Using a Live-Cell High-Throughput Time-Resolved FRET Assay to Discover Enzyme Modulators

Abstract

We developed a high-precision high-throughput live-cell assay to screen for small molecules that activate the non-muscle sarco/endo-plasmic reticulum calcium ATPase (SERCA2b) using a “2-color” SERCA2b biosensor and a fluorescence-lifetime plate reader (FLT-PR). The 2-color SERCA2b (h2CS2b) biosensor was derived from a human pancreatic SERCA isoform, in which red fluorescent protein (mRuby2) was fused to the N terminus and green fluorescent protein (Clover) to an interior loop. A HEK cell line expressing the h2CS2b biosensors maintained stable expression level of the biosensors for several tens of passages, ideal for large scale HTS campaigns. This biosensor, upon binding of SERCA2b effectors, translates the minute SERCA structural rearrangements into small changes of intramolecular FRET that can only be reliably detected using fluorescence lifetime (FLT). The h2CS2b biosensor provided dose-dependent FRET changes in response to SERCA inhibitors such as thapsigargin, CPA, and BHQ, and SERCA activators previously discovered in this lab. Two instruments developed by Fluorescence Innovations were crucial for this work: a FLT plate reader, which provides a 30-fold increase of precision compared to conventional intensity-based instruments, and a high-speed fluorescence-spectra plate reader, which was used to filter out artifacts due to compound fluorescence. We screened a commercial chemical library of 50000 compounds and identified 94 compounds that significantly affect 2-color SERCA fluorescence lifetime. Functional assays are underway to evaluate the biochemical properties of these small molecules, and their potential for therapeutic application in diabetes and heart failure. This type of assay is being applied to several other drug targets, with promising results. This work was funded by NIH grants to DDT (R01 GM27906, P30 AR0507220), and to GDG (STTR,5R41DA037622-02).