Discovery of enzyme modulators via high-throughput time-resolved FRET in living cells

J Biomol Screen. 2014 Feb;19(2):215-22. doi: 10.1177/1087057113510740.

Abstract

We have used a "two-color" SERCA (sarco/endoplasmic reticulum calcium ATPase) biosensor and a unique high-throughput fluorescence lifetime plate reader (FLT-PR) to develop a high-precision live-cell assay designed to screen for small molecules that perturb SERCA structure. A SERCA construct, in which red fluorescent protein (RFP) was fused to the N terminus and green fluorescent protein (GFP) to an interior loop, was stably expressed in an HEK cell line that grows in monolayer or suspension. Fluorescence resonance energy transfer (FRET) from GFP to RFP was measured in the FLT-PR, which increases precision 30-fold over intensity-based plate readers without sacrificing throughput. FRET was highly sensitive to known SERCA modulators. We screened a small chemical library and identified 10 compounds that significantly affected two-color SERCA FLT. Three of these compounds reproducibly lowered FRET and inhibited SERCA in a dose-dependent manner. This assay is ready for large-scale HTS campaigns and is adaptable to many other targets.

Keywords: FRET; HEK; LOPAC; SERCA2a; enzyme activation; screening.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Biosensing Techniques / methods*
  • Fluorescence Resonance Energy Transfer*
  • Green Fluorescent Proteins / chemistry
  • HEK293 Cells
  • Hepatocytes / metabolism
  • High-Throughput Screening Assays*
  • Humans
  • Luminescent Proteins / chemistry
  • Sarcoplasmic Reticulum Calcium-Transporting ATPases / antagonists & inhibitors
  • Sarcoplasmic Reticulum Calcium-Transporting ATPases / isolation & purification*
  • Sarcoplasmic Reticulum Calcium-Transporting ATPases / metabolism
  • Small Molecule Libraries

Substances

  • Luminescent Proteins
  • Small Molecule Libraries
  • red fluorescent protein
  • Green Fluorescent Proteins
  • Sarcoplasmic Reticulum Calcium-Transporting ATPases