High-Throughput FRET Screening in Living Cells Based on Lifetime Detection to Identify Small-Molecule Effectors of Serca

Abstract

The development of an effective high-throughput screening (HTS) paradigm using FRET biosensors in living cells has enabled us to identify multiple classes of small-molecules that alter the activity of the cardiac sarco/endoplasmic reticulum calcium ATPase (SERCA2a). We aim to discover small-molecule activators that improve SERCA's function for the treatment of heart failure. We have validated an intramolecular SERCA FRET biosensor (red and green fluorescent proteins fused to distinct SERCA domains) by screening a small test library using novel plate readers (PRs) that detect the fluorescence lifetime (FLT) (Schaaf et al, 2017). Here we describe an FLT-PR that scans a 1536-well plate in ≤2.5 min. This instrument is equipped with multichannel FLT detection at two emission wavelengths, used to filter out fluorescent compounds that would be falsely identified as hits. False hits were further eliminated using FRET biosensors based on non-specific peptide linkers, and using spectral analysis. FRET biosensors were developed and tested based on two different isoforms of SERCA: the SERC2a cardiac-specific isoform and SERCA2b non-muscle isoform were used to discover compounds with specificity toward SERCA2a. Concentration-response studies were performed to determine potency of compounds. Hits were assessed by two separate functional assays to determine how they alter ATPase activity and calcium-uptake. The majority were found to be inhibitors, but some were activators, and some compounds affected the coupling of SERCA's ATPase and calcium transport activities Our future focus is to evaluate the structure-function relationships of the newly discovered SERCA activators, using medicinal chemistry to optimize drug-like properties. These new HTS methods are being applied to a wide range of drug targets.