Discovery of Enzyme Modulators via High-Throughput Time-Resolved FRET in Living Cells

Abstract

We have used a “2-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 ten compounds that significantly affected 2-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.

High-throughput screen, using time-resolved FRET, yields actin-binding compounds that modulate actin–myosin structure and function

“…FM-labeled donor actin was excited with a 473-nm microchip laser (Bright Solutions, Cura Carpignano, Italy), and emission was filtered with 488-nm long pass and 517/20-nm band pass filters (Semrock, Rochester, NY) (13,18). This instrument enables high-throughput fluorescence lifetime detection at high precision by utilizing a unique direct waveform-recording technology (40).…”

“…Fluorescence lifetime measurements were carried out by a high-precision FLTPR (Fluorescence Innovations, Inc., Minneapolis, MN) (13)(14)(15). FM-labeled donor actin was excited with a 473-nm microchip laser (Bright Solutions, Cura Carpignano, Italy), and emission was filtered with 488-nm long pass and 517/20-nm band pass filters (Semrock, Rochester, NY) (13,18).…”

“…We hypothesize that this TR-FRET approach can be used as a tool to screen for compounds that rescue defects in actomyosin structure and function. Small-molecule modulators of actomyosin structural dynamics represent potential leads for future drug development, and this search is greatly facilitated by recent developments in high-throughput FRET-based screening methods, which measure the effects of compounds on the distance between donor and acceptor probes on interacting proteins (1,(12)(13)(14)(15).…”

Phospholamban phosphorylation, mutation, and structural dynamics: a biophysical approach to understanding and treating cardiomyopathy

“…However, all of those compounds were direct SERCA2a activators, rather than SERCA2a-PLB uncouplers. Therefore, a subsequent screen was developed, in which FRET is measured within SERCA2a, using a 2-color fluorescent fusion construct of the enzyme (Gruber et al 2014) (Figure 8). This assay was further enhanced by employing a novel fluorescence lifetime plate-reader.…”

“…Translation from animals to humans may also present unforeseen problems as with PLB ablation, which is compatible with life in mice (Schmidt et al 2001) but lethal in humans (Hou et al 2008; Kelly et al 2008). Thus next-generation structurally designed PLB mutants, designed to increase SERCA2a affinity but also decrease inhibitory potency, while maintaining PKA-mediated phosphorylation, hold great promise (Gruber et al 2014; Lockamy et al 2011). Direct small-molecule activation of SERCA2a is also an attractive approach, since it is likely to preserve β-adrenergic control and variation of dosage is more feasible than for gene therapy (Gruber et al 2014).…”