ATPase activity.

Main Results:

• The screen identified 43 initial hits, with a 0.2% hit rate.
• 72% of initial hits were false-positives, highlighting challenges in steady-state fluorescence assays.
• Six compounds significantly activated SERCA Ca-ATPase activity (up to 60%) and improved cardiomyocyte contractility.
• Preliminary data suggest a novel fluorescence lifetime plate reader offers improved precision for future HTS.

Conclusions:

• The FRET-based HTS successfully identified compounds that activate SERCA by reversing PLB inhibition.
• The study validates the FRET approach for identifying SERCA modulators, despite challenges with false-positives.
• Identified compounds represent promising leads for medicinal chemistry and preclinical development for heart failure treatment.
• Improved HTS instrumentation is crucial for enhancing the quality and efficiency of drug discovery efforts.